Calcium Signalling in Cancer [1st ed.] 0849309425, 9780849309427, 9781420041293

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Calcium Signalling in Cancer

Calcium Signalling in Cancer Gajanan V. Sherbet Cancer Research Unit The Medical School University of Newcastle upon Tyne,UK and The Institute for Molecular Medicine Huntington Beach, CA USA

CRC Press Boca Raton London New York Washington, D.C.

Library of Congress Cataloging-in-Publication Data Sherbet, G. V. (Gajanan V.) Calcium signalling in cancer : the role of calcium binding proteins in signal transduction, cell proliferation, invasion, and metastasis / Gajanan V. Sherbet. p. cm. Includes bibliographical references and index. ISBN 0-8493-0982-4 (alk. paper) 1. Carcinogenesis. 2. Calcium-binding proteins. 3. Metastasis. 4. Cancer invasiveness. I. Title. [DNLM: 1. Neoplasm Proteins—physiology. 2. Calcium Signaling—physiology. 3. Calcium-Binding Proteins—physiology. 4. Cell Differentiation—physiology. 5. Neoplasm Invasiveness. 6. Neoplasm Metastasis. QZ 200 S551c 2000] RC268.5 .S53 2000 616.99′407—dc21 00-062115

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© 2001 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0982-4 Library of Congress Card Number 00-062115 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper

Dedication to Shri Sai Baba

Preface

The perfect messenger commands respect, delivers the message uncorrupted, at the right time, at the right place, with integrity. Thiru Valluvar (Tamil poet, second century, India) Thirukkural, Chapter 69, verse 688

Calcium signalling occupies a preeminent position in the signal transduction system of the cell by virtue of its participation in a wide range of physiological functions and in the biological events associated with genetic expression, cell proliferation and apoptosis, and cell differentiation and morphogenesis. Calcium signalling is an important feature of cell adhesion and motility, and of cancer invasion and metastasis. The calcium ion virtually unifies several pathways of signal transduction. It has rightly been described as a second messenger. Calcium-binding proteins are an important link in the calcium-signalling pathway. These proteins not only maintain the integrity of the calcium signal, but they are also responsible for transmitting the message in a temporally and spatially coordinated manner. It follows therefore that the integrity of the calcium binding proteins themselves is a basic requirement of normal biological function. This concept is encapsulated most eloquently in the Tamil quotation given above. If their integrity is compromised or lost from abnormal or inappropriate expression, or by genetic changes, that could lead to a profound deregulation of signal transduction with dramatic and wide-ranging effects on the life of the cell and its biological behaviour. That is the simple and singular justification for focusing on this protein species in this book. This volume is a natural sequel to my previous works. They are: The Biochemical and Biophysical Characterisation of the Cell Surface (Academic Press, 1978), The Biology of Tumour Malignancy (Academic Press, 1982), The Metastatic Spread of Cancer (Macmillan, 1987), and The Genetics of Cancer (Academic Press, 1997). In these one can discern the evolution of thought relating to the biological behaviour of cells and the pathogenesis of cancer, from inception of the tumour to its progression to the overt metastatic state. This book has not been written with the specialist only in mind. The discussion of topics is self-contained, as far as it was practicable, and I believe, therefore, it would be useful to students and research scientists alike.

Also, the presentation and discussions cater to the needs of scientists in related but peripheral fields of discipline. I had considerable help from my colleagues in the preparation of this book. Professor P.A. Riley of the University College School of Medicine and Dr. M.S. Lakshmi very kindly read the entire manuscript. Their comments and criticisms have been taken and consequently the manuscript has greatly improved in presentation and coverage. Dr. Lakshmi also kindly provided the Tamil quotation and its translation. I wish to thank them most sincerely for their help. It is needless to say, however, that the responsibility for errors and omissions rests solely with me. I thank Paula Rutter of the Audio-visual Centre for preparing the figures and for the patience and skill that she has displayed. CRC Press LLC received this book most enthusiastically and with great fervour. It has been a considerable pleasure working with them, especially with Fequiere Vilsaint, on this project. Finally, the North of England Cancer Research Campaign largely supported the research work in my laboratory and I wish to record my gratitude to them. Gajanan V. Sherbet Cancer Research Unit The Medical School University of Newcastle upon Tyne, UK and The Institute for Molecular Medicine Huntington Beach, CA, USA

Abbreviations βAPP ABP ACTH ACV aFGF ALS ANN APC AR BA-1p BAE BDNF bFGF BMD bp BPH BrDU CAI CaM cAMP CAMPK Capn CAR CBD CBD9K CBP cdk cGMP CH CHO CK CNS CP CRE CREB CS DAG DAGK db-cAMP

β-amyloid precursor protein Amyloid-β protein Adrenocorticotropic hormone Acyclovir Acidic fibroblast growth factor Amyotrophic lateral sclerosis Artificial neural network Adenomatous polyposis coli (protein) Androgen receptor Bone-specific alkaline phosphatase Bovine aortic endothelial (cell) Brain-derived neurotropic factor Basic fibroblast growth factor Becker muscular dystrophy Base pair(s) Benign prostatic hyperplasia Bromo-deoxyuridine Carboxyamido-triazole Calmodulin Cyclic 3′,4′-adenosine monophosphate Calcium/calmodulin-dependent protein kinase Calpain Cancer-associated retinopathy Calbindin D-28K Calbindin D-9K Calcium-binding protein Cyclin-dependent protein kinase Cyclic guanosine monophosphate Calponin homology (domain) Chinese hamster ovary (cell line) Casein kinase Central nervous system (Actin) capping protein Cyclic AMP response element Cyclic AMP response element binding protein Cowden’s syndrome 1,2-Diacylglycerol Diacylglycerol kinase Dibutyryl cyclic AMP

DD DMD DMSO EAE EC ECM EDC EFABP EGF EGFr eNOS Ep-ICAM ER ER ERK ES FC FN FGF FLG FSP1 GABA GAP GC GCAP GDNF GDP GFAP GRP GRPr GTP GTPase 4-HNE HO-1 HSP HUVEC ICAM ICE ICTP IF IFN IGF IL IP3 IP3R IP3R-P

Darier’s disease Duchenne muscular dystrophy Dimethyl sulphoxide Experimental allergic encephalomyelitis Endothelial cell Extracellular matrix Epidermal differentiation complex Epidermal-type fatty acid-binding protein Epidermal growth factor Epidermal growth factor receptor Endothelial nitric oxide synthase Epithelial intercellular adhesion molecule Endoplasmic reticulum Oestrogen receptor Extracellular signal-regulated receptor kinase (or MAPK) Embryonic stem (cells) Follistatin-like (domain) Fibronectin Fibroblast growth factor Profilaggrin Fibroblast-specific protein 1 (S100A4) Gamma-amino butyric acid GTPase-activating protein Guanylate cyclase Guanylate cyclase-activating protein Glial cell-derived growth factor Guanosine diphosphate Glial fibrillary acidic protein Gastrin-releasing peptide Gastrin-releasing peptide receptor Guanosine triphosphate Guanosine triphosphatase 4-Hydroxynonenal Heme oxygenase-1 Heat shock protein Human umbilical vein endothelial cell Intercellular adhesion molecule Interleukin-1β-converting enzyme Pyridinoline cross-linked telopeptide of type I procollagen Intermediate filaments Interferon Insulin-like growth factor Interleukin Inositol 1,4,5-trisphosphate Inositol 1,4,5-trisphosphate receptor Phosphorylated form of inositol 1,4,5-trisphosphate receptor

JAK LAK LGMD L-NAME LOH LPS MAP MAPK MB-40 MBP MBPK 5-MC MDBK MHC MHC MLC MLCK MMP MMTV MS MSH MTase NAD NCAM NCBP NDP NF2 NF-AT NFT NGF NMDA NOS NSCLC NT3 NMU OA OM ORF OSE OSE-bp OT PA PAI PARP PCNA PCR

Janus tyrosine kinase Lymphokine-activated killer (cells) Limb girdle muscular dystrophy NG-Nitro-l-arginine methyl ester Loss of heterozygosity Lipopolysaccharide Microtubule associated protein Mitogen-activated protein kinase (or ERK) Basement membrane-40 protein (SPARC, osteonectin) Myelin basic protein Myelin basic protein kinase 5-Methylcytosine Madin-Darby bovine kidney (cell line) Major histocompatibility complex Myosin heavy chain Myosin light chain Myosin light chain kinase Matrix metalloproteinases Murine mammary tumour virus Multiple sclerosis Melanocyte-stimulating hormone Methyltransferase Nicotinamide adenine dinucleotide Neural cell adhesion molecule Neural calcium binding protein Nucleoside diphosphate Neurofibromatosis type 2 Nuclear factor of activated T cells Neurofibrillary tangles Nerve growth factor N-Methyl-d-aspartate Nitric oxide synthase Non-small cell lung carcinoma Neurotropin-3 N-methyl-N-nitrosourea Osteoarthritis Oncomodulin Open reading frame Osteonectin silencer element Osteonectin silencer element-binding protein Oxytocin Plasminogen activator Plasminogen activator inhibitor Poly (ADP-ribose) polymerase Proliferating cell nuclear antigen Polymerase chain reaction

PDE PDGF PDI PEP1 PEST PGE PgR PH PHF PI3K PICP PIIINP PIP PIP2 PIP3 PIP4 PKA PKC PLA2 PLC PMA PMCA PMN PP-1A/PP-1B PS PSA PSA-NCAM PT PTTH PV RA RA rb RBP RCN RGS RP rptn RSV RT RTK RT-PCR RV RXR RyR SCCA

Phosphodiesterase Platelet-derived growth factor Protein disulphide isomerase Profilaggrin endopeptidase 1 Pro, Asp/Glu, Ser, Thr sequence (in proteins) Prostaglandin E Progesterone receptor Pleckstrin homology [domain] Paired helical filaments Phosphoinositide-3 kinase C-Terminal peptide of type I procollagen N-Terminal peptide of type III procollagen Phosphatidyl inositol-3 phosphate Phosphatidyl inositol 4,5-bisphosphate Phosphatidyl inositol 3,4,5-trisphosphate Phosphatidyl inositol 1,3,4,5-tetrakisphosphate Protein kinase A Protein kinase C Phospholipase A2 Phospholipase C Phorbol 12-myristate 13-acetate Plasma membrane Ca2+-ATPase Polymorphonuclear leukocyte Protein phosphatases 1A and 1B Presenilin (genes 1 and 2 in Alzheimer’s disease) Prostate-specific antigen Polysialylated NCAM Demyelinating paralytic tremor rabbit mutant Prothoracicotropic hormone Parvalbumin Retinoic acid Rheumatoid arthritis Retinoblastoma susceptibility (gene product) Retinol-binding protein Recoverin Regulator of G-protein signalling (proteins) Retinitis pigmentosa Repetin Rous sarcoma virus Reverse transcriptase Receptor tyrosine kinase Reverse transcriptase polymerase chain reaction Rubella virus Retinoid X receptor Ryanodine receptor Squamous cell lung carcinoma antigen

SCLC SDK SERCA SLE SMS SPARC SR STAT SVZ Tβ TCR TGF THH TIMP TK Tn TNF TPA tPA uPA VASP VCAM VD3 VDR VDRE VEGF VGCC VILIP VP WAS WASP

Small cell lung carcinoma Sphingosine-dependent kinase Sarcoplasmic–endoplasmic reticulum Ca2+-ATPase Systemic lupus erythematosus Smith–Magenis syndrome Secreted protein, acidic, rich in cysteine (osteonectin; BM-40) Sarcoplasmic reticulum Signal transducer and activator of transcription factors Subventricular zone Beta thymosins T-cell antigen receptor Transforming growth factor Trichohyalin Tissue inhibitor of metalloproteinase Thymidine kinase Troponin Tumour necrosis factor 12-O-Tetradecanoyl phorbol 13-acetate Tissue plasminogen activator Urokinase-type plasminogen activator Vasodilator-stimulated phosphoprotein Vascular cell adhesion molecule Vitamin D3 Vitamin D3 receptor Vitamin D response element Vascular endothelial growth factor Voltage-gated calcium channel Visinin-like protein Vasopressin Wiskott–Aldrich syndrome Wiskott–Aldrich syndrome protein

Author Dr. Gajanan Sherbet is a professor at the Institute for Molecular Medicine, Huntington Beach, CA. He received his D.Sc. and M.Sc. degrees from the University of London, and Ph.D., M.Sc., and B.Sc. degrees from the University of Poona. Dr. Sherbet was reader in Experimental Oncology and deputy director of the Cancer Research Unit in the Medical School of the University of Newcastle upon Tyne, England. Previous to this Dr. Sherbet was a staff member of the Chester Beatty Research Institute, Institute of Cancer Research, and University College Hospital Medical School in London. He has held prestigious fellowships such as the Beit Memorial and Williams Fellowship of the University of London. He held a career fellowship awarded by the North of England Cancer Research Campaign. For a brief period, he was a fellow of Harvard University, Cambridge, MA. Dr. Sherbet is a fellow of the Royal College of Pathologists (FRCPath), Royal Society of Chemistry (FRSC), and The Institute of Biology (FIBiol) of U.K. He served as editor of Oncology and Experimental Cell Biology and was senior editor of Pathobiology. Currently, he is a member of the editorial boards of Pathobiology and Anticancer Research. Dr. Sherbet’s major interest is in cancer metastasis. In recent years he has been investigating the role of the calcium binding protein S100A4 in cell proliferation, cancer invasion, and metastasis, focusing mainly on melanomas, neuroectodermal tumors, and breast cancer. He recently demonstrated the potential value of S100A4 as a marker for assessing the progression of breast cancer. He is also studying the potential of artificial neural networks in the management of breast cancer, especially the analysis of expression of cancer markers and image cytometric data of breast cancer by using artificial neural networks. Dr. Sherbet has published numerous scientific papers in international journals. He has written and edited several books on cancer. Notable among them are The Metastatic Spread of Cancer (Macmillan, 1987), The Biology of Tumour Malignancy (Academic Press, 1982), and The Biochemical and Biophysical Characterisation of the Cell Surface (Academic Press, 1978). His latest book, The Genetics of Cancer (Academic Press, 1997), was co-authored with Dr. M.S. Lakshmi. He was guest editor of Retinoids: Their Physiological Function and Therapeutic Potential (1997). Dr. Sherbet is co-editor of Artificial Neural Networks in Cancer Diagnosis, Prognosis, and Patient Management to be published soon by CRC Press LLC.

Contents Preface Abbreviations Chapter 1 Introduction................................................................................................................1 Chapter 2 The Calcium Signalling Pathway..............................................................................5 Homeostasis of Cell Calcium...........................................................................5 The Plasma Membrane Ca2+-ATPase Pump ........................................5 The Sarcoplasmic-Endoplasmic Reticulum Ca2+-ATPase Pump .....................................................................................................7 Voltage-Gated Calcium Channels.........................................................8 The Deregulation of Calcium Homeostasis as a Primary Event in Carcinogenesis .......................................................................9 Phospholipid Signalling..................................................................................10 PTEN Phosphatase in the Regulation of Lipid Signalling ............................11 The Protein Kinase C Pathway ......................................................................13 Protein Kinase C and Its Isoforms in Signal Transduction ...........................14 Inositol Phosphates in Calcium Signal Transduction ....................................16 Deregulation of Inositol 1,4,5-Trisphosphate Pathway and Its Consequences ............................................................................................18 Ryanodine and Related Receptors in Calcium Mobilisation.........................20 Cyclic AMP in Calcium Signalling ...............................................................21 Architectural Aspects of the Signal Transduction Machinery.......................25 The Role of Caveolae in Signal Transduction ...................................25 Caveolin Expression in Cancer ..........................................................28 Chapter 3 Calcium Binding Proteins and Their Natural Classification ..................................29 Chapter 4 Non-EF-Hand Calcium Binding Proteins ...............................................................35 Annexins .........................................................................................................35 Structure..............................................................................................35 Biologic Functions..............................................................................36 Annexins in Cancer Growth and Progression....................................38 Annexins in Morphogenesis and Differentiation ...............................39

The Gelsolin Family of Calcium Binding Proteins .......................................40 Gelsolin in Severing and Capping of Actin Filaments ......................40 Gelsolin in Embryonic Development and Morphogenesis ................41 Gelsolin Expression in Amyloidosis ..................................................42 Gelsolin in Cancer ..............................................................................42 Severin and Cytoskeletal Reorganisation...........................................44 Villin in Differentiation and Neoplasia..............................................44 Calreticulin and Its Functional Diversity .......................................................46 Structure and Molecular Features of Calreticulin..............................46 Regulation of Calreticulin Expression ...............................................46 Phosphorylation of Calreticulin..........................................................47 Intracellular Distribution of Calreticulin............................................48 Calreticulin in Intracellular Calcium Storage ....................................48 Calreticulin and Calnexin as Molecular Chaperones.........................49 Calreticulin in Cell Proliferation and Differentiation ........................50 Calreticulin in Cell Adhesion .............................................................50 Calreticulin in Neoplasia ....................................................................51 Immunological Implications of Calreticulin Function.......................52 Calsequestrin and Intracellular Calcium Storage...........................................53 Osteocalcin in Bone Metabolism and Osteotropism of Cancer ....................54 The Biology of Osteocalcin ...............................................................54 Calcium-Binding Properties of Osteocalcin.......................................55 Osteocalcin Gene Structure and Function..........................................55 Regulation of Osteocalcin by Vitamin D3 .........................................56 Osteocalcin in Cell Proliferation and Differentiation ........................57 Osteotropism of Metastatic Dissemination ........................................60 Chapter 5 The EF-Hand Calcium-Binding Proteins ................................................................63 Molecular Organisation of Calcium Binding EF-Hand Proteins ..................63 Calcium Binding and the Molecular Configuration of Calcium-Binding Proteins ...........................................................................................................65 The Structure and Organisation of S100 Family Genes................................68 Alternatively Spliced Variants of S100A4 .....................................................68 Functional Significance of Alternatively Spliced Isoforms ...........................69 Regulation of Expression of S100 Family Genes..........................................71 Transcriptional Regulation of S100 Genes ........................................71 Regulation of Gene Expression by DNA Methylation......................72 DNA Methylation in Cancer ..............................................................72 Regulation of S100 Gene Transcription by Methylation...................74 Chapter 6 The Calmodulin Family of Calcium Binding Proteins...........................................75 Calmodulin and Its Physiological Function...................................................75 Structure and Mode of Action of Calmodulin ...................................75

Calmodulin-Mediated Signal Transduction........................................76 Calmodulin and Cell Proliferation .....................................................77 Calmodulin in Neoplasia ....................................................................78 Recoverin Subfamily of Neural Calcium-Binding Proteins and Their Function ..........................................................................................................79 The G-Protein Signalling Pathway.....................................................79 Recoverin and Its Function ................................................................80 Mode of Action of Recoverin.............................................................82 Post-translational Modification of Recoverin ....................................82 Recoverin and Cancer-Associated Retinopathy .................................83 Recoverin and Cancer-Associated Retinopathy in Small Cell Lung Cancer ......................................................83 Retinopathy Associated with Other Forms of Human Cancer .....................................................................84 Is Recoverin Involved in Retinitis Pigmentosa? .................85 Guanylate Cyclase-Activating Proteins..........................................................85 Chapter 7 The Structure of Contractile Proteins .....................................................................87 The Actin Component of Contractile Machinery of the Cell........................87 Actin Isoforms ....................................................................................87 Regulation of Actin Dynamics ...........................................................88 Cofilin in the Regulation of Actin Dynamics ....................................88 Profilin in the Regulation of Actin Dynamics ...................................90 Rho GTPases in Actin Dynamics and Signal Transduction ..............90 Interaction of Formin with Profilin and Rho GTPases......................92 The Role of Thymosin Family Actin-Binding Proteins in Actin Dynamics ..............................................................................................93 Sequestration of Actin by Thymosins ................................................93 Effects of Thymosins on Cell Proliferation .......................................93 Thymosins and Cell Motility and Differentiation .............................94 Expression of Thymosins in Embryonic Development .....................94 Potential Role of Thymosins in Cancer Progression.........................95 The Fimbrin Family of Actin-Binding Proteins............................................. 96 Molecular Features of Fimbrin...........................................................96 Function of Fimbrin in Cytoskeletal Organisation ............................97 Regulation of Fimbrin Expression .....................................................99 Is Fimbrin Involved in Cancer?........................................................100 Modulation of Actin Dynamics and Cancer Cell Dissemination ...................................................................................101 α-Actinin.......................................................................................................102 Molecular Structure of α-Actinin ....................................................102 α-Actinin Isoforms ...........................................................................103 Function of α-Actinin.......................................................................104

Actinins in Cell Adhesion, Motility, and Signal Transduction........104 The Cadherin–Catenin Complex in Signal Transduction and Cell Adhesion ...................................................................................104 Myosin Filaments .........................................................................................110 Myosin Heavy Chain (MHC) Isoforms ...........................................111 Actomyosin Assembly......................................................................112 Myosin Light Chain (MLC) Phosphorylation and Function ...........115 Troponins and Tropomyosins in the Regulation of Muscle Cell Contraction....................................................................................................118 The Regulatory Role of Troponins and Tropomyosins in Muscle Contraction........................................................................................118 Tropomyosin Isoforms in Benign and Malignant Cells ..................119 The Regulatory Role of Caldesmon.................................................121 Calponin: Its Function and Regulation ............................................122 Caltropin-Mediated Reversal of Myosin-ATPase Inhibition by Caldesmon and Calponin..................................................................124 Chapter 8 Structure and Biology of Calbindin ......................................................................125 Calbindin in Neuronal Populations ..............................................................125 Neural Cell Lineage and the Regulation of Calbindin Expression .............125 Calbindin Expression in Embryonic Development and Ageing..................126 Physiological Function of Calbindin............................................................127 Neuroprotective Function of Calbindin........................................................129 Calbindin Expression and the Metastatic Phenotype ..................................129 Chapter 9 Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker......................................................................................................131 Calretinin and Its Alternatively Spliced Isoforms........................................131 Regulation of Calretinin Expression ............................................................132 Calretinin Expression in Cell Proliferation and Differentiation..................133 Calretinin and Its Possible Neuroprotective Property..................................133 Calretinin as a Potential Tumour Marker.....................................................133 Chapter 10 Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading ................135 Molecular Features of Calcineurin...............................................................135 Calcineurin in Cell Proliferation and Adhesion-Related Phenomena .........136 Putative Role of Calcineurin in Cell Cycle Progression .................136 The Effects of Calcineurin on Cell Adhesion and Motility ............138 Calcineurin in Alzheimer’s Disease .............................................................140 Calcineurin in Immunosuppression..............................................................141

Chapter 11 Centrins (Caltractins) and Their Biological Functions .........................................145 Chapter 12 Reticulocalbin Family of EF-Hand Proteins.........................................................149 Molecular Features of Reticulocalbin Homologues ....................................150 Putative Functions of Reticulocalbin and Its Homologues .........................150 Chapter 13 Calpains in Normal and Aberrant Cell Physiology ..............................................153 The Calpain Family of Calcium-Binding Proteins ......................................153 Molecular Organisation of Calpains ............................................................154 Regulation of Physiological Events by Proteolytic Function......................155 Involvement of Calpains in Development and Differentiation....................157 Calpains in Cell Proliferation and Apoptosis ..............................................158 Calpains in Cell Spreading and Migration ..................................................160 Calpains in Integrin-Mediated Cell Adhesion and Signal Transduction..................................................................................................161 Calpains in Cancer Growth and Progression ...............................................162 Calpains in Myelodegenerative Diseases .....................................................163 Calpains in Muscular Dystrophy..................................................................165 Association of Calpains with Duchenne Muscular Dystrophy .......165 Calpains and Limb Girdle Muscular Dystrophy..............................166 Chapter 14 Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia .................169 Caspases in Apoptotic Cell Death................................................................169 Poly (ADP-Ribose) Polymerase as a Marker of Apoptosis ........................172 Caspase-Mediated Apoptosis and Cell Growth Inhibition in Tumour Expansion........................................................................................173 Caspase-Mediated Proteolysis of Fodrin: Implications for Apoptosis, Cell Adhesion, Cell Migration, and Neoplastic Transformation .................176 Caspases and Neuronal Loss in Alzheimer’s Disease .................................177 Chapter 15 Parvalbumins in Neuronal Development, Differentiation, and Proliferation .......181 Chapter 16 Osteonectin in Cell Function and Behaviour........................................................183 Molecular Structure of Osteonectin .............................................................183 Functions and Functional Domains of Osteonectin.....................................184 Regulation of Osteonectin Expression .........................................................184 Osteonectin in the Remodelling of the Extracellular Matrix ......................186

Osteonectin in Embryonic Development and Differentiation .....................187 Modulation of Cellular Adhesion, Cell Shape, and Motility by Osteonectin ..............................................................................................188 Modulation of Cell Proliferation by Osteonectin ........................................190 Effects of Osteonectin on Angiogenesis ......................................................191 Osteonectin Expression in Cancer Development and Progression..............193 Osteonectin Involvement in Other Disease States .......................................196 Osteonectin Homologues and Their Putative Tumour Suppressor Properties ......................................................................................................197 Chapter 17 S100 Proteins: Their Biological Function and Role in Pathogenesis ..................199 S100 Proteins in Cell Differentiation, Motility, and Cancer Invasion ........202 Profilaggrin (FLG) in Keratinocyte Differentiation.........................202 The Molecular Characteristics of Profilaggrin..................202 Trichohyalin (THH)..........................................................................205 Effects of S100 Proteins on Cell Deformability and Cellular Morphology ..................................................................................................205 Cell Adhesion and Invasive Potential of Cancer Cells ....................210 S100 Proteins in Remodelling of the Extracellular Matrix.............213 S100 Proteins in Cell Proliferation ..................................................214 Cell Cycle-Related Expression of S100 Proteins ............................217 Postulated Mechanism of Cell Cycle Control by S100A4..............219 S100A Isoforms............................................................................................222 S100A2 as a Putative Tumour Suppressor...................................................223 S100A3 Expression in Cell Differentiation and Neoplasia.........................224 Molecular Features of S100A3 ........................................................224 S100A3 Expression in Cell Differentiation and Human Gliomas .............................................................................................225 S100A4 in Cancer Development and Progression.......................................225 S100A4 Expression and Metastatic Potential of Cancers ...............225 Clinical Potential of S100A4 as a Marker for Cancer Prognosis...........................................................................................230 S100A4 in Human Breast Cancer ....................................................230 S100A4 in Other Forms of Human Cancer .....................................234 S100A6 (Calcyclin) in Cancer .....................................................................235 The Biological Properties of S100A7 (Psoriasin) .......................................236 Structure and Molecular Properties of S100A7...........................................236 S100A7 in Skin Pathology ...............................................................237 S100A7 in Neoplastic Disease .........................................................238 S100A8 and S100A9 Proteins in Inflammatory Diseases ...........................239 S100A11 (S100C) and Possible Modes of its Function..............................239 S100P in Cancer Progression .......................................................................241 S100P and Its Putative Functions.....................................................241

Potential Value of S100 Proteins as Markers of Cancer Progression and Prognosis.......................................................................................................243 Epilogue ................................................................................................................245 References .............................................................................................................249 Index......................................................................................................................349

1

Introduction

Calcium-binding proteins (CBPs) are a family of unique importance in normal and aberrant cell biology, by virtue of their participation in a wide spectrum of physiological processes. These proteins appear to function from the inception of life, participating in sperm maturation and motility and implantation of the fertilised ovum, to cell differentiation and morphogenesis. Among other important physiological functions of CBPs are their involvement in Ca2+ transport and the maintenance of intracellular Ca2+ levels, as well as metabolic processes such as nucleotide metabolism. Signal transduction is another area of cellular physiology in which CBPs are heavily involved. Defective signal transduction is often associated with aberrant cell cycle regulation, which in turn can lead to the development and progression of cancer. Besides, several CBPs, especially those belonging to the S100 protein family, have recently been demonstrated to be actively engaged in the regulation of the cell cycle in normal as well as in aberrant cell proliferation and in apoptotic cell death. It follows, therefore, that CBPs may be associated with the pathogenesis of several diseases, including neoplastic disease. Directly relevant in the context of carcinogenesis is the accumulation of a substantial body of evidence demonstrating that CBPs participate in the formation and emergence of neoplastic foci of cells and in the invasive and metastatic spread of these cells constituting the progressive phase of the disease. A major pathway by which CBPs affect neoplastic progression is by altering cell proliferation via the modulation of the process of transduction of proliferative signals imparted by extracellular growth factors and hormones, and thus the regulation of the cell cycle traverse and apoptotic cell death. Influencing cell adhesion and motility by modulating the function of enzymes implicated in the remodelling of the extracellular matrix, leading to metastatic dissemination of cancer cells, are also important features of CBP function in cancer. CBPs are associated with other pathological conditions of the central nervous system (CNS) and skin, such as amyloidosis, Alzheimer’s disease, and Smith–Magenis syndrome (SMS), among others. The purview of the present volume is to review, analyse, and assimilate these apparently diverse functions of CBPs into a coherent picture. The conceptual basis of this book, summarised in Figure 1, is to recognise, redefine, and establish CBPs as second messengers themselves. Intracellular calcium levels modulate in response to extracellular signals and calcium transport across the cell membrane. CBPs play a crucial role in calcium homeostasis, by calcium buffering, calcium transport, and release of calcium from intracellular stores. They carry the information downstream to activate the phosphorylation of target proteins, which leads to enzyme function and metabolism, muscle contraction, and a host of other physiological functions. They also influence cytoskeletal dynamics, actively participate in the remodelling of the extracellular matrix, and consequentially affect cell morphology, motility, and intercellular interactions. A recent development is the 1

2

Calcium Signalling in Cancer

FIGURE 1 Summary of the conceptual basis of this book, providing an outline of the postulate that forms the core of this work: calcium binding proteins (CBPs) are second messengers participating in the calcium signalling cascade. CBPs not only regulate intracellular calcium levels, but they also are involved in the regulation of normal cell physiology, as well as cell behaviour, proliferation, apoptosis, tumorigenesis, and the secondary spread of cancer.

demonstration that certain CBPs may actively control the progression of the cell cycle in consort with other proteins such as p53 and stathmin. Apoptosis or programmed cell death is a calcium-dependent phenomenon, and CBPs can activate several pathways of molecular degradation leading to apoptosis. In light of these multifarious functions, the thesis developed here is that CBPs are an integral component of the mechanisms of cell population homeostasis and tumour growth. Genetic activation is the coup de grace of signal transduction. The inappropriate expression, temporally and spatially, of some CBPs induces invasive behaviour and

Introduction

3

secondary spread of immortalised cells. These genetic and phenotypic changes induced by CBPs are integrated here to provide a postulate for the growth and progression of cancers. The formulation of this postulate underpins the evolution and development of new strategies for controlling the growth of the tumour and its metastatic spread.

2

The Calcium Signalling Pathway

HOMEOSTASIS OF CELL CALCIUM Ion channels are involved in a large number of cell functions and in the induction of cellular responses to extracellular stimuli. There are three types of voltagesensitive ion channels: the sodium channel, the potassium channel, and the calcium channel. These function in a coordinated fashion in determining cellular responses to extraneous signals. The Na+ channel is involved in the generation of action potentials and the K+ channel is involved in the regulation of membrane potential and is significantly involved in synaptic plasticity. The regulation of intracellular levels of calcium is an important element in the signalling process mediated by calcium as a second messenger. There are several mechanisms by which calcium levels in the cell are exquisitely controlled and calcium homeostasis is achieved. These are listed together with their characteristic features and properties in Table 1. The Ca2+-ATPase pumps in the plasma membrane and the sarcoplasmic–endoplasmic reticulum play a highly significant part in the regulation of calcium homeostasis. Calcium homeostasis also involves the ligandgated and voltage-gated channels. Calcium-binding proteins are of special interest in the present context because of their ability to regulate intracellular calcium levels.

THE PLASMA MEMBRANE CA2+-ATPASE PUMP The plasma membrane Ca2+-ATPase extrusion pump (PMCA) and the sarcoplasmic–endoplasmic reticulum Ca2+-ATPase pump (SERCA) are specifically targeted to the two membrane systems of the cell. The N-terminal region of the SERCA molecule contains a domain that has an endoplasmic reticulum (ER)-retention signal sequence. Certain mutations of the PMCA can cause it to become localised to the ER (Guerini et al. 1998). There are four genes which code for four isoforms. These isoforms show differential localisation in different organs and the subcellular compartment. PMCA-1 and PMCA-2 are expressed ubiquitously, but PMCA-3 and PMCA-4 occur specifically in neural cells (Carafoli et al. 1996). The expression of PMCA genes seems to be developmentally regulated. Thus, in mouse development, PMCA genes show an overlapping, but, nonetheless, a distinctive pattern of expression (Zacharias and Kapper, 1999). Furthermore, PMCA and SERCA genes are co-ordinately regulated. The differentiative effects of nerve growth factor (NGF) on PC12 cells are an example of this. NGF not only up-regulates PMCA but also down-regulates SERCA expression (Keller and Grover, 2000).

5

6

Calcium Signalling in Cancer

TABLE 1 Mechanisms of Homeostasis of Cell Calcium Mode of Homeostasis Calcium Pumps Na+/Ca2+ exchanger PMCA Ca2+-ATPase pump SERCA Ca2+-ATPase pump Ligand-gated calcium channels Voltage-gated calcium channels (VGCCs) Subtypes: T L N P/Q R Calcium binding proteins

Characteristics

Plasma membrane–associated low affinity mechanism; pumps out large quantities of Ca2+ Plasma membrane calcium-ATPase pump involved in calcium extrusion High affinity ER-located mechanism; leads to luminal accumulation of Ca2+ and reduces cytosolic levels Initiation of calcium influx upon binding of ligands to respective receptors; direct influx of Ca2+ or mediated by IP3 Ca2+ influx/release from intracellular stores

Transient currents in response to membrane depolarisation Prolonged currents in response to depolarisation Inactivate in response to moderate, not severe depolarisation; not sensitive to intracellular cations Nerve terminals; initiation of neurotransmitter release Regulation of VGCC, PMCA and SERCA (e.g., by CaM); NMDA receptor channel by CaM and references cited in the text

Note: CaM, calmodulin; ER, endoplasmic reticulum; IP3, inositol 1,4,5-triphosphate; NMDA, N-methyl-D-aspartate; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarcoplasmic–endoplasmic reticulum Ca2+-ATPase. Source: Collated from Tsien et al. (1988), Tsunoda (1993), Campbell et al. (1988), Marin et al. (1998); Pascale and Etcheberrigaray (1999); Tsien et al. (1991); Dunlap et al. (1995); Ehlers et al. (1996), and references cited in the text.

Membrane depolarisation can also differentially regulate PMCA and SERCA expression (Guerini et al. 1999). Kuo et al. (1997) had previously reported that their expression might indeed be interdependent. The N-terminal region of PMCA molecule is highly variable and is essential for enzymatic activity. There seems to be an absolute requirement of a specific amino acid sequence for its function (Talgham and Adamo, 1999). The transcription of the PMCA isoforms is controlled by calcium itself. Calciumbinding proteins are intricately connected with this process. In developing neurones calcineurin seems to regulate PMCA expression (Carafoli et al. 1999). Besides, PMCA isoforms show a marked differential sensitivity to calcium, calmodulin (CaM), ATP, and kinases. The calcium pump activity appears to be regulated by phosphorylation, and phosphorylation might be the means by which different

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7

isoforms become functional in a temporal dimension (Monteith et al. 1998). PMCA is activated by Ca2+/CaM, and the isoforms also show marked differences in their sensitivity to Ca2+/CaM (Elwess et al. 1997, 1998). Human PMCA-4a is phosphorylated at a serine residue that occurs within the CAM-binding domain (Verma et al. 1999; Carafoli, 1997; Carafoli et al. 1996). Phosphorylation appears to prevent CaM binding and lead to the inactivation of the pump (Enyedi et al. 1997; Penniston and Enyedi, 1998).

THE SARCOPLASMIC–ENDOPLASMIC RETICULUM CA2+-ATPASE PUMP SERCA is one of the modes that controls the homeostasis of intracellular calcium. SERCA2 transports Ca2+ into the lumen of the reticulum by an ATP-dependent mechanism. By virtue of this ability, SERCA2 plays an important part in normal physiological function such as the contraction and relaxation cycle of cardiac muscle, as well as in the pathogenesis of certain diseases. In addition, the correct localisation of the SERCA protein in the ER is of the utmost importance. As stated above, the localisation signal occurs in a stretch of 28 amino acids at the N-terminus of the molecule (Guerini et al. 1998). Ankyrin, a cytoskeletal protein, also seems to be involved in the targeting of SERCA. This is indicated by its abnormal localisation in ankyrin –/– mice. In these mutant mice, the absence of ankyrin also affects the localisation of ryanodine and inositol, 1, 4, 5-trisphophate (IP3) receptors (IP3R), both involved in calcium mobilisation (Tuvia et al. 1999). The ATP2A2 gene encodes the sarcoplasmic–endoplasm reticulum ATPase isoform type 2. Periasamy et al. (1999) have demonstrated the loss of calcium sequestering activity and consequent impairment of cardiac function in heterozygous ATP2A2 gene mutants. Mutations of the ATP2A2 gene have been associated with the pathogenesis of Darier’s disease (DD) (keratosis follicularis) (Sakuntabhai et al. 1999a, 1999b). DD is an autosomal dominantly inherited skin disorder. It is characterised by the presence of keratotic papules, and in histology is distinguished by acantholytic dyskeratosis. The keratotic papules occur mainly in the upper trunk, scalp, and palmar pits. Nail dystrophy is also a prominent feature of DD (Soroush and Gurevitch, 1997). Because ATP2A2 controls calcium homeostasis in the cell, it is conceivable that its deregulation by mutation of the ATP2A2 gene might represent an early event in carcinogenesis. It was demonstrated many years ago that thapsigargin, an inhibitor of SERCA2, not only alters intracellular calcium levels, but also functions as a tumour promoter (Hakii, 1986; Thastrup et al. 1990). With the demonstration that ATP2A2 mutations are associated with the genesis of DD, there have also been several attempts to investigate the incidence of neoplasia in association with DD. Soroush and Gurevitch (1997) have pointed out that basal cell carcinomas and other skin neoplasms occur in patients with DD. Downs et al. (1997) described the occurrence of a subungual squamous cell carcinoma in a DD patient. However, in this case the human papilloma virus might have been the aetiological agent. In general, neoplasia may be associated only infrequently with DD. There is an obvious common feature between DD and neoplasia that deserves a mention. This concerns the abnormal expression of the calcium-binding transmembrane glycoprotein cadherin. Some isoforms of cadherin have been regarded as

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Calcium Signalling in Cancer

invasion suppressors, because there is a significant body of evidence that associates the acquisition of invasive ability by tumour cells with the loss of cadherins. E-type cadherin is found in the cell membrane of epidermal cells. E-cadherin is distributed in the plasma membrane of keratinocytes, in the intercellular space between desmosomes. Interestingly, this cadherin is expressed at greatly reduced levels in the acatholytic cells of DD and Hailey–Hailey disease (benign pemphigus) (Furukawa et al. 1997). Desmosomes provide the adhesion machinery in most epithelia, and abnormalities associated with them may be a key feature of inherited diseases such as DD and Hailey–Hailey disease. Indeed, the loss of cadherin may be the determining factor that causes the characteristic loss of adhesion between epidermal cells encountered in DD. Furukawa et al. (1997) also point out that both E- and Pcadherins are absent in squamous carcinomas, malignant melanomas and in Paget’s disease. But basal cell carcinomas, which are noninvasive, do not show a loss of cadherin. The above discussion underscores the importance of the SERCA2 calcium pump and intracellular calcium homeostasis in the calcium-signalling events associated with cell differentiation and dedifferentiation, and in the pathogenesis of DD and neoplasia.

VOLTAGE–GATED CALCIUM CHANNELS The Voltage-gated calcium channels (VGCCs) are located in the plasma membrane. The high voltage-activated channel subtypes L,N, P, Q, and R occur as heterodimers of four subunits. The largest of the subunits is the α1 subunit, which spans the plasma membrane and the auxiliary subunits, β, γ, and α2δ (Isom et al. 1994). The properties of the calcium channel appear to be determined by the differential expression of α1, which is the pore-forming subunit. The β subunit seems to regulate channel properties and the targeting of α1. The β subunit, of which four isoforms (β1–4) have been identified, is said to interact with α1. This interaction is mediated by certain highly conserved domains (Pragnall et al. 1994; De Waard et al. 1994). The interaction between these subunits appears to regulate channel activity and the diversity of calcium currents (Varadi et al. 1991; Isom et al. 1994; Olcese et al. 1994). The β subunits are believed to modulate the kinetics of channel activation and inactivation by means of phosphorylation. Calcium-binding proteins feature prominently in calcium homeostasis as important regulators of calcium channel activity. The elevation of intracellular levels of calcium can provide a negative feedback and close the channel. On the other hand, the feedback can have a positive element or facilitation, which opens the calcium channel. Zuhlke et al. (1999) have shown that CaM functions as a sensor for both negative and positive regulation of L-type channel activity. CaM appears to do this by binding to the consensus sequence called the IQ motif that occurs in the Cterminal α1C subunit of the channel protein. They substituted the isoleucine residue with alanine and reduced Ca2+-dependent inactivation and enhanced facilitation of the channel. Both inactivation and facilitation were abrogated when the isoleucine residue was converted to glutamate. CaM binds to the IQ motifs of other channel subtypes N, P/Q, and R as well (Peterson et al. 1999). Indeed, calcium-mediated

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9

modulation of P/Q channel subtypes also seems to involve CaM function. Again the channel activity is regulated CaM binding to the IQ motif of the α1A channel subunit (A. Lee et al. 1999).

THE DEREGULATION OF CALCIUM HOMEOSTASIS AS A PRIMARY EVENT IN CARCINOGENESIS The operation of ligand-gated calcium channels and the role that calcium binding proteins play in calcium homeostasis and as messengers of the calcium signal provide the major focus for this work. There is much justification for the view held by many investigators that the deregulation of calcium homeostasis in the cell might be a primary event in cell transformation and carcinogenesis. Indeed, several agents that block or retard the influx of calcium into cells appear to be able to inhibit the invasive ability of cancer cells, alter their adhesion properties, and inhibit tumour growth and stabilise the disease. Carboxyamido-triazole (CAI) is one such compound. CAI has been reported to be able to inhibit the invasive behaviour of breast carcinoma cell lines in vitro. The matrix metalloproteinases associated with these cells also appear to be inhibited in parallel (Lambert et al. 1997). Not surprising, therefore, is the finding that CAI inhibits the adhesion of glioma cells to collagen type IV coated substrata and also inhibits their invasion in vitro. CAI also appears to be capable of inhibiting cell proliferation (Jacobs et al. 1997). The antiproliferation and anti-invasion properties of CAI have also been described by Wasilenko et al. (1996) using human prostate cancer cell lines. These are clear indications of the variety of downstream pathways activated by calcium influx that are inhibited by CAI. Finally, CAI has been reported to stabilise the disease in patients with a variety of refractory solid tumours (Kohn et al. 1996). Verapamil, a selective L-type calcium channel blocker, also has demonstrable anti-proliferative and anti-invasive properties. These were recognised some years ago. Thus verapamil inhibits tumour growth in vitro (Brocchieri et al. 1996) and seems to arrest cells in the G0G1 phase of the cell cycle (Zeitler et al. 1997). The antiproliferative activity has been confirmed by Hoffman et al. (1998) on retinal pigment cells. Verapamil is able to inhibit the migration of CD4+ and CD8+ T lymphocytes across the endothelium (Blaheta et al. 2000). Other calcium antagonists such as mibefradil can also inhibit the adhesion and diapedesis of lymphocytes across the endothelium (Blaheta et al. 1998). Earlier Hailer et al. (1994) had noticed that the expression of endothelial adhesion molecules was unaltered by verapamil. It may be too simplistic to interpret these findings merely in terms of blocking of calcium channels. Nonetheless, some of these publications also indicate that the Factin levels of the lymphocytes are reduced by the treatment. This could be due to changes in cytoskeletal dynamics that occur after verapamil-mediated modulation of intracellular calcium levels. This in turn could alter cellular motility. Other factors will have serious effects on the permeability of the endothelium. For instance, CD4+ lymphocytes form two subsets known as Th1 and Th2 cells. Upon activation both subsets produce a wide range of cytokines. Th1 cells secrete interleukin (IL)-2 and (IFN)-γ, and Th2 cells secrete IL-4, 5, 10, and 13. Some of these lymphokines are

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Calcium Signalling in Cancer

bound to affect endothelial permeability. An additional factor that must be reckoned with is the generation of nitric oxide, which is known to inhibit the adhesion and spreading of endothelial cells. Nitric oxide also affects the formation of stress fibres in the cells, which is bound to influence cellular motility. Nitric oxide also actively influences angiogenesis. IFN-γ is a powerful inducer of nitric oxide synthase (NOS), whereas IL-4 from Th2 cells inhibits NOS. It would be well to remember here that endothelial NOS is a Ca2+/CaM-inducible enzyme. It is activated when it is binds the Ca2+/CaM complex. More recently, the effects of verapamil have been tested on the local invasion and metastasis of a murine carcinoma cell line in BALB/C mice. Farias et al. (1998) found that it inhibited local invasion and also produced a 50% reduction in both spontaneous and experimental metastasis. In common with CAI, the cytostatic and anti-invasive effects may be due to the inhibition of membrane-associated proteinases such as matrix metalloproteinases and urokinase-type plasminogen activator (Farias et al. 1998). These events are virtually terminal events in the process of inhibition of adhesion and invasiveness by verapamil. More upstream in the pathway, verapamil has been shown to down-regulate the expression of S100A4 (Parker and Sherbet, 1992), which, as discussed elsewhere in this book, is closely allied with p53 and stathmin genes in the control of cell cycle progression. Furthermore, S100A4 expression is closely linked with the remodelling of the extracellular matrix.

PHOSPHOLIPID SIGNALLING A host of regulatory molecules alter cell behaviour and physiological events. Thus extracellular signals imparted to the cells regulate their growth, proliferation, and apoptosis. They also regulate intercellular adhesion and cell adhesion to substrata, and consequently cell migration and invasive behaviour. A family of second messengers, the inositol phospholipids, transduces the regulatory signals across the membrane. Phospholipids (phosphatides) are a major structural component of the cell membrane and also subserve important functions of transducing the extracellular signals. The second messengers phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and IP3 are generated from these phospholipids by the action of phospholipases A and C (PLC), which are activated by the extracellular signals, and kinases. An intermediary in this process is the phosphatidylinositol (PI) transfer protein, which presents the PI metabolites to lipid kinases. The phosphorylation of the inositol ring at the 3-position gives rise to phosphoinositides PIP (phosphatidylinositol), PIP2, and PIP3. From here the transduction of the signal occurs by the interaction of these second messengers with target proteins that possess specific phosphoinositide-binding domains, such as an FYVE domain or pleckstrin homology (PH) domain. PIP2 and PIP3 show specific binding to PH domains (see Leevers et al. 1999). The second pathway is the generation of IP3 from PIP2 by PLC. As discussed below, IP3 then mediates the mobilisation of calcium from intracellular stores. The depletion of the intracellular stores triggers the so-called capacitative entry of calcium into the cell. Naturally, a deregulation of these pathways will have serious consequences in terms of differentiation, intercellular relationships, adhesion, and

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cellular morphology. Such profound changes might indeed lead to the development of cancers, their invasion, and the formation of metastases.

PTEN PHOSPHATASE IN THE REGULATION OF LIPID SIGNALLING Phosphoinositide-3 kinase (PI3K) is responsible for the phosphorylation and generation of PIP2 and PIP3. This phosphorylation is antagonised by a phosphatase that is encoded by the gene called PTEN/MMAC1/TEP1 located on chromosome 10q23. The phosphatase, referred to here as PTEN, shows sequence homology to tensin (Steck et al. 1997). The phosphatase activity is essential for the normal functioning of PTEN (Tamura et al. 1999b). PTEN regulates the levels of the phosphoinositol phosphates by dephosphorylation of the phosphate group at the 3-position of the inositol ring (Maehama and Dixon, 1998). This seems to inhibit cell proliferation and migration (Furnari et al. 1997; J. Li et al. 1998; Ramaswamy et al. 1999; Tamura et al. 1999c; Podsypanina et al. 1999). Ramaswamy et al. (1999) transfected cells that lack PTEN with wild-type PTEN cDNA and found that cells were arrested in the G1 phase of the cell cycle. Similar transfection studies have been carried out on the glioma cell line U373, which lacks the PTEN gene. In these cells, wild-type PTEN inhibits growth and reduces saturation density of U373 cultures. However, PTEN mutants lacking phosphatase activity have the opposite effect and they indeed enhance cell growth in soft agar (Morimoto et al. 1999), which merely confirms the potential suppressor function of PTEN. PTEN also seems to be able to regulate the apoptotic pathway. PIP3 is known to activate the akt serine/threonine kinase, which has anti-apoptotic properties. It has been shown to inhibit akt kinase activity (Maehama and Dixon, 1999; Ramaswamy et al. 1999), which could push cells into the apoptotic pathway. For instance, extraneous PTEN induces apoptosis of breast cancer cells, which correlates with the down-regulation of akt. Furthermore, akt can rescue cells from PTENinduced apoptosis (J. Li et al. 1998). PTEN also increases the expression of p27kip1 cyclin-dependent kinase (cdk) inhibitor (Y.L. Lu et al. 1999). The cdk inhibitors, kip1 and kip2, inhibit the phosphorylation of rb protein and this prevents the entry of the cells into S-phase. This is consistent with the recent finding of Paramio et al. (1999) that PTEN cannot produce growth arrest in rb-deficient cells, but it induces growth arrest when functional rb is reintroduced. This could provide an alternative mechanism by which PTEN induces cell arrest in the G1 phase. The overall effects of growth arrest could be a combination of G1 arrest and apoptosis (Weng et al. 1999), but Paramio et al. (1999) believe that apoptosis might not contribute to this. It appears that PTEN might regulate cell adhesion and alterations in cell shape and motility by influencing the downstream of the integrin-mediated signalling pathway (Tamura et al. 1999c). Tamura et al. (1999a) have reported that PTEN also may interact with focal adhesion kinase and regulate its phosphorylation and focal adhesions. A further suggestion is that PTEN function might take the nitrogenactivated protein kinase (MAPK) pathway in the regulation of motility (Gu et al. 1999). It ought to be recognised, nonetheless, that these pathways may not be

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Calcium Signalling in Cancer

mutually exclusive in facilitating the flow of information, and one or more pathways may be involved in the process. Consistent with this, Morimoto et al. (2000) have shown that PTEN not only affects PLC-mediated signalling, but also inhibits the kinases that regulate integrin function as well as the anti-apoptosis kinase akt. PTEN is required at certain stages of embryonic development. Its loss appears to be lethal in early embryogenesis and to lead to abnormalities of the skin, the gastrointestinal tract, and the pancreas. PTEN –/– cells have shown marked changes in their ability to differentiate into cell types characteristic of the three germ layers (i.e., the ectoderm, the endoderm, and the mesoderm) (Di Cristofano et al. 1998). Presumably, inactivation of the gene affects the state of “competence” of the cells to respond to differentiation-inducing stimuli (see Sherbet, 1982). Thus the abnormalities in the various organs seem to have an early developmental history. The PTEN gene is mutated, or there is a consistent loss of heterozygosity (LOH) at this gene locus, in many forms of human cancer. The mutations tend to occur mainly in the phosphatase domain (Steck et al. 1997; J. Li et al. 1997; W.G. Liu et al. 1997; Guldberg et al. 1997; Cairns et al. 1997; Tashiro et al. 1997; Tang et al. 1997; Kong et al. 1997; Okami et al. 1998; Ali et al. 1999). The seemingly consistent association of inactivating mutations and LOH of PTEN with cancers suggests the possibility that the lipid-signalling pathway could be seriously deregulated in the pathogenesis of neoplasia, and the PTEN gene has been suggested as a putative tumour suppressor gene. This is suggested unambiguously by the ability of PTEN to inhibit rasmediated cell transformation, with a concomitant dephosphorylation of PIP-1,3,4,5tetrakisphosphate (Tolkacheva and Chan, 2000). Germline mutations of PTEN also occur with the pathogenesis of Cowden’s syndrome (CS) and the Bannayan-Zonana syndrome (Liaw et al. 1997; Nelen et al. 1997; Tsou et al. 1998; Marsh et al. 1998a, 1988b). CS is an autosomal dominant inherited syndrome. Several benign and malignant neoplastic conditions are associated with this syndrome. Women with CS are believed to be more prone to develop breast cancer, and most of them have been found to develop benign fibrocystic disease. The CS mutations tend to be in exon 5, which contains the phosphatase core motif (Marsh et al. 1998b; Nelen et al. 1997). Although PTEN germline mutations are frequent in the Bannayan–Zonana syndrome, none of them have been found in exon 5 (Marsh et al. 1998a). An overview of the pattern of mutations suggests that those occurring in tumours could differ in nature from germline mutations (see Ali et al. 1999). Perhaps it is premature to speculate whether the generation of these mutations in different tumours is related to their pathogenesis. On the other hand, Guldberg et al. (1997) have found the same missense mutation in both primary and metastatic melanomas. Although further confirmation of this is essential, this has a considerable implication for metastatic dissemination. Nonetheless, while considering the potential tumour suppressor function of PTEN, it should be borne in mind that in PTEN abnormalities do not occur as a generalised event of tumour development. Yeh et al. (2000) examined human hepatocellular carcinomas for aberrant expression of PTEN, but encountered none. Neither did they find any abnormalities associated with the gene itself. Furthermore, the PTEN gene does not seem to be involved in the pathogenesis of acute myeloid leukaemia (T.C. Liu et al. 2000). Therefore, it might be prudent to reserve judgment regarding this issue. On

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The Calcium Signalling Pathway

the other hand, PTEN overexpression might be a safeguard against progression. In laryngeal papillomas, for example, PTEN is overexpressed and the anti-apoptotic akt kinase is inhibited (P. Zhang and Steinberg, 2000). This should have an overall controlling effect on cell population size, and as a consequence upon progression, because more often than not, tumour progression is related to the rate of tumour expansion.

THE PROTEIN KINASE C PATHWAY Growth factors and hormones are known to increase the intracellular Ca2+ (Ca [i]), either by releasing Ca2+ from intracellular stores or by promoting Ca2+ influx. Ca2+[i] controls several biological functions, mainly by mediating the phosphorylation of cellular effector proteins. Calcium-binding proteins transduce the calcium signals to specific subcellular compartments. The generation of second messengers such as IP3, 1,2-diacylglycerol (DAG), and cyclic 3′,4′-adenosine monophosphate (cAMP) is a major mechanism of activation of the Ca2+ signalling pathway. IP3 and DAG are formed through the agency of an inositide specific phospholipase, which is stimulated by biological response modifiers. This PLC hydrolyses PIP2 into DAG and IP3. IP3 activates the release of Ca2+ from intracellular stores. The activation of appropriate protein kinases is a downstream event, which occurs in the transduction pathway. The major kinases involved are protein kinase C (PKC), the calcium/calmodulin-dependent protein kinases (CAMPKs) and protein kinase A (PKA). The CAMPKs can in turn activate the cAMP-mediated pathway by activating adenylyl cyclase, resulting in an increase in cAMP levels (Figure 2). 2+

Ca

2+

L

L

L

R

R G

R G

PL C

AC cAMP

IP3

PIP 2

2+

DAG

Ca [i]

PK C

CAMP K

PK A

FIGURE 2 The activation of signal transduction pathways upon binding of a ligand (L) to its appropriate receptor (R). AC, adenylyl cyclase; cAMP, cyclic 3′,4′-adenosine monophosphate; CAMPK, calcium/calmodulin-dependent protein kinase; DAG, 1,2-diacyglycerol; G, G-protein; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidyl 4,5-bis phosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.

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Calcium Signalling in Cancer

Calcium ions and DAG successively activate PKC and induce its translocation to the plasma membrane or the nucleus. Both intracellular calcium and DAG binding to PKC maintain it in a sustained activated state (Oancea and Meyer, 1998). DAG itself is regulated by phosphorylation by DAG kinase (DAGK). DAG phosphorylation seems to reduce PKC activity (Sakane and Kanoh, 1997) (summarised in Figure 3). It may be noted that as many as eight isoforms of DAGK have been identified. These have the characteristic zinc finger structures and PH domains. PH domains are found in many proteins and consist of approximately 120 amino acid residues. They are electrostatically polarised and show binding to G-protein subunits, PKCs, and phospholipids, thus linking PH domains as a characteristic feature of signal transduction proteins. The presence of these domains in DAGK isoforms suggests that these kinases are a component of the signal transduction network. Besides, DAGK-α, -β, and -γ also possess two consecutive EF-hand calcium-binding domains (Sakane et al. 1990; Yamada et al. 1997; Sakane and Kanoh, 1997). Therefore, DAGK isoforms show calcium-dependent activity. This effect is variable, which has been attributed to differences in calcium affinities of and the conformational changes occurring in the EF-hand domains as a response to calcium binding (Yamada et al. 1997). Upon activation, DAG kinases also translocate to the nucleus and remain tightly associated with nuclear matrix (Wada et al. 1996). Some DAGK isoforms, such as DAGK -δ, -ε, -ζ, have no EF-hand domains (Sakane et al. 1996; W. Tang et al. 1996; Bunting et al. 1996). So calcium-mediated activation is not a universal phenomenon in the activation of these kinases.

PROTEIN KINASE C AND ITS ISOFORMS IN SIGNAL TRANSDUCTION DAG, upon being activated by the calcium binding protein DAGK, now activates PKC by enhancing its affinity for Ca2+ and altering its enzymatic activity. This leads to the phosphorylation of substrate proteins, thus effectively transducing the calcium signal into a physiological response. Some CBPs may inhibit PKC-mediated phosphorylation of protein substrates. In other words, CBPs can actively regulate PKC function (see Sherbet, 1987; Sherbet and Lakshmi, 1997a, 1997b, for review). They may also be directly associated with the coupling of calcium signalling with gene regulation. This role is discussed in detail elsewhere in this book. Several isoforms of PKC have been identified and it is believed that they may subserve specific functions in signal transduction. It is well known that PKCs are translocated from cytoplasmic to nuclear location when cells are stimulated by growth factors. In Swiss 3T3 cells, although a number of isoforms are detectable in the cytoplasm, only PKCα is found in nuclei isolated from cells that have been exposed to growth factors (Neri et al. 1994). In HL-60 cells that were induced by all-trans-retinoic acid to differentiate, the translocation of PKCα and ζ to the nucleus has been observed (Zauli et al. 1996). This differential translocation has been attributed to possible linkage with the nuclear inositol lipid cycle (Neri et al. 1994). Further support for this view comes from the observation by Mizukami et al. (1997) concerning the

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The Calcium Signalling Pathway

Ligand binding

Receptor

Calcium spikes

DAG activation

DAG kinase

PKC activation FIGURE 3 The flow of information of ligand binding to its membrane receptor via intracellular calcium increases and DAG-to-PKC activation and its translocation to specific intracellular sites are illustrated. DAG is in turn regulated by DAG kinases, which are EFhand proteins.

translocation of PKCζ. They reported that translocation of this PKC isoform to the nucleus could be inhibited by inhibiting phosphatidylinositol-3 kinase. With the plethora of PKC isoforms discovered over the years, their occurrence is being rationalised by the postulate that these isoforms might be specifically associated with identifiable signal transduction pathways and signal transduction in different physiological functions in a tissue-specific manner. In PC12 cells, the epidermal growth factor (EGF) signal either is mitogenic or induces differentiation. The flow of information and the phenotypic effect appear to depend on the PKC isoform. EGF induces neurite formation and arrests cell proliferation if the cells overexpress PKC-ε, but not PKC-α or δ. Neurite differentiation is accompanied by phosphorylation of mitogen-activated protein kinase, and inhibition of this phosphorylation event also inhibits neurite differentiation as well as arrest of proliferation. In sharp contrast, overexpression of PKC-α and -δ is strongly associated with the mitogenic function of EGF (Brodie et al. 1999). There are also examples of tissue-specific functioning of PKC isoforms. For instance, calcium-independent vascular smooth muscle contraction seems to involve PKC-ε rather than PKC-ζ, both calcium-independent isoforms of PKC (Walsh et al. 1996). Such specificity of function might reflect the substrate specificity of the kinase. Walsh et al. (1996) suggested the possibility that calponin is the musclespecific protein that might be a substrate of this PKC isoform. DAGKs that activate

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Calcium Signalling in Cancer

PKCs show considerable tissue-specific distribution themselves. It may be that the tissue-specific function of PKCs might flow from the distribution of the DAGKs.

INOSITOL PHOSPHATES IN CALCIUM SIGNAL TRANSDUCTION The transduction of signals imparted to the cell by the binding of extracellular ligands to their respective membrane receptors generates repetitive calcium spikes or oscillations, and these transient increases of intracellular calcium are a result of the production of IP3 by the mediation of PLC (see Figure 3). The repetitive nature of these oscillations is believed to be a consequence of a positive feedback loop in which calcium induces its own release, and a negative feedback effect which stops calcium release. Such calcium oscillations occur in many physiological phenomena. An increase in intracellular calcium is a critical event in the activation of the egg (ovum) in a wide variety of species. Experimental enhancement of intracellular calcium of mouse eggs using calcium ionophores or ethanol results in activation. Conversely, when intracellular calcium levels are reduced egg activation is prevented (Cuthbertson, 1983; Ducibella et al. 1988; Kline and Kline, 1992). That IP3 mediates egg activation is indicated by the demonstration that microinjection of IP3 produces calcium release from intracellular stores and initiates some of the events associated with activation (Kurasawa et al. 1989; Ducibella et al. 1993). IP3 levels are increased by fertilisation of the egg (Kline, 1996). An increase is also known to take place in the number of IP3 receptors (Mehlmann et al.1996). IP3 receptors occur in the anterior acrosomal region of mammalian sperm, and by implication IP3 is involved in the acrosome reaction of mammalian sperm (Walensky and Snyder, 1996). IP3 regulates intracellular Ca2+ levels by mobilising calcium from intracellular ER-associated stores and probably also by stimulating Ca2+ influx into cells. Only a proportion (up to 50%) of the ER Ca2+ pool is IP3 sensitive, and the remainder can be released by calcium ionophores. The IP3-sensitive calcium pool is clearly associated with IP3R function. IP3R is an intracellular calcium release channel on the ER. IP3 stimulates IP3R, which results in calcium mobilisation from intracellular stores. Jayaraman et al. (1995) stably transfected antisense IP3R cDNA into Jurkat T cells. The transfected cells, which then lacked functional IP3R, failed to elevate Ca2+[i] in response to antigen-specific activation. However, the depletion of intracellular calcium stimulated calcium influx, which suggests that IP3R are required for IP3-mediated mobilisation of calcium from intracellular stores, but not for calcium influx. The function of IP3R seems to be regulated by phosphorylation. Both cyclic guanosine monophosphate (cGMP) and cAMP are known to inhibit the mobilisation of intracellular calcium, and this has been found to be a consequence of the phosphorylation of IP3R by cGMP-dependent kinase (Komalavilas and Lincoln, 1994, 1996) (Figure 4). Komalavilas and Lincoln identified serine 1755 of IP3R as the phosphorylation site. Nonreceptor tyrosine kinases have also been shown to generate IP3, activate IP3R, and raise intracellular calcium levels. Jayaraman et al. (1996) demonstrated the association between nonreceptor tyrosine kinase Fyn and IP3R

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17

FIGURE 4 Possible mechanisms involved in IP3-mediated mobilisation of intracellular calcium. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; cGMPK, cGMP kinase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; IP3R-P, phosphorylated forms of IP3R.

following T-cell receptor stimulation. Furthermore, phosphorylation of tyrosine residues was found to be reduced in thymocytes derived from fyn (–/–) mice. These studies showed that phosphorylation of tyrosine might also regulate IP3R function. It would appear that the phosphorylation of serine and tyrosine might have antagonistic regulatory effects. This situation is somewhat analogous to the activation or inactivation of p34cdc2 kinase, which is involved with the phosphorylation of the retinoblastoma gene product in the regulation of cell cycle progression (see Sherbet and Lakshmi, 1997b). Although the molecular mechanisms by which IP3R raises Ca2+[i] are not fully understood to date, it is obvious that the process of phosphorylation of IP3R is an important event in IP3-mediated calcium signalling. The occurrence of IP3R was recognised many years ago (Supattapone et al. 1988; Snyder and Supattapone, 1989). There are three types of IP3R. Three IP3R genes have been cloned. One of them, the IP3R1, is expressed predominantly in Purkinje neurones of the cerebellum. IP3R1 and IP3R2 genes have been found to be expressed at low levels in several tissues studied to date. The distribution of the mRNA of the three receptor genes appears to display a definable pattern, leading to the suggestion that the different receptor types may have distinct functions (Fujino et al. 1995; T.X. Yang et al. 1995). Indeed, multiple isoforms of IP3R genes may be expressed in cells. Alternatively spliced isoforms could be fulfilling different cellular requirements, that is, they may be functionally different (Iida and Bourguignon, 1995, 1996; Katusic and Stelter, 1995). IP3R1 may be associated with intracellular calcium mobilisation, because IP3R1-deficient T-lymphocytes show a lowered IP3-mediated calcium release by T-cell receptor stimulation (Jayaraman and

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Calcium Signalling in Cancer

Marks, 1997). The same study also implicated IP3R1 in apoptosis, by the observation that IP3R1-deficient T lymphocytes are resistant to dexamethasone-induced apoptosis. IP3R3 genes have been credited with the capacitative entry of calcium influx, when intracellular Ca2+ is depleted. In Xenopus oocytes IP3R3 was associated with the plasma membrane, which facilitated the influx of calcium (Putney, 1997). The transduction of signals imparted by a variety of biological response modifiers occurs via cytoskeletal structures. Compatible with this generalisation, the signal transduction pathway of IP3 has been linked with cytoskeletal dynamics. Ribeiro et al. (1997) found that the disruption of cytoskeletal structures of NIH3T3 cells by treatment with cytochalasin D resulted in the abolition of intracellular calcium mobilisation normally induced by platelet-derived growth factor (PDGF). The depolymerisation of the tubulin network similarly abolished calcium mobilisation. However, PDGF continued to stimulate the generation of IP3 even in the presence of cytochalasin. This suggests that the cytoskeleton is closely involved in the IP3-mediated transduction of the PDGF proliferative signal. It is of interest to note that Bourguignon and Jin (1995) had previously identified a C-terminal amino acid sequence of GGVGDVLRKPS in IP3R that possesses sequence homology to the ankyrin-binding domain of the intercellular adhesion molecule CD44. They further had demonstrated that an 11 amino acid synthetic peptide with that sequence showed specific binding to ankyrin, which is a cytoskeletal protein.

DEREGULATION OF INOSITOL 1,4,5-TRISPHOSPHATE PATHWAY AND ITS CONSEQUENCES It is hardly surprising that interference with signal transduction pathways and their deregulation should have serious consequences for biological responses in developmental systems as well as in pathogenesis. Ecdysone, a steroid hormone, regulates a variety of developmental processes and metamorphosis in insects. It also influences neuronal responses, apoptosis, and histiolysis in a variety of larval tissues, binds to nuclear receptors, which show tissue- and stage-specific expression (Kamimura et al. 1997); and regulates ecdysone-responsive genes (No et al. 1996; Truman, 1996). The metamorphic changes involve “early” and “late” genes, with the early genes encoding proteins that regulate the expression of late genes (Baehrecke, 1996). Although our knowledge of the mechanics of ecdysone signal transduction is still rudimentary, it is clear that the pathway involves calcium signalling. The prothoracicotropic hormone (PTTH) stimulates the synthesis of ecdysone in the prothoracic gland of Galleria larvae (Birkenbeil, 1996). PTTH increased intracellular Ca2+ levels of prothoracic gland in vitro. This increase could be abolished by removing extracellular calcium and by calcium channel inhibitors. The PTTH-stimulated increase of intracellular calcium was not abolished by agents, which inhibit mobilisation of calcium from intracellular stores. Recently, Venkatesh and Hasan (1997) found that disruption of IP3 receptor gene function in Drosophila larvae grossly affected metamorphosis and ecdysone synthesis and secretion. They generated IP3R gene (itpr) mutants. The mutations delayed moulting and were lethal at early larval stages.

The Calcium Signalling Pathway

19

Feeding ecdysone to mutant larvae partially counteracted the phenotypic effects of the itpr mutation, and down-regulated ecdysone-inducible genes. General or specific abnormalities of signal transduction pathways (e.g., occurrence of abnormalities in one or more components of the transduction pathway) may be associated with disease processes, including the invasive behaviour and metastatic progression of cancers. Such disruptions of the signal transduction cascade in human cancers has been studied by measuring the levels of three enzymes involved in the generation of IP3, namely, phosphatidylinositol 4-kinase, phosphatidyl 4-phosphate 5 kinase, and PLC. Weber et al. (1996) found that all three enzymes were up-regulated with tumour progression. In rat hepatomas the enzyme levels rose by two- to eight-fold compared with normal liver. A three- to four-fold increase in PIP kinase and PI kinase was found in ovarian epithelial cancers. Weber et al. (1996) found a dramatic 16- and 96-fold increase of these enzymes in human breast carcinoma cells compared with normal breast parenchymal cells. These changes clearly correlated with enhanced IP3 expression and proliferative potential. Further evidence has also been reported by Singhal et al. (1997), who investigated the connection between elevated IP3 and DAG with proliferation of the breast cancer cell line MDA-MB-435 and the ovarian cancer cell line OVCAR-5. They demonstrated that quercetin treatment of the cells inhibited PI kinase and reduced IP3 before any inhibition in cell proliferation was evident. The expression of these kinases seems to be up-regulated by differentiation-inducing agents. Thus Ai et al. (1995) found enhanced expression of PI kinase as well as IP3 in murine erythroleukaemia cells induced toward erythroid differentiation by dimethyl sulphoxide (DMSO) treatment. Another line of circumstantial evidence may be cited. For instance, monoterpenes such as limonene and perillyl alcohol have been reported not only to prevent tumour initiation and promotion, but also to inhibit tumour progression. These monoterpenes appear to inhibit the isoprenylation of G-proteins (Gould, 1997), which are a component of the signal transduction machinery (Figure 5). Similarly, abnormalities in the related pathway involving DAG and PKC may also be associated with cancers, as demonstrated by Hoelting et al. (1997) using the PKC agonist TPA (12-O-tetradecanoyl phorbol 13-acetate). They reported a 15% increase in the invasive ability of a follicular thyroid cancer cell line. In contrast, PKC inhibitors such as staurosporine, chelerythrine and calphostin C reduced invasion by 62%. CAI which inhibits calcium influx into cells, has been reported to inhibit the proliferative and invasive capacity of cell lines derived from human prostate cancer (Wasilenko et al. 1996). The neuroendocrine cells of prostate cancer express bombesin and gastrin-releasing peptide (GRP). In vitro bombesin and GRP signals invoke cell proliferation via bombesin receptors of which there are three subtypes: GRP receptors (GRPr), neuromedin B receptor, and bombesin receptor subtype 3. Transcripts of GRPr only, but not neuromedin B or bombesin receptor subtype 3, have been found in androgen-insensitive prostate cancer cell lines PC-3 and DU-145, suggesting the very important role GRPr play in signal transduction in these cells (Aprikian et al. 1996). In all lines derived from advanced prostate cancer, several agents such as GRP, bombesin, and bradykinin, among others, raised intracellular calcium levels.

20

Calcium Signalling in Cancer

FIGURE 5 The cAMP/Ca2+-signalling pathway. The binding of extracellular ligands to specific receptors regulates adenylyl cyclase activity via the linking G-proteins. The cAMP generated activates protein kinases that phosphorylate substrate proteins. Another route starts with the raising of intracellular calcium levels, which in turn regulate adenylyl cyclase activity.

However, bombesin did not influence calcium levels of an immortalised prostate cell line (Wasilenko et al. 1997). The authors have, therefore, suggested not only that there might be multiple receptors that can mediate increases in intracellular calcium levels of androgen-independent prostate cancer cell lines, but also that GRPr expression may be associated with the progression of prostate cancer. Calcitonin-like peptides may also function as neuropeptides in prostate cancer. The peptide binds to high-affinity receptors in the plasma membrane and induces a rapid and marked increase in intracellular calcium. Furthermore, the calcitonin signal is transduced into a clear proliferative response. The pattern of calcium response is biphasic in the form of a spike, and plateau is regarded as indicative of the phospholipid/calciumsignalling system (Shah et al. 1994).

RYANODINE AND RELATED RECEPTORS IN CALCIUM MOBILISATION Besides IP3R, other receptor-mediated pathways are available to the cell for calcium mobilisation, and the activation and availability of the pathways may be developmentally regulated. In embryonic development, the IP3-dependent calcium signalling is switched to an IP3-independent mode of signal transduction. The ryanodine receptor (RyR) is also capable of regulating intercellular calcium. In the early stages of development, IP3R are ubiquitously expressed. But in subsequent development RyR begin to be expressed. Eventually, although most cell types do continue to

The Calcium Signalling Pathway

21

express IP3R, in excitable cells including muscle cells, RyR becomes the major Ca2+ release channel (Rosemblit et al. 1999). Ryanodine receptors, of which there are several isoforms, are activated by calcium influx via voltage-gated Ca2+ channels of the plasma membrane. Recently, cyclic ADP-ribose has been identified as an activator of RyR (Petersen and Cancela, 1999). The fatty acid metabolic products, the fatty acyl-CoA esters, can also activate RyR in skeletal muscle (Fulceri et al. 1994; El-Hayek et al. 1993; Dumonteil et al. 1994). This seems to be mediated by the acyl-CoA binding protein (Fulceri et al. 1997). A reference to the multifunctional receptor called megalin would not be out of place here. Megalin is a member of the low-density lipoprotein receptor family, which function as endocytic receptors. The megalin glycoprotein of rat kidney is approximately 330 kDa in size. The 550-kDa human homologue of megalin is found in the luminal surface of cells of the renal proximal tubule and epididymis. It is expressed in mammary epithelia, thyroid follicular cells, and the ciliary body of the eye (Lundgren et al. 1997). It is also found in parathyroid and trophoblast cells. Mackrill et al. (1999) raised antibodies against RyR from rabbit muscle. These antibodies recognised not only skeletal RyR but also another high-molecular-weight protein (k-HMW) in kidney microsomes thought to be a rabbit homologue of megalin. Antibodies raised against k-HMW were unable to recognise RyR. Furthermore, this protein showed partial sequence homology to RyR. Megalin functions as a receptor for thyroglobulin (Marino et al. 1999; G. Zheng et al. 1998), lipoprotein (a) (Niemeier et al. 1999), and insulin (Orlando et al. 1998). It is implicated in the transport of retinol. Megalin-deficient mice show greatly increased urinary excretion of retinol and retinol-binding protein (RBP). Furthermore, the uptake of retinol and RBP is partially inhibited by antibodies against megalin (Christensen et al. 1999). Megalin also serves as a receptor for aminoglyocoside antibiotics, such as gentamycin, and mediates the endocytosis and accumulation of the antibiotic in renal tubule cells (Decorti et al. 1999). There is a preliminary report that the nephrotoxicity of gentamycin is related to the loss of megalin receptors (Vaamonde et al. 1996). The megalin receptor has also been attributed with a putative role in calcium homeostasis (Friedman, 1999; Christensen et al. 1998). This seems reasonable in light of its similarity to RyR. The deregulation of this function might be responsible for its apparent involvement in pathogenesis. Megalin might function as an autoimmune antigen in the pathogenesis of membranous glomerulonephritis (Mackrill et al. 1999). Its dysfunction might occur also in neoplasia. Apparently, the expression of megalin is down-regulated in hyperplasia of the parathyroid.

CYCLIC AMP IN CALCIUM SIGNALLING A majority of hormones, neurotransmitters, and growth factors generate cellular responses by means of a signal transduction machinery that is composed of three elements. These agents bind to specific receptors, which are linked to the effector component by means of G-proteins (Iyengar and Birnbaumer, 1990a, 1990b). Gilman (1989) identified these as components of the machinery regulating adenylyl cyclase

22

Calcium Signalling in Cancer

activity. A large number of hormones modulate cAMP levels by regulating adenylyl cyclase activity. The G protein family encompasses a number of heterotrimers composed of subunits encoded by three G-protein-type genes, α, β, and γ genes. Several Gα, Gβ, and Gγ genes have been cloned (Iyengar and Birnbaumer, 1990b). The G-protein linker GS mediates stimulatory effects, whereas GI mediates inhibitory effects of hormones. Most G-protein intervention follows the activation of the α subunit by binding to GTP, and this activation occurs upon the receptor being bound by the appropriate ligand. Numerous biological responses to extracellular stimuli are mediated by cAMP synthesised by adenylyl cyclase activity, and in turn cAMP activates cAMP-dependent protein kinase, which phosphorylates a variety of cellular substrate proteins (Krebs, 1989). This pathway involves an elevation of intracellular calcium levels either by increased uptake of extracellular calcium or release of calcium from intracellular stores. Calcium can modulate adenylyl cyclase activity. There is a considerable body of evidence that CBPs such as calmodulin may regulate adenylyl cyclase activity. It has been shown that Ca2+/CaM can stimulate adenylyl cyclase activity (Minocherhomjee et al. 1988), but in some tissues the activity of this enzyme is insensitive to Ca2+/CaM. Furthermore, adenylyl cyclase activity may be related to intracellular distribution, and this may or may not be affected by activated GS. The cellular response generated by biological response modifiers may depend on the G-protein and the appropriate effector component being expressed by cells (Iyengar and Birnbaumer, 1990b). The control of cellular response to external signalling ligands requires that the levels of the second messengers themselves be regulated strictly. Enzymes such as phosphodiesterases that degrade these molecules regulate cAMP. Besides adenylyl cyclase, phosphodiesterases, protein kinase II, and protein phosphatase are also CaM-dependent enzymes. Receptor proteins and kinases may amplify second messenger signals. Indeed, this mechanism, involving their amplification on the one hand and degradation on the other, maintain the steady state levels of second messengers (see Figure 5). Several biological responses have been identified in which the cAMP/calcium pathway of signal transduction is followed upon the binding of specific extracellular ligands. The processes of aggregation and development and chemotactic response of Dictyostelium discoideum involve the cyclic nucleotides cAMP and cGMP. The chemotactic response appears to be a function of the elevation of intracellular Ca2+, which has been shown to correlate with the association of myosin with the cytoskeleton (Menz et al. 1991). This suggests a close involvement of calcium uptake with the process of cell motility. cAMP has been implicated in sperm motility (Garbers and Kopf, 1980). In fact, high extracellular levels of calcium have been known to increase cAMP levels in sperm cells (Garbers et al. 1982), and possibly Ca2+ could be activating adenylyl cyclase activity. Functionally it seems possible to differentiate between influxed calcium and Ca2+ released from intracellular stores. This is illustrated by the modulation of S100A4 expression in B16 murine melanoma cells by agents that modulate intracellular calcium levels. Melanocyte-stimulating hormone (MSH) increases calcium influx and cAMP levels. Verapamil is a calcium channel blocker that reduces cAMP levels (Atlas and Adler, 1981; Janis et al. 1987). Thapsigargin is a sesquiterpene lactone that raises intracellular calcium levels by releasing calcium from intracellular

The Calcium Signalling Pathway

23

stores, by inhibiting the ER-associated Ca2+-ATPase (Takemura et al. 1989; Thastrup et al. 1990). MSH has been reported to up-regulate S100A4, whereas verapamil does the opposite. In contrast, thapsigargin down-regulates its expression despite effecting an increase in intracellular calcium levels (Parker and Sherbet, 1992) (Figure 6). This is compatible with the suggestion by Haverstick et al. (1991) that Ca2+ influx and release from intracellular stores have independent roles. It has also been suggested that a low micromolar range of intracellular calcium levels inhibits adenylyl cyclase, in contrast to calmodulin-stimulated adenylyl cyclase which may be mediated by calcium influx (Cooper, 1991). The release of calcium from intracellular stores may therefore activate specific components of the calcium-signalling pathway (Figure 7). The transcription factor, the cAMP response element binding protein (CREB), is phosphorylated by Ca2+/CaM-dependent protein kinase PKII. The phosphatases PP-2A and -2B (calcineurin) dephosphorylate the phosphatase inhibitor (PI-1), which in turn inhibits the phosphatase PP-1. The inactivation of PI1 by PP-2A or PP-2B may allow the phosphatase PP-1 to dephosphorylate CREB, thus rendering the latter inactive. Calcineurin is reported to have an affinity for CaM two to three magnitudes greater than that of PKII. Therefore, low levels of intracellular calcium, such as those resulting from its release from intracellular stores, might preferentially activate calcineurin.

FIGURE 6 The differential regulation of S100A4 expression by calcium influx into cells as compared with the release of Ca2+ from intracellular stores. Calcium influx up-regulates S100A4. Verapamil, which blocks calcium influx, down-regulates its expression. In sharp contrast, thapsigargin, which raises intracellular calcium levels by releasing Ca2+ from intracellular stores, down-regulates S100A4 expression. This suggests that calcium released from intracellular stores may be functionally differentiated from influxed calcium and may be activating different components of the calcium signalling system. (Based on Parker and Sherbet, 1992). cAMP cyclic AMP; MSH, melanocyte-stimulating hormone.

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Calcium Signalling in Cancer

FIGURE 7 Summary of cAMP and Ca2+/CaM-mediated regulation of “immediate early” gene expression. cAMP, cyclic AMP; CREBP, cyclic AMP response element binding protein; PI-1, phosphatase inhibitor l; PKA, protein kinase A; PKII protein kinase II; PP-1, protein phosphatase 1; PP2B, calcineurin.

Several extracellular agents are known to activate the cAMP- and Ca2+-signalling pathway. There are also instances where the calcium-signalling pathway is independent of an upstream event of cAMP activation. cAMP and calcium signalling mediate the biological responses of several hormones. Adrenocorticotropic hormone (ACTH) stimulates uptake of Ca2+ and elevates cAMP levels in lymphocytes. Splenic lymphocytes that possess ACTH receptors show ACTH-dependent calcium uptake, but thymocytes, which lack these receptors, do not show enhanced calcium uptake (Clarke et al. 1994). In adrenocortical cells maintained in culture, ACTH exerts a biphasic effect, inhibiting cell proliferation in the initial phase followed by a stimulatory phase of cell differentiation, and synthesis and secretion of corticosterone and 18-OH-deoxycorticosterone. cAMP and 8-bromo-cAMP can induce adrenocortical cells to differentiate and to secrete steroid hormones, but they do not inhibit cell proliferation. This suggests that although cAMP mediates the mitogenic, differentiative, and steroid stimulatory signals of ACTH, it is not involved in the process of inhibition of cell proliferation by ACTH (Arola et al. 1993). Similarly, although the stimulation of bone resorption in foetal limb bud bones by tumour necrosis factor (TNF) appears to involve cAMP, the latter may not be directly associated with TNF signalling (Shankar and Stern, 1993). Calcitonin and calcitonin-like substances induce cell proliferation in prostate cells in culture and possibly also in the genesis of carcinoma of the prostate. Calcitonin induces a dose-dependent enhancement of cAMP and intracellular levels

The Calcium Signalling Pathway

25

of Ca2+ (Shah et al. 1994). The alterations in intracellular calcium levels in Sertoli cells induced by follicle-stimulating hormone (FSH) involve the generation of endogenous cAMP in response to this hormone treatment (Gorczynska et al. 1994). Hypothalamic neurohormones that control a variety of normal biological processes, such as cell proliferation and differentiation as well as hyperplasia and neoplastic processes, transduce their signals by the cAMP/Ca2+ pathway (Spada et al. 1997). The expression of cytokines in T lymphocytes is a highly regulated feature, involving cAMP. Benbernou et al. (1997) treated activated Jurkat cells with dibutyryl cAMP or pentoxifylline and found that IFN-γ mRNA expression was inhibited but that of IL-10 was unaffected. Cholera toxin and prostaglandin E (PGE)-2 also inhibited IFN-γ but greatly enhanced IL-10 mRNA expression. Using immunofluorescence techniques to detect intracellular cytokines, these authors confirmed the inhibition of IFN-γ by dibutyryl cAMP and the enhancement of expression of IL-10 by PGE2. This clearly shows that a differential control is exerted by cAMP at both the transcriptional and the protein levels. Downstream events such as the induction of class II major histocompatibility complex (MHC) genes by IFN-γ, on the other hand, can be inhibited by agents that trigger the cAMP signal transduction pathway (Ivashkiv et al. 1994). The endpoint of the pathway is obviously the activation of responsive genes. There are certain regulatory regions called cAMP response elements (CREs) that interact with CREB. Besides CREB, the transcription factor known as ATF1 also mediates the transcription of responsive genes upon transduction of the extracellular signal via the cAMP/Ca2+-signalling pathway. Both these transcription factors are phosphorylated by the mediation of cAMP. CREB and ATF may integrate the signals flowing from the cAMP/Ca2+ pathway. As stated before, cAMP activation can either inhibit or enhance biological responses. This has been suggested to be a consequence of blocking or activation of CREB. P.Q. Sun et al. (1994) showed by transient transfection studies with the Ca/CaM-dependent protein kinases CAMKIV and CAMKII that the former is a more potent activator of CREB than the latter. This they found was due to the nature of CREB phosphorylation. CAMKIV phosphorylated CREB at serine residue 133, but CAMKII phosphorylated serine 142 in addition to serine 133. The studies also showed that whilst phosphorylation of serine 133 activated CREB, phosphorylation of serine 142 inhibited CREB activation. In ATF1, CAMKII phosphorylates the positive regulatory serine 63, corresponding to serine 133 of CREB, but not serine 72, which corresponds to the negative regulatory serine 144 of CREB (Shimomura et al. 1996). Thus, although CREB and ATF share substantial sequence homology, it may be that these two transcription factors may differentially transactivate the responsive gene (Figure 8).

ARCHITECTURAL ASPECTS OF THE SIGNAL TRANSDUCTION MACHINERY THE ROLE

OF

CAVEOLAE

IN

SIGNAL TRANSDUCTION

The signal transduction machinery contains several elements along which information flows. A wide variety of signals are imparted to the cell and these need to be

26

Calcium Signalling in Cancer

FIGURE 8 Phosphorylation of the transcription factors CREB (cyclic AMP response element binding protein) and ATF in the negative and positive regulation of genetic activity. The Ca2+/CaM-activated protein kinases CAMKIV and CAMKII phosphorylate specific positive and negative regulatory serine residues of the transcription factors, which therefore differentially transactivate the responsive genes.

targeted and conveyed to specific subcellular compartments for eliciting cellular responses. This inevitably means that there should be an optimal organisation of the various elements to achieve a coordinated functioning of signal transduction. Certain invaginations involving specialised microdomains of the plasma membrane called caveolae were identified in epithelial and endothelial cells many years ago (Simionescu, 1993; Anderson, 1998). Caveolae do occur in many cell types. They have been identified with the endocytosis of macromolecules and in signal transduction and calcium homeostasis. The status of caveolae as a dynamic structural component of signal transduction is underlined by the presence within these membrane microdomains of a variety of signal transduction molecules. They contain G-protein-coupled receptors, ras family proteins, receptor tyrosine kinases (RTKs), and isoforms of PKC (Shaul and Anderson, 1998). Also identified with them are adenylyl cyclase and CBPs such as CaM and annexin. Caveolin, a cholesterol and lipid-binding protein (Murata et al. 1995; Trigatti et al. 1999), of which a number of isoforms are now known, is an integral membrane protein of caveolae. The caveolins are synthesised in the ER and are transported to the plasma membrane. Specific domains of the molecule take part in the movement of caveolins from the ER to the cell surface (Machleidt et al. 2000). The N-terminal cytoplasmic domains of caveolin-1 interact with G-proteins (S. Li et al. 1995), RTKs (Couet et al. 1997), and endothelial nitric oxide synthase (eNOS) (Okamoto et al. 1998).

The Calcium Signalling Pathway

27

eNOS is also a Ca2+/CaM-dependent enzyme. The increases in intracellular calcium levels induced by extracellular signals and the binding of Ca2+/CaM complex with eNOS seem to lead to its activation. eNOS also appears to be negatively regulated by caveolin-1. The interaction between eNOS and caveolin seems to reduce nitric oxide production (Shah et al. 1999). The vascular endothelial growth factor (VEGF) increases endothelial cell permeability as well as endothelial cell (EC) migration and proliferation. The VEGF signal appears to involve the production of nitric oxide by eNOS (Yiyu et al. 1999), and this mediated by the Flt-1 receptor (Ahmed et al. 1997). The up-regulation could be attributed also to posttranscriptional changes leading to the stability of eNOS mRNA (Bouloumie et al. 1999). In any event, the serine/threonine kinase Akt phosphorylates the serine residue 1179 of eNOS and activates the enzyme (Fulton et al. 1999). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME) inhibits VEGF-mediated EC migration and proliferation (Shizukuda et al. 1999). Nitric oxide is able to inhibit focal adhesion and spreading of endothelial cells and affects the formation of stress fibres (Goligorsky et al. 1999). Human tumour cells have been transfected with the NOS gene. The transfectant cells have shown enhanced invasive ability, growth rate, and vascular density, with parallel constitutive expression of NOS (Thomsen and Miles, 1998). The mechanisms involved in the nitric oxide–mediated increase in invasive and proliferative capacity have not yet been addressed, but there are suggestions that nitric oxide might up-regulate matrix metalloproteinases and block their endogenous inhibitors (Lala and Orucevic, 1998). IFN-γ and lipopolysaccharide (LPS) induce nitric oxide synthesis in C3-L5 murine adenocarcinoma cells as well as enhance their invasive ability in vitro. This is also accompanied by an upregulation of matrix metalloproteinase (MMP)-2. Furthermore, endogenous nitric oxide seems to down-regulate TIMP-2 and TIMP-3 (tissue inhibitors of metalloproteinases). This has led to the suggestion that nitric oxide alters the balance between MMPs and their inhibitors (Orucevi et al. 1999). VEGF is a potent inducer of angiogenesis. This angiogenic signal also involves nitric oxide production. L-NAME inhibits angiogenesis in vitro (Pipili-Synetos et al. 1995) as well as tumour-induced angiogenesis in vivo (Jadeski and Lala, 1999). Norrby (1998) supports the view that nitric oxide suppresses angiogenesis, but states that L-NAME did not suppress VEGF-induced angiogenesis. Nitric oxide synthase is also inducible. The inducible form (INOS) is found in large quantities in malignant prostate epithelium and in bladder cancer epithelium, but normal epithelial cells are only weakly positive. eNOS is expressed in the endothelial cells of tumour stroma, but not in stroma of normal bladder tissue (Klotz et al. 1998, 1999). In breast and gastric cancers also INOS occurs predominantly in the stromal component (Thomsen and Miles, 1998). This suggests a possible functional differentiation between INOS and eNOS. A marked up-regulation of NOS isoforms has also been reported in non–small cell lung cancers in relation to their progression (Ambs et al. 1998). Also of much interest is the finding by Gallo et al. (1998) that NOS activity in head and neck tumours with lymph node metastasis was higher than in tumours that had shown no metastatic spread. This has received some further support from Thomsen and Miles (1998) who state that NOS activity correlates with tumour grade in breast cancer.

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Calcium Signalling in Cancer

It is possible that VEGF might follow two different pathways to increase endothelial permeability, endothelial cell migration, and proliferation, and induce angiogenesis. Whereas nitric oxide production is essential for effecting an increase in endothelial permeability, it appears to negatively regulate the angiogenic signal. This is partly borne out by the demonstration that VEGF reduces the activity of PKC isoforms, especially PKC-δ, and this could be blocked by pretreatment with LNAME (Shizukuda et al. 1999). Overall, it seems that there is a switching mechanism operating here, which apparently is distinct from the transduction pathway involving eNOS activation. This is likely to be a very rewarding area of investigation in the context of cancer spread and metastasis. The presence of IP3-like receptors (Patel et al. 1999; Fujimoto et al. 1995) and Ca2+-ATPase (Fujimoto, 1993) amply testifies to the participation of caveolae in calcium homeostasis. Caveolae seem to sequester extracellular calcium into the vesicles and release it into intracellular locations, presumably by the mediation of IP3 and Ca2+-ATPase. Furthermore, with possible lateral mobility of caveolae one can envisage a regulation of entry and release of calcium to appropriate cellular compartments.

CAVEOLIN EXPRESSION

IN

CANCER

On account of the highly significant participation of caveolae in signal transduction, the expression of caveolin has been the focus of some investigation in cancer progression. Caveolin expression appears to decrease with tumour progression, which is compatible with the view that there is a serious deregulation of signal transduction in neoplasms. Caveolin-1 and -2 genes, which occur at chromosome 7q31, are frequently deleted in tumours (Fra et al. 1999; Hurlstone et al. 1999). Caveolin expression is lower in breast cancer cell lines as compared with normal epithelial cells of the breast. Furthermore, caveolin expression seems to be inhibitory of growth (S.W. Lee et al. 1998). S.W. Lee et al. (1998) also transfected caveolin cDNA into breast cancer cells that normally show no detectable caveolin and showed that its overexpression in the transfectants resulted in a 50% reduction in growth rate and colony forming ability. Pflug et al. (1999) grew LNCaP prostate cancer cells in androgen-depleted culture medium, and from these they derived tumorigenic androgen-independent cell clones. These clones also showed greatly reduced levels of caveolin expression. In contrast, benign prostate epithelial cells express caveolin at high levels. On the face of it, caveolin seems to function as a tumour suppressor. However, metastatic tumours also overexpress caveolin (Thompson, 1998). It would seem therefore that although its overexpression can lead to androgen resistance, its association with metastatic progression could be a secondary event. Caveolin-3, which is believed to be a muscle-specific isoform, is often mutated in patients with limb girdle muscular dystrophy, and the mutations have been suggested to be associated with the pathogenesis of the disease (McNally et al. 1998a; Minetti et al. 1998).

3

Calcium Binding Proteins and their Natural Classification

Calcium-binding proteins comprise a large family of proteins that can be subdivided into two subfamilies based on their molecular organisation. A large number of these proteins have been identified and characterised, and their physiological functions have been investigated (Tables 2 and 3). The subfamily of CBPs, referred to here as non-EF-hand CBPs, binds to certain phospholipids in a calcium-dependent fashion (Heizmann and Hunziker, 1990). They do not possess the characteristic feature of the other subfamily, popularly described as the EF-hand CBPs. The EF-hand protein subfamily in turn can be divided into two groups (Da Silva and Reinach, 1991). The members of one group function as Ca2+ sensor proteins. These are inactive at calcium concentrations in the low range of 10–7 to 10–8 M. They are activated into playing their regulatory role when the Ca2+ concentration increases to around 10–5 to 10–6 M. The second group comprises EF-hand proteins that are mainly concerned with calcium buffering and transport. Calcium ions function as second messengers par excellence in many pathways of cellular response. The translation of the calcium signal into biological function is mediated by CBPs that occur ubiquitously in intracellular locations as well as in the extracellular matrix. In order to be able to mediate the transduction of calcium signalling into cellular responses, CBPs need to recognise and interact with downstream target proteins. The binding of Ca2+ to CBPs produces conformational changes in these molecules and these changes appear to endow the CBPs with the ability to recognise and interact with their target molecules. This is discussed in greater detail in a later section. Both EF-hand and non-EF-hand CBPs participate in a host of normal physiological functions and consequently they are also associated with an array of pathological conditions (Tables 2 and 3). Although many EF-hand proteins have been studied for their involvement in normal and aberrant physiology and in pathogenesis, only a small number of them have been studied extensively. Consequently, the information currently available about some EF-hand proteins has tended to be somewhat fragmentary. Nonetheless, there is an indisputable theme that underlies the molecular features of CBPs as associated with their function in normal physiology and in pathogenesis. Their functions appear to be linked closely with the molecular organisation of the EF-hands, and furthermore, to the characteristic conformational changes that they undergo and the new molecular configuration that they assume upon Ca2+ binding.

29

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Calcium Signalling in Cancer

TABLE 2 Non-EF-Hand Calcium Binding Proteins Non-EF-Hand CBP Annexins (individual annexins also known as chromobindin, endonexin, lipocortin, etc.)

Gelsolin family proteins Gelsolin

Severin Villin

Function Membrane-related functions; adhesion; fusion; exocytosis; possible cell-cycle-related expression; morphogenesis; differentiation

Martin and Creutz (1987, 1990); Hamman et al. (1988); Martin et al. (1989); Creutz et al. (1992, 1994); further references cited in text

Cytoskeletal dynamics; severing and capping of actin filaments; cell migration, cancer progression; amyloidosis Sequence homology with gelsolin in actin-interacting domains Regarded as a marker of differentiation; occurs in intestinal and brush border epithelia, and in Barrett’s metaplasia and adenocarcinoma

Cited in text

Cap G Fragmin Calreticulin

Calsequestrin Osteocalcin

Regucalcin [senescence marker protein-30 (SMP-30)]a

Ref.

ER-associated CBP; molecular chaperone; intracellular calcium storage; cell adhesion; proliferation; autoimmune diseases Muscle homologue of calretinin, intracellular calcium storage Extracellular matrix (ECM) protein; bone metabolism; cell proliferation; differentiation; cancer invasion; osteotropism of cancer metastasis Implicated putatively in cell proliferation; regulation activity of protein phosphatases and Ca2+/CaM-dependent kinases; adenosine triphosphate (ATP)dependent transport of calcium across the plasma membrane

Cited in text Cited in text

Mishra et al. (1994) Van Ginkel et al. (1998) Constantin et al. (1998) Khanna et al. (1987); Smith et al. (1989); Milner et al. (1991); Heilmann et al. (1993); N. Liu et al. (1993) Cited in text Cited in text

Fujita (1999); M. Yamaguchi (1998); M. Yamaguchi et al. (1996, 1997); T. Murata et al. (1997); Murata and Yamaguchi (1998); Kurota and Yamaguchi (1997); Hanahisa and Yamaguchi (1998); H. Takahashi and Yamaguchi (1997), M. Yamaguchi and Kanayama (1996)

31

Calcium Binding Proteins and their Natural Classification

TABLE 2 (CONTINUED) Non-EF-Hand Calcium Binding Proteins Non-EF-Hand CBP Calelectrin Calcimedin

a

Function

Ref. Morse and Moore (1988) Moore and Dedman (1984); Moore et al. (1984); Morse and Moore (1988)

A large body of literature about regucalcin, mostly from the laboratory of M. Yamaguchi, has appeared in the past few years. The references cited indicate that it is a non-EF-hand CBP that shows tissuespecific expression mainly in the liver and to a lesser extent in the kidney. It is also found in hepatoma cell lines. The mouse regucalcin gene is described as spanning 18 kbp of the genome and containing seven exons and six introns. The human regucalcin gene has been provisionally mapped to chromosome Xp11.3–q11.2.

32

Calcium Signalling in Cancer

TABLE 3 Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology EF-Hand Protein

S100 family members

Calmodulin

Function Regulatory Proteins Modulation of enzyme function; cytoskeletal dynamics; binds to various cellular target proteins; control of cell cycle traverse and cell proliferation; apoptosis; intercellular communication, signal transduction modulation of cell shape; motility and invasion; metastasis; ageing; Alzheimer’s disease Regulation of enzyme activity; inhibition of transcription; cell proliferation; invasion

Troponin C

Muscle contraction

Myosin light chains Recoverin

Muscle contraction Activation of guanylate cyclase; phototransduction Calcium buffering and transport; neuronal function; motor coordination; neuroprotective action

Calbindins, 9 and 28 kDa

Calretinin

Guanylate cyclase (GC)-activating protein Centrin (caltractin)

Calnuc (nucleobindin) Reticulocalbin family (reticulocalbin, calumenin, ERC-55, Cab-45) DAG kinases α, β, and γ

Calpains

Calcium buffering and transport; phosphorylation; cell proliferation and differentiation; tumour marker Photoreceptor-specific CBP

Ref.

Cited in text

James et al. (1995); Corneliussen et al. (1994) J.D. Potter and Johnson (1982); Wnuk (1988) Stryer (1991); Gorczyca et al. (1995) Bronner et al. (1986); Chard et al. (1993); further references cited in text Rogers (1987); further references cited in text Cited in text

Microtubule dynamics; cell polarity; cell motility; duplication of microtubule organising centre; cell proliferation; response to environmental signals Associated with luminal surface of Golgi membrane ER-associated proteins; membrane trafficking of macromolecules DNA supercoiling (?)

Cited in text

Phosphorylation of DAG and activation of PKCs

Cited in text; Sakane et al. (1990); Sakane and Kanoh (1997); Yamada et al. (1997) Cited in text

Keratin filament aggregation (proteolytic processing of profilaggrin)

P. Lin et al. (1998, 1999) Cited in text

33

Calcium Binding Proteins and their Natural Classification

TABLE 3 (CONTINUED) Functional Grouping of Major EF-Hand Proteins and Their Involvement in Normal and Aberrant Physiology EF-Hand Protein Osteonectin

Calcineurin B Calmyrin

β-Parvalbumin (oncomodulin) α-Parvalbumin

Calbindins, 9 and 28 kDa Calretinin

Function Embryonic development and differentiation; extracellular matrix (ECM) remodelling; modulation of cell adhesion; cell shape; angiogenesis; tumour development and progression Protein phosphatase B regulatory subunit Calcineurin B- and CaM-related 191 amino acids; two C-terminal EF-hands; binds to cytoplasmic domain of platelet integrin αII bβ3; putative regulation of the integrin function Buffer Proteins Lymphocyte maturation Muscle contraction; electrophysiology of neurones

Calcium buffering and transport Calcium buffering and transport; phosphorylation

Ref. Cited in text

Cited in text Naik et al. (1997)

Brewer et al. (1990) Heizmann (1984); Heizmann and Berchtold (1987); Heizmann and Braun (1990) Bronner et al. (1986); Chard et al. (1993) Rogers (1987)

Note: The S100 protein family is separately listed in Table 17. Calretinin has been advisedly included as both a regulatory and buffer protein, for although it was regarded preeminently as a buffer protein, its participation in cell proliferation and differentiation and utility as a tumour marker were considered as providing a strong basis for classifying it with other calcium-binding regulatory proteins. EF-hand proteins have also been identified in prokaryotes. An example of this is the Escherichia coli lytic transglycosylase Slt 35. The EF-hand domain of this enzyme binds Ca2+ as well as Na+, albeit in different molecular configurations. Ca2+ ions are bound preferentially, and this appears to be conducive to the stability of the enzyme (Van Asselt and Dijkstra, 1999). Source: Based on B.W. Schafer and Heizmann (1996) and references cited in the text in the course of discussions relating to their respective physiologic functions.

4

Non-EF-Hand Calcium Binding Proteins

Several non-EF-hand calcium-binding proteins have been studied in the past few years with regard to their participation in normal physiological processes and their apparent ability to modulate biological behaviour of cells (see Table 2). Of these, annexins, gelsolin, calreticulin, and osteocalcin have been extensively investigated. Although they are grouped together here as non-EF-hand CBPs, the pathways of their approach to the modulation of cell physiology and function are distinctively different. Annexins are calcium-dependent membrane binding proteins that participate in membranerelated processes of fusion, adhesion, exocytosis, and the remodelling of the plasma membrane. Calreticulin also subserves a membrane-associated function (e.g., the intracellular storage of calcium). Osteocalcin is an extracellular matrix protein and is involved in bone metabolism. It is apparently also significantly implicated in invasive behaviour of cancers, their proliferation, and osteotropic metastasis. In contrast, gelsolin activity is mainly targeted on the cytoskeletal dynamics.

ANNEXINS STRUCTURE Several non-EF-hand CBPs have been identified and sequenced. Of these, annexins have been studied extensively. Annexins form a family of structurally related and highly conserved proteins that bind phospholipids in a Ca2+-dependent manner. Several annexins have been identified and some of these have been well characterised, e.g., annexins I, IV, V, and XII. A 40-kDa annexin, distinct from annexin XII, has been reported from Hydra vulgaris (Schlaepfer et al. 1992a). Although basically annexins are intracellular proteins, it has been postulated that these proteins may be inserted into the phospholipid bilayer of the plasma membrane. Integration of the molecules into the plasma membrane is essential for their postulated functions of membrane trafficking, fusion, and the formation of ion channels. The crystal structure of annexins is characterised by a common fold that consists of four domains. Each domain has five helices with a short interconnecting loop. The loops between helices A and B (domain 3) and helices D and E (domain 2) are attributed with the ability to bind phosphatidyl serine in a Ca2+-dependent manner. It has been suggested that annexin XII forms a trimer on the surface of the membrane (Luecke et al. 1995). Furthermore, it appears that an intramolecular Ca2+-binding site participates in the formation of the trimer, and this is quite distinct from the Ca2+ site involved in interaction with phospholipid (Mailliard et al. 1997). Langen et al. (1998) have postulated that, at low pH, annexin is inserted into the membrane as a continuous transmembrane helix that is formed, in a reversible manner, from

35

36

Calcium Signalling in Cancer

the helix–loop–helix configuration. They regard this latter configuration as being conducive to ion channel formation. Tyrosine phosphorylation of the tetramer may occur following the interaction of the tetramer with phospholipid. This is suggested by the stimulation of pp60src RTK following the binding of the annexin II tetramer to plasma membrane or phosphatidyl serine vesicles (Bellagamba et al. 1997).

BIOLOGICAL FUNCTIONS Annexins are regarded generally, although not exclusively, as intracellular proteins with predominantly intracellular functions. Several biological functions have been attributed to annexins, (e.g., membrane fusion, calcium-channel formation, etc.) that involve cAMP as the second messenger in signal transduction. Thus membranebound annexins could be subserving a different class of function. Annexin II, for instance, can be found in both soluble and membrane-bound states. It has been identified on the cell surface, and in this location it could be functioning as a receptor for extracellular signalling ligands (Siever and Erickson, 1997). This argument is further reinforced by the apparent interaction of annexin V with the actin cytoskeleton. Membrane preparations of activated platelets indicate the presence of an 85kDa complex, which contains annexin V. Besides, this complex also contains actin (Tzima et al. 1999). This suggests the presence of a link between the plasma membrane and the actin cytoskeleton, which is potentially of considerable significance in defining the function of annexins as an early link in the transmembrane signal transduction machinery. Annexins also show a major involvement down-stream in the flow of information in PKC-mediated transduction of growth factor signals. It has been demonstrated that annexin V is able to inhibit PKC and this in turn results in the inhibition of phosphorylation of annexin I (Schlaepfer et al. 1992b, 1992c). Annexin I has phosphorylation sites in the hinge region, which is believed to be important for cell proliferation and differentiation. Annexin XII is also a high-affinity substrate for phosphorylation by PKC (Schlaepfer et al. 1992b). This could be a reason why they show cell proliferation related expression (Schlaepfer and Haigler, 1990). Another aspect of PKC-mediated regulation of annexin function is the phosphorylation of certain amino acid residues that are involved in the interaction with EF-hand proteins of the S100 family. Annexin II is known to interact with S100A10 (p11) protein. There is a serine residue in the N-terminal region of annexin. If this residue is phosphorylated by PKC, annexin II interaction with S100A10 appears to be inhibited. Other amino acid residues have been implicated in similar interactions between annexin I and S100A11 (S100C) (Seemann et al. 1996). Annexin XI-A has been shown to bind to S100A6 (calcyclin) by means of specific hydrophobic residues on the surface of its α-helix (Sudo and Hidaka, 1999). It seems, however, that annexin V does not inhibit the phosphorylation of annexin XII by the epidermal growth factor RTK (Schlaepfer et al. 1992c). On the other hand, annexin II, which is also involved with cell proliferation is a substrate for RTKs. Hubaishy et al. (1995) demonstrated that the phosphorylation of annexin II is effected by RTKs. They reported that phosphorylation negatively regulates annexin

Non-EF-Hand Calcium Binding Proteins

37

II function, because it inhibited the binding of annexin II to and the formation of F-actin bundles. The annexins show a definable pattern of intracellular distribution. Barwise and Walker (1996) studied the distribution of annexins I, II, IV, V, and VI in human foreskin fibroblasts. Annexins IV and V were predominantly located in the cytoplasm and annexin VI was partly associated with the endoplasmic reticulum, whereas annexins I and II were associated with the plasma membrane. Furthermore, annexins I, IV, and V occurred in the nucleus at higher concentrations than in the cytoplasm. When the cells were treated with calcium ionophores and C2+[i] was raised, a marked relocation of the annexins occurred. Annexins II, IV, and V translocate from the nucleoplasm to the nuclear membrane. In MG-63 cells a relocation of intranuclear annexins IV and V to the nuclear membrane occurs within 40 s after treatment (Mohiti et al. 1995). Annexins located in the cytoplasm may be translocated to the plasma membrane. Human osteosarcoma MG-63 cells grown under normal serum conditions show high levels of annexin V in the nucleus. Upon serum starvation, however, the cells lose 72% of the nuclear annexin. When serum levels are restored nuclear annexin levels are also restored (Mohiti et al. 1997). These authors also observed that annexin phosphorylation is essential for the relocation of annexins. The translocation of annexin V appears to be inhibited when tyrosine kinase activity is blocked. Whether PKC-mediated phosphorylation plays any part in the translocation is unclear. PKC itself shows intracellular translocation upon being activated. It would have been of considerable interest also to determine PKC translocation, especially in light of the role PKC plays in the regulation of annexin function. A different pattern of annexin II and V translocation has been described in human neuroblastoma SH-SY5Y cells. Blanchard et al. (1996) found that both annexins were associated with membranes. Annexin II showed a uniform association before elevation of intracellular calcium levels, but upon treatment with calcium ionophores, it was relocated to discrete patches. Annexins take part in the process of calcium-dependent aggregation of liposomes. Annexins I through IV, but not V or VI, seem to mediate liposome aggregation (L. Liu et al. 1996). From this functional viewpoint, there is evidence of cAMP involvement in calcium-dependent aggregation of phosphatidyl serine liposomes and bovine chromaffin granules and in calcium channel activity (Cohen et al. 1995). Blanchard et al. (1996) showed that membrane-associated annexin II is reorganised into discrete patches. It seems preferentially to partition into cholesterol-rich domains of the plasma membrane. Furthermore, the transmembrane adhesive glycoprotein CD44 also partitions into the same type of domain, with the result that CD44 clusters at the external membrane surface and annexin II at the cytoplasmic surface (Oliferenko et al. 1999). Whether this seemingly fortuitous partitioning of annexin II has a functional provision in CD44-mediated cell adhesion is unclear at present. Nevertheless, with the demonstration by Tzima et al. (1999) that annexin II might be coupled with the actin cytoskeleton, this is highly suggestive of its involvement in membrane-mediated functions of intercellular adhesion and cell fusion. Several mammalian annexins positively influence the process of exocytosis (Creutz et al. 1992). Such a function seems to be strengthened by the observation by Oliferenko et al. (1999) that the redistribution of CD44 and annexin II was accompanied by changes in the cytoskeletal organisation. CD44 patching is induced

38

Calcium Signalling in Cancer

by overexpression of the S100A4 protein, and this appears to relate to the ability of S100A4 to depolymerise cytoskeletal elements (Lakshmi et al. 1993, 1997). Therefore, there is the distinct possibility that annexins and S100A4 might cooperate in generating the membrane-mediated behavioural changes of cells. The intracellular translocation of annexins in the human osteosarcoma cell line MG-63 has been found to be phosphorylation dependent. Serum depletion results in a reduction of nuclear annexin V levels. With serum stimulation these levels increase. Inhibition of tyrosine kinases has been found to reduce nuclear translocation of the annexin (Mohiti et al. 1997), which suggests that the relocation of annexins requires tyrosine phosphorylation.

ANNEXINS

IN

CANCER GROWTH

AND

PROGRESSION

The influence of annexins on cell growth was recognised some years ago. The expression of annexin VII has been reported to inhibit the growth and viability of some secretory mutants of Saccharomyces cerevisiae at semipermissive temperatures. In contrast, annexin IV mitigated the growth defect of the sec2 secretory mutant (Creutz et al. 1992). Recent studies have in fact elucidated in some detail the significant part that annexins play in cell proliferation. Raynal et al. (1997) demonstrated convincingly that annexins exhibit a cell cycle-related expression. Apparently, annexins II, V, and VI are, on the whole, uniformly expressed at a low level at all phases of the cell cycle. Annexin IV, on the other hand, showed a 50% increase in the S-phase of the cycle. Annexins I and V decreased by 40% in early G2M phase, although the corresponding gene expression did not vary. These observations show that there are specific patterns of occurrence of annexins and the expression may be regulated at the transcriptional level. The apparent cell cycle-related expression of annexins and the deregulation of PKC-mediated signal transduction with consequent deregulation of proliferation has prompted the investigation of annexins in relation to the proliferative state and progression of human cancers. Masaki et al. (1996) found that annexin I levels were greatly increased, at both the transcriptional and the protein levels, in human hepatocellular carcinomas as compared with normal liver tissue. Annexin II was reported to be overexpressed in human pancreatic carcinomas compared with normal pancreatic tissue (Vishwanatha et al. 1993). Furthermore, these authors found that the expression of annexin II was associated with proliferating ductal adenocarcinomas and especially that its expression colocalised with that of proliferating cell nuclear antigen (PCNA). Roseman et al. (1994) also found some association between levels of annexin II and the BrDU (bromo-deoxyuridine) proliferation index. Compatible with this is the reported histological grade-related expression of annexin II in gliomas (Roseman et al. 1994). They found that annexin II levels were greater in glioblastoma multiforme than in anaplastic astrocytomas, which in turn showed higher levels than did astrocytomas. With variations of annexin levels seemingly in consonance with growth deregulation in human cancers, there has been some effort directed at examining whether variations in expression might relate to the stage of progression of cancers. As stated before, annexin levels have been found to correlate with histological grade of gliomas

Non-EF-Hand Calcium Binding Proteins

39

(Roseman et al. 1994). Annexin I does not occur in ductal luminal cells of normal breast tissue, but it is expressed in a range of breast cancers including noninvasive ductal carcinomas as well as invasive and metastatic breast cancer (Ahn et al. 1997). Annexin VI, in contrast, has been shown previously to inhibit cell proliferation. Theobald et al. (1994) transfected A431 cells with annexin VI. The growth of these cells, in normal serum concentration, was only moderately inhibited by the expression of annexin VI. When grown in low serum, their doubling time seemed to increase. However, Theobald et al. (1994) found that annexin VI-expressing cells grown in low serum conditions showed arrest at the G1 phase of the cell cycle. Furthermore, annexin VI expression inhibited the growth of A431 cells injected subcutaneously into nude mice (Theobald et al. 1995). It appears that the inhibitory effect is exerted only by the larger of the two splice variants of annexin VI. Fleet et al. (1999) found that the larger splice variant inhibited the Ca2+ mobilisation and cell proliferation induced by EGF. The shorter splice variant of annexin VI was not capable of such inhibition. Francia et al. (1996) identified several mRNAs that are differentially expressed in the murine melanoma cells B16F-10 and an immortalised melanocyte cell line called Melan A. Among these was an mRNA that was found to be identical to the 3′ region of murine annexin VI. The expression of this mRNA was reduced in B16F-10 melanoma cells. Melan A may not be a proper control cell line with which to compare the mRNA profile of B16 melanomas, and the low metastasis variant B16F1 might have been a more appropriate control cell line. Nonetheless, Francia et al. (1996) followed this up with a study of the expression of annexin VI in human melanomas. In these tumours also a reduced or loss of annexin expression was encountered during progression. The expression of annexin V has been found to be markedly suppressed in carcinomas of the uterine cervix and the endometrium (Karube et al. 1995). Thus, most of the annexins studied to date have related directly, however indecisively, to cell proliferation and progression; however, annexin VI seems to differ from the rest. If confirmed, the reported tumour suppressor function of annexin VI does add a new dimension to the biology of annexins. But clearly, more work needs to be done to examine whether the putative suppressor function is associated with the larger splice variant and whether the second shorter variant of the annexin might possess some compensatory and antagonistic function. Overall, in spite of the cell cycle-related expression of some of the annexins, no clear relationship has emerged between their expression and the neoplastic disease process. Annexin II, which seems to relate to histological grade and differentiation of gliomas, tends to be expressed uniformly in the cell cycle. The expression of annexins I and V seems to change in opposite directions in the context of the cell cycle. Both are down-regulated in their expression at the G2M point in the cell cycle. Perhaps such comparisons are unhelpful because of the differences in the biology and histogenesis of the tumours studied.

ANNEXINS

IN

MORPHOGENESIS

AND

DIFFERENTIATION

The expression patterns of annexins have been studied with respect to cell differentiation. Rahman et al. (1997) reported that annexins V and VI are not detectable in

40

Calcium Signalling in Cancer

undifferentiated mesenchymal cells of foetal rat limb buds. The progenitor cells of skeletal muscle, which appear in the limb bud on day 14, express annexin VI on the cell surface upon differentiation from myogenic cells into myotubules. Annexins V and VI also are associated with the differentiation of chondrocytes. King et al. (1997) have confirmed that annexin V is required for the formation of the pericellular matrix of chondrocytes.

THE GELSOLIN FAMILY OF CALCIUM-BINDING PROTEINS Actin-binding proteins constitute a large family of proteins that actively participate in actin bundling, nucleation, actin capping, and severing. Gelsolin, severin, and villin are three important proteins of the gelsolin family of actin-binding proteins that will be discussed here, for they possess the remarkable property of rapidly reorganising the cytoskeleton and also rapidly modulating, in a Ca2+-dependent manner, cell shape, substrate adhesion, and cell locomotion.

GELSOLIN

IN

SEVERING

AND

CAPPING

OF

ACTIN FILAMENTS

Gelsolin was identified as an 82-kDa actin-binding protein that is involved in Ca2+dependent severing of actin filament and capping of filaments. The kinetics of polymerisation of actin filaments, namely, the association and dissociation of monomeric subunits, essentially involves F-actin elongation by the association of ATPbound monomers with the barbed end of the filament and a slower loss of ADPactin from the pointed end (Sheterline and Sparrow, 1994). Actin polymerisation is altered by the sequestration of monomeric elements and by filament capping, which prevents the addition of subunits. The exchange of subunits at the filament ends is controlled by gelsolin and gelsolin-related proteins. They bind to the filaments and inhibit the addition of actin subunits. Several proteins that participate in the regulation of cytoskeletal organisation have been identified (A. Schafer and Cooper, 1995). A large number (>60) of actin-binding proteins are known (Pollard et al. 1994) and the new ones are continually being identified and reported. A group of proteins composed of the gelsolin family and of tensin, and profilin bind to barbed ends. Others bind to pointed filament ends, whereas some proteins bind alongside the filament and yet successfully influence filament assembly. Certain isoforms of tropomyosin have been found to protect actin filaments from gelsolin-mediated severing. This protective function may be further enhanced by caldesmon (Ishikawa et al. 1989a). Tropomyosins appear to be able to anneal the actin fragments and again caldesmon is said to accentuate this process (Ishikawa et al. 1989b). The Drosophila flightless-I gene and the homologous human FLII gene encode gelsolin-like actin-binding proteins (H.D. Campbell et al. 1997). Constantin et al. (1998) have isolated a gelsolin-like protein from Physarum polycephalum that affects cytoskeletal integrity when introduced into mammalian cells. Tropomodulin and spectrin bind to pointed filament ends. Two isoforms of gelsolin had been identified: a cytoplasmic form and a secreted isoform called plasma gelsolin. A third isoform has now been reported; all three isoforms arise by alternative splicing of the gelsolin

41

Non-EF-Hand Calcium Binding Proteins

transcript (Vouyiouklis and Brophy, 1997). The FLII maps to chromosome 17p11.2 (Chen et al. 1995), in the region that is consistently deleted, and is associated with Smith-Magenis Syndrome (SMS). Cytoplasmic-free calcium and inositol 4,5-bisphosphate regulate the function of gelsolin family proteins. Gelsolin binding to PIP2 has been found to be modulated by Ca2+ (K.M. Lin et al. 1997). Several functional domains that regulate actin filament length have been identified in the gelsolin molecule. The N-terminal domain (S1) is able to inhibit actin polymerisation. The inhibitory activity has been attributed to a small peptide sequence. The interaction of the C-terminal domains (S4–6) with actin filaments occurring during the severing process has been localised with residues 112 to 120 of actin subdomain 1 (Feinberg et al. 1997a, 1997b). By deletion mutagenesis, residues close to the S2 domain have also been implicated in actin filament binding, capping and as well as interaction with phosphoinositides (H.Q. Sun et al. 1994). Cofilin is another protein that binds actin and promotes its depolymerisation. There are important sequence homologies between cofilin and gelsolin. The actin-binding peptide of cofilin has been found to compete with gelsolin segment S2 for binding to actin, suggesting that they share a binding site on the actin filament (Van Troys et al. 1997). Interaction with gelsolin induces conformational changes in actin, and nucleation of actin polymerisation may be promoted by these conformational changes (Khaitlina and Hinssen, 1997).

GELSOLIN

IN

EMBRYONIC DEVELOPMENT

AND

MORPHOGENESIS

Gelsolin and related proteins have been shown to be actively involved in normal embryonic development and morphogenesis as well as in pathogenesis of diseases such as amyloidosis and cancer. The gelsolin gene has been mapped to the chromosome 17p11.2 region, which is a critical region deleted in SMS which encompasses short stature, brachydactyly, dysmorphic features, retarded development, and behavioural problems. Gelsolin may also participate in the process of programmed cell death or apoptosis. Gelsolin shows a definable pattern of expression in the developing CNS. It is found predominantly in oligodendrocytes and Schwann cells. It is also found in the myelin sheath. In oligodendrocytes, gelsolin is found in the soma and in cultured cells it is detectable in the branched cell processes (Lena et al. 1994). The protein is detectable in the brain of newborn rats. The expression of gelsolin gradually increases at 8 to 10 days after birth, reaching a maximum at 20 to 30 days when myelin formation is actively occurring. However, subsequently, its levels decrease even though myelin basic protein levels continue to rise until 6 months after birth. In Schwann cells gelsolin is found in the cytoplasm and in compact myelin (J. Tanaka and Sobue, 1994). It would appear therefore that gelsolin may be involved in the wrapping of myelin sheaths around the axons by promoting a process of motility generated by means of its effects on the cytoskeleton (J. Tanaka and Sobue, 1994; Lena et al. 1994). The gelsolin-like protein, fragmin, which has been isolated from Physarum, has been shown to interact with actin in a Ca2+-dependent fashion, when introduced into PtK2, CV1, and NIH3T3 cells. This has been reported to cause cytoskeletal disruption and bring about changes in cellular morphology (Constantin

42

Calcium Signalling in Cancer

et al. 1998). Fibroblast migration has been found to be dependent on the actinsevering activity of gelsolin (P.D. Arora and McCulloch, 1996). Furthermore, gelsolin has been implicated in cytoskeleton-mediated transduction of EGF signal and EGF-induced cell motility (P. Chen et al. 1996). The Drosophila melanogaster flightless-I gene product is a gelsolin-like protein that is also involved in embryonic development and in the structural organisation of the indirect flight muscle. However, it should be pointed out that gelsolin-null transgenic mice appear to develop normally, albeit associated with certain abnormalities such as prolongation of bleeding time caused by abnormalities of platelet shape, and also inhibition of neutrophil migration. The transgenic mice also show increased stress fibre formation in dermal fibroblasts together with reduced motility and increased contractility (Witke et al. 1995).

GELSOLIN EXPRESSION

IN

AMYLOIDOSIS

The strong association of gelsolin with CNS components has initiated studies of possible alterations in its expression in CNS-associated abnormalities. CNS abnormalities in patients with familial amyloidosis may conceivably be related to gelsolin expression (Kiura et al. 1995). Paunio et al. (1997) recently measured the mRNA levels of both intracellular and secreted gelsolin isoforms in tissues of human subjects. Intracellular gelsolin mRNA has been found to be a major component in all tissues. Most adult tissues can show mRNAs of both isoforms, with muscle and skin tissues showing especially high levels of expression. The high levels of skin gelsolin may be related to skin amyloidosis. The levels of expression may differ between adult and infant tissues. Patients with gelsolin-related amyloidosis had higher serum levels of gelsolin but did not show increased gene expression.

GELSOLIN

IN

CANCER

A major feature of cancer development is the perceived derangement of the cell signalling system and the inappropriate acquisition of cell motility. The process of actin filament severing and capping brings in its wake alterations in cellular motility. The involvement of gelsolin in the remodelling of the cytoskeleton has naturally led to the investigation of the expression and possible involvement of gelsolin and related proteins in cancer development and progression. Neoplastic transformation alters cell behaviour, morphology, and pattern of growth. These changes are indeed attributable to the loss of stress fibres and focal adhesion. Accompanying cell transformation, modulation of the expression of cytoskeleton-associated proteins is observed, and among them is gelsolin. Van de Kerckhove et al. (1990) showed some time ago that gelsolin expression is down-regulated in transformed human fibroblasts and epithelial cells. Transformed cells also show a down-regulation of tropomyosin. Upon restoration of tropomyosin status, cells return to the normal modes of spreading and organisation of stress fibres. These events are far from being coincidental, for the higher molecular weight forms of tropomyosin appear to protect actin filaments from the severing activity of gelsolin (Matsumura et al. 1985; Ishikawa et al. 1989a).

Non-EF-Hand Calcium Binding Proteins

43

Although only a few studies have been carried out to date, gelsolin appears to be consistently down-regulated in cancers. Moriya et al. (1994) found that gelsolin expression was down-regulated in several tissue culture cell lines derived from human gastric carcinomas. Gelsolin levels were found to be markedly reduced in human breast cancer cell lines as compared with normal breast epithelial cells and cell lines derived from benign breast diseases. Gelsolin loss was also found in 70% of 30 sporadic invasive breast carcinomas as well as in all chemically induced murine and rat mammary tumours (Asch et al. 1996). In human transitional cell bladder carcinoma cell lines as well as tumour tissues (14 out of 18 samples) gelsolin was not detectable or detectable only at low levels compared with normal bladder epithelium (M. Tanaka et al. 1995). M. Tanaka et al. (1995) also transfected gelsolin cDNA into the human bladder cancer cell line UMUC-2. The transfectants showed a greatly reduced colony forming ability as well as tumorigenicity. One can see from these studies the emergence of the concept of tumour suppression by gelsolin. However, it is paradoxical that gelsolin on the one hand can enhance cellular motility and on the other suppress tumorigenicity. Tumorigenicity and cellular motility can be dissociated temporally, and therefore one can envisage a situation where the loss of gelsolin could lead to tumour development and be involved in a positive way at a later stage of tumour progression by conferring invasive properties on tumour cells by altering cytoskeletal dynamics. There is little doubt that this is a fruitful avenue to explore. One needs to investigate the pattern of gelsolin alteration at various stages of tumour progression. The colon carcinoma model immediately comes to mind. The progression of colon carcinoma has been described in clearly identifiable phenotypic phases, and there is considerable evidence that different genetic changes are associated with the different phases of progression and that the development of carcinoma is a cumulative effect of these genetic alterations (Sherbet and Lakshmi, 1997b). It would be interesting to see whether alterations of gelsolin or related gene alterations have a place in this picture. There is also the need to glean more information about the intracellular localisation of these proteins in the context of the alterations in cytoskeletal dynamics occurring in proliferating and transformed cells. Other questions can be raised. For example, are gelsolin and related proteins associated with cell proliferation and apoptosis? We have noted earlier that EGFinduced cell motility involves gelsolin. The alteration of cytoskeletal dynamics is an essential ingredient of cell proliferation. There is compelling evidence that other Ca2+-binding proteins such as the S100A4 (18A2/mts1), which also causes perturbation of cytoskeletal dynamics (Lakshmi et al. 1993), can control cell cycle progression by interfering with G1S and G2M checkpoint control exerted by the p53 suppressor gene and stathmin (Parker et al. 1994a, 1994b; Cajone and Sherbet, 2000). Therefore, if the gelsolin story is to be comprehended it is of the utmost importance to determine whether gelsolin is associated with cell proliferation. There are indications that gelsolin may indeed influence cell population dynamics by inhibiting apoptotic death of cells (Ohtsu et al. 1997).

44

Calcium Signalling in Cancer

SEVERIN

AND

CYTOSKELETAL REORGANISATION

Severin is a 40-kDa Ca2+-activated actin-binding protein that was isolated from the amoeba Dictyostelium discoideum, and of this, mammalian homologues have also been identified. Severin severs F-actin, nucleates actin assembly, and caps actin filaments. It is a gelsolin-related protein and shares sequence homology with gelsolin and profilin in certain conserved functional domains that are involved with actin interaction (Schnuchel et al. 1995). Severin possesses two actin-binding sites located next to each other, and these participate in both severing and nucleation functions. The third actinbinding site, situated near the N-terminus, suffices for the filament capping function (Eichinger et al. 1991). Eichinger et al. (1998) recovered a protein kinase from cytosolic extracts of D. discoideum. This kinase phosphorylates severin, but phosphorylation is reduced in the presence of calcium. Eichinger et al. (1998) have therefore suggested that phosphorylation might be another regulatory mechanism. Severin is known to alter rapidly the organisation of the cytoskeleton and as a consequence influence cell adhesion to the substratum, pseudopodial activity, and cell motility. Early work had suggested that mutants deficient in severin did not affect the motility of Dictyostelium (Andre et al. 1989). But Schindl et al. (1995), who studied mutants that were deficient in severin, found the protrusion of pseudopodia was slower and the pseudopodia were shorter in the mutants. As noted in the previous section, the loss of gelsolin has been associated by some with cell motility, especially in neoplastic transformation. Folger et al. (1999) have reported that severin replaces gelsolin in murine Lewis lung carcinoma (LL2) cells. No severin is detected in normal epithelial lung tissue, but gelsolin is expressed at high levels. Folger et al. (1999) suggested tentatively that severin takes over from gelsolin the function of actin fragmentation in transformed cells. In light of the paradoxical effects of gelsolin of suppressing tumorigenicity on the one hand, and stimulating invasive behaviour on the other, further work on the expression of gelsolin and severin in a temporal relationship to tumour progression seems warranted.

VILLIN

IN

DIFFERENTIATION

AND

NEOPLASIA

Villin is another actin-binding protein that shows sequence homology with gelsolin with respect to certain conserved sites involved in actin-binding. It occurs in intestinal and kidney brush border epithelia. Villin is known to take part in actin bundling, nucleation, filament capping, and severing in a Ca2+-dependent manner, in vitro. Villin cDNA has been transfected into cells that do not synthesise the protein, and it has been found that this induces the growth of microvilli and the reorganisation of the submembranous actin cytoskeleton of the transfectant cells. This process is inhibited by cytochalasin (Friederich et al. 1993). Friederich et al. (1999) have provided further experimental evidence for the involvement of villin in actin bundling. Villin not only induces the growth of microvilli, but also in parallel induces actin bundling. This actin bundling ability seems to depend on the KKEK motif

Non-EF-Hand Calcium Binding Proteins

45

occurring at the C-terminal end of the F-actin-binding site. However, some uncertainty about these events has arisen with the report by Ferrary et al. (1999) that in villin-null mice there is no disruption or disorganisation of microvilli, nor are there any changes noticed in the localisation of actin-binding and membrane proteins of the intestinal brush border in these null animals. Nevertheless, because the expression of villin appears to be a marker of differentiation, it has been investigated as a potential marker of tumour progression. Villin has been employed as a means of differentiating between primary pulmonary adenocarcinomas and pulmonary metastases of neoplasms of the bronchial or digestive tract. Nambu et al. (1998) examined 57 primary pulmonary adenocarcinomas for villin expression and found that 31% of the cancers expressed villin. Of the positives, 10.5% showed a diffuse cytoplasmic distribution of villin and 21% showed cytoplasmic distribution with minor brush border staining for villin. In contrast, metastatic lung tumours showed mainly primary brush border localisation that is characteristic of villin. The findings of J.Y. Tan et al. (1998) are similar. These authors found that villin, together with cytokeratin 7 and 20, is useful in differentiating between primary pulmonary adenocarcinomas and pulmonary metastases of colon carcinomas. Villin is expressed in Barrett’s metaplasia as well as in Barrett’s adenocarcinoma. According to Regalado et al. (1998), all metaplasias and 93% (30) of adenocarcinomas were villin positive. Furthermore, Northern analyses revealed the presence of both 3.5- and 2.7-kb villin mRNAs. Thus, overall, villin seems to be expressed in most adenocarcinomas, metaplasias, as well as other oesophageal tumours. Villin also has been investigated as a possible marker of neuroendocrine tumours of the gastrointestinal tract and also of hepatic tumours (P.J. Zhang et al. 1997; Velazquez et al. 1998), but definitive reports of these studies have not been published. Another actin-regulatory protein called Cap G, which belongs to the gelsolin family, has been investigated somewhat superficially. Cap G is an actin-capping but not -severing protein. The Cap G gene is located on the short arm of chromosome 2 and consists of ten exons and nine introns. The open reading frame has nine exons and eight introns (Mishra et al. 1994). Van Ginkel et al. (1998) found that this gene is differentially expressed between normal uveal melanocytes and uveal melanomas and cell lines derived from these tumours. In summary, there seems to be ample evidence that villin gene function is involved in the organisation of the brush border cytoskeletal assembly and in the formation of microvilli. Therefore, the use of this protein as an indicator of brush border differentiation, with an implicit relationship to neoplastic transformation, seems fully justified. However, as a marker, villin will need to perform more stringently and with greater specificity than merely providing a means of differentiating between primary tumour of the lung and tumours metastatic to the lung. There is much scope for combining villin expression with other proteins such as ezrin and moesin, which link the plasma membrane with the cytoskeleton. These proteins not only localise with the actin cytoskeleton, but they are also specifically associated with the formation of microvilli and membrane ruffles.

46

Calcium Signalling in Cancer

CALRETICULIN AND ITS FUNCTIONAL DIVERSITY STRUCTURE

AND

MOLECULAR FEATURES

OF

CALRETICULIN

Two types of CBPs are associated with the endoplasmic reticulum (ER), namely reticulocalbin (an EF-hand CBP whose localisation and function are discussed in Chapter 12 and calreticulin). Calreticulin is a non-EF-hand CBP that is ubiquitously associated with the ER of a variety of tissues. It is regarded as the nonmuscle equivalent of calsequestrin, which is associated with the sarcoplasmic reticulum (SR). Calreticulin, which was identified as a CBP and isolated from SR (Ostwald and MacLennan, 1974), is believed to be a homologue of calnexin, which is a 64kDa transmembrane protein (Hammond and Helenius, 1995). It is a highly conserved protein. Calreticulin obtained from rat liver has a molecular size of 60 kDa. In some cell-free systems a 62-kDa protein has been detected, which is probably processed into 60-kDa calreticulin (Denning et al. 1997). Hershberger and Tuan (1998) have cloned a full-length cDNA for calreticulin from mouse trophoblast coding for a 57kDa protein showing a high degree of sequence homology to calreticulin. Yamamoto and Nakamura (1996) isolated and sequenced the ER-associated calnexin from Rana rugosa, which shared 77% sequence homology with calreticulin. Two distinct isoforms of calretinin have been isolated from spinach leaves and from the pollen of Liriodendron tulipifera L. and Ginkgo biloba L. (Navazio et al. 1995, 1998; Nardi et al. 1998). These are reported to show very low sequence homology with animal calreticulin. However, maize calreticulin is said to be a 48-kDa protein (as deduced from its cDNA sequence), highly acidic in nature, sharing 77 to 92% sequence homology with other plant calreticulins, and have approximately 50% homology with animal calreticulin (Dresselhaus et al. 1996). Calreticulin consists of 400 amino acid residues. It has three distinct domains: an N-terminal domain containing 180 amino acid residues, a C-terminal domain with acidic residues and lysines, and a middle domain (P-domain) that is composed of three repeats of a 17 amino acid motif. The C-domain binds calcium with high capacity but low affinity. In contrast, the P-domain binds calcium with low capacity but high affinity. Overall, calreticulin may be seen as a high-capacity but low-affinity CBP. The mouse calreticulin gene spans 4.2 kbp of genomic DNA and contains nine exons and eight introns (Waser et al. 1997). In the mouse the gene is located on chromosome 8 (Rooke et al. 1997). It should be pointed out here that P. Lin et al. (1998) state that nucleobindin, a mammalian protein showing a high degree of homology to calreticulin, is an EF-hand CBP. This protein is found in the cytosol and is associated with membranes, in the latter case mainly with the luminal surface of Golgi membranes.

REGULATION

OF

CALRETICULIN EXPRESSION

The expression of calreticulin appears to be regulated by intracellular calcium levels. The promoter region of the calreticulin gene contains elements responsive to the calcium ionophore A23187 and to agents such as thapsigargin, which can raise intracellular calcium levels by releasing calcium from intracellular stores. Both A23187 and thapsigargin have been shown to increase transcription of the calreticulin gene. It is not

Non-EF-Hand Calcium Binding Proteins

47

affected by changes in extracellular or cytoplasmic levels of calcium. This has led to the suggestion that loss of calcium levels in the intracellular stores could induce gene transcription (Waser et al. 1997). Calreticulin gene expression is also up-regulated by other stimuli such as heat shock and heavy metals like zinc and cadmium (Nguyen et al. 1996; Szewczenko-Pawlikowski et al. 1997). A number of heavy metal ions, e.g., Ni2+, Zn2+, Cu2+, and La3+, stimulate the release of calcium from intracellular stores (McNulty and Taylor, 1999). Hyperthermia also raises intracellular calcium levels by calcium mobilisation from intracellular stores as well as by simulating its influx into the cell (Itagaki et al. 1998). Therefore, the up-regulation of calreticulin gene expression might, again, be mediated by the stress response of depletion of calcium held in the intracellular stores. This is in sharp contrast with the effects of hyperthermia and thapsigargin on the expression of S100A4. Both treatments have been shown to downregulate S100A4 expression (Parker and Sherbet, 1992; Albertazzi et al. 1998a). This might suggest a different mode of regulation from that of calreticulin. The thesis that the calreticulin gene is regulated by androgen has been advocated vigorously by N. Zhu et al. (1998). This is based on the finding that androgen ablation down-regulates and its restoration up-regulates both calreticulin mRNA and protein expression in the prostate. Calreticulin is expressed at higher levels in the prostate than in the seminal vesicle and other organs and muscle. The regulation by androgen occurs only in the prostate and seminal vesicles. In vitro, the induction of calreticulin by androgen is not inhibited by inhibitors of protein synthesis, hence the suggestion that the calreticulin gene might be regulated by androgen. Furthermore, in androgen-sensitive LNCaP prostate cancer cell lines, androgen is able to block apoptosis induced by the calcium ionophore A23187. This effect of androgen can be negated by antisense calreticulin oligonucleotides (N. Zhu et al. 1999).

PHOSPHORYLATION

OF

CALRETICULIN

Calreticulin phosphorylation occurs under different physiological conditions. However, it is unclear at present whether this is a physiological mechanism by which calreticulin activity is regulated. A phosphorylated form of calreticulin is detectable in cells infected with rubella virus (RV). It has been reported that the binding of phosphorylated calreticulin to the 3′-end of RV genomic RNA is necessary for initiating RNA replication (Singh et al. 1994). Phosphorylation is a requirement also in the binding of calreticulin to Leischmania donovani RNA (Joshi et al. 1996). Calreticulin from spinach leaves is phosphorylated by protein kinase (casein kinase) CK2, but those of animal origin are not substrates for this kinase. It appears that this is due to structural differences between the two types of calreticulin (Baldan et al. 1996). CK2 was identified as an ER-associated kinase involved in the phosphorylation of calreticulin (Ou et al. 1992). CK2 might be localised in the lumen of the ER (N.G. Chen et al. 1996). The in vitro phosphorylation of calreticulin and other proteins in plasma membrane-enriched fractions has been attributed also to other protein kinases susceptible to inhibition by the PKC inhibitor staurosporine (Droillard et al. 1997). CK2 also phosphorylates calnexin in vitro as well as in vivo. Serine residues that occur in the C-terminal half of the cytosolic domain of calnexin were exclusively phosphorylated (Wong et al. 1998). Sphingosine-dependent kinases

48

Calcium Signalling in Cancer

(SDKs) are another kinase variety that have been shown recently to phosphorylate calreticulin. SDK1 has been found specifically to phosphorylate calreticulin and protein disulphide isomerase (PDI) (Megidish et al. 1999). Heat shock proteins are also substrates of SDKs. Calreticulin, heat shock proteins, and PDI have a common function as molecular chaperones. Furthermore, calreticulin and PDI both occur in the ER. Therefore, these observations might be the closest researchers have come to suggesting a relationship between calreticulin function and phosphorylation. In spite of these various findings, how phosphorylation of these molecules regulates their participation in their apparently diverse physiological functions is yet to be elucidated. At present, no changes in the state of calreticulin phosphorylation have been found to correlate with a specific function. Furthermore, it has been suggested that calreticulin could occur in a constitutively phosphorylated form (Cala, 1999). This would deny the process of phosphorylation any regulatory control over calreticulin function. Nevertheless, the induction of apoptosis of HL-60 cells by geranylgeraniol has been shown to be accompanied by a decrease in calreticulin as well as a decrease in the phosphorylation of another protein of 36 kDa molecular size. These reductions occur prior to DNA fragmentation (Nakajo et al. 1996). The occurrence of these events in parallel suggests that calreticulin may influence the phosphorylation of the 36-kDa protein.

INTRACELLULAR DISTRIBUTION

OF

CALRETICULIN

As stated earlier, calreticulin shows a predominant association with the intracellular membrane system, the ER. The molecule contains KDEL/HDEL sequence at the Cterminal, which is the targeting signal for its localisation at the ER. Calreticulin occurs also in the SR and membranes of the Golgi bodies. In protoplasts of plants, it is localised in the ER (Opas et al. 1996b). In animal as well as in some plant cells, calreticulin is found in the lumen of the ER, nucleus, and nuclear membrane, on the surface of the cells, and in the cytoplasm (Opas et al. 1991; Dedhar, 1994; White et al. 1995; Dresselhaus et al. 1996).

CALRETICULIN

IN INTRACELLULAR

CALCIUM STORAGE

Calreticulin is prominently associated with the endoplasmic reticulum, as its nomenclature implies. Over the past few years, it has been found to participate in several biological phenomena. With its occurrence in the ER, calreticulin has been linked empirically with the cellular faculty of intracellular storage of calcium. This role has been confirmed by a variety of experimental data. There are several lines of evidence on this score. Using reporter gene constructs carrying the promoter of calreticulin gene, Waser et al. (1997) showed that the promoter can be transactivated by agents such as bradykinin and thapsigargin, which are known to release calcium held in intracellular stores. They have identified two regions of the promoter that are responsive to thapsigargin as well as the calcium ionophore A23187. Furthermore, both these agents have been shown to be able to enhance the transcription of calreticulin gene in NIH3T3 cells.

Non-EF-Hand Calcium Binding Proteins

49

Mery et al. (1996) transfected a mouse fibroblast cell line with an expression vector carrying a calreticulin cDNA insert. In the transfectant cells, a 1.6-fold overexpression of calreticulin was found, and concurrently, intracellular calcium levels rose to nearly two-fold. Furthermore, most of this calcium originated from intracellular stores, as indicated by thapsigargin sensitivity. A study of the intracellular distribution of calreticulin in motor neurones of rat spinal cord has revealed that it is localised not only in the ER but also in “coated” vesicles (Copray et al. 1996). Copray et al. (1996) suggested that these vesicles may be counterparts of calciosomes, which are calcium storage vesicles found in liver cells and in cerebellar Purkinje cells. It ought to be pointed out, nonetheless, that Coppolino et al. (1996) found that the intracellular calcium stores were unaffected in cells in which the calreticulin gene had been knocked out.

CALRETICULIN

AND

CALNEXIN

AS

MOLECULAR CHAPERONES

A second function attributed to calreticulin is that of a molecular chaperone. The assembly of the T-cell antigen receptor (TCR) complex involves several genes, including calreticulin. Calnexin, which shares considerable sequence homology with calreticulin, is known to be involved with the chaperoning of newly synthesised TCR proteins. Similarly, calreticulin seems to be involved in the chaperoning of nascent TCR-α and -β proteins (Van Leeuwen and Kearse, 1996). Indeed, on the one hand, both calnexin and calreticulin seem to bind to and promote correct folding of proteins. They prevent aggregation, delay oligomerisation, and suppress degradation (Herbert et al. 1996). On the other hand, they possess the ability to bind to glycoproteins of the ER. Calnexin was shown to interact with and be involved in the assembly of class I MHC (major histocompatibility complex) molecules (Degen and Williams, 1991). Calreticulin has also been shown to interact with class I MHC molecules and probably with greater specificity than calnexin (Zhang and Salter, 1998; Harris et al. 1998). Both calnexin and calreticulin are lectin-like proteins and are able to interact with newly synthesised glycoproteins that have been partially trimmed of the N-linked oligosaccharides and assist in their folding and assembly (Helenius et al. 1997; Zhang and Salter, 1998). Receptors for insulin and insulinlike growth factor (IGF)-1 are receptor tyrosine kinases that have to dimerise before they are exported to the ER. The receptor monomers form dimers through disulphide bonding, when the newly synthesised monomers are associated with calreticulin or calnexin (Bass et al. 1998). Some of these studies implicate oligosaccharide-dependent binding to calnexin and calreticulin in a direct way in protein folding, assembly, and secretion. However, there are instances in which a lack of binding to these proteins has been of little consequence. It is possible that the chaperoning function of calreticulin might have implications for gene function. Calreticulin-deficient cells show an inability to import transcription factors belonging to the NF-AT (nuclear factor of activated T cells) family into the nucleus (Mesaeli et al. 1999). This could be a reflection of the part played by calreticulin-mediated chaperoning of the transcription factors. The NFAT transcription factors are substrates for calcineurin, and thus calreticulin might indirectly influence the expression of genes regulated by these factors. (See Figure

50

Calcium Signalling in Cancer

19.) This view is compatible with the intracellular distribution of calreticulin. An overall increase in the levels of calreticulin has often been associated with cancers. Yoon et al. (2000) have reported that the total calreticulin content of hepatocellular carcinomas is similar to that of corresponding normal tissues, but carcinomas differ from normal tissues in their intracellular distribution. Calreticulin occurs more abundantly in the nuclear matrix of hepatocellular carcinomas as compared with controls. Whether this might reflect an enhanced transport of substances such as transcription factors into the nucleus of cancer cells is an interesting subject for speculation. Such altered chaperoning of materials into the nucleus could greatly alter the pattern of gene expression.

CALRETICULIN

IN

CELL PROLIFERATION

AND

DIFFERENTIATION

There is very little evidence at present that calreticulin is involved in cell proliferation and differentiation. A retinoblastoma susceptibility (rb) regulatory element has been identified in the promoter region of the calreticulin gene (Valente et al. 1996). The rb gene is a negative regulator of cell cycle progression (see Sherbet and Lakshmi, 1997b). This observation provides some basis for implicating the calreticulin gene in cell proliferation. Dresselhaus et al. (1996) found an enhancement in the expression of calreticulin cDNA in the maize zygote during cell division. It has also been reported that calreticulin reduces intimal hyperplasia following arterial injury (Dai et al. 1997). There is considerably more information with regard to the involvement of calreticulin in cell adhesion and morphology, which, by implication, may be deemed to support the view that it might influence also cell proliferation. However, its expression has been related to differentiation in NG108-15 neuroblastoma/glioma hybrid cells (N. Liu et al. 1996; Johnson et al. 1998). Higher levels of calreticulin expression were observed in these cells, when induced to differentiate by exposure to dibutyryl cAMP (db-cAMP). Although there could be a true effect on cell proliferation, calreticulin, in general, does not seem to affect the proliferative capacity of the cell. However, cell numbers might increase merely by the ability of calreticulin to protect cells from apoptosis (see below).

CALRETICULIN

IN

CELL ADHESION

Two other examples of the physiological involvement of calreticulin are in the process of cell adhesion and cell spreading and in disease states engendered by autoimmune conditions. The influence exerted by calreticulin on cell adhesion to substratum was appreciated from early experiments of White et al. (1995). They demonstrated that anticalreticulin antibodies inhibited the binding of murine B16 melanoma cells to a substratum covered with laminin. They also showed the antibodies bound to the cell surface. The interaction of cells with the substratum also appeared to involve mannoside residues. Further, cell spreading could be inhibited by adding purified calreticulin to the medium, which presumably competed with cell surface calreticulin and denied the cells interactive binding with the substratum. Incidentally, this also confirms the lectin-like properties of calreticulin.

Non-EF-Hand Calcium Binding Proteins

51

An exciting avenue has opened in the continued search for potential functions of calreticulin. It seems to possess the ability to influence cell adhesion mediated by integrins. The walk along this avenue is exciting because integrins are a class of transmembrane heterodimeric glycoproteins that function as receptors for adhesion mediating macromolecules. The integrin family of cell surface receptors has been extensively studied and it has been established that their mediation of signal transduction as well as cell adhesion is accomplished by linking up with the cytoskeletal machinery. Vinculin, α-actinin, and talin provide the linkage to the actin cytoskeleton. This involves a specific interlinking of the three proteins as follows: integrin/talin/vinculin:α-actinin/actin. There is considerable evidence that adhesiondependent phenomena such as invasion and metastatic deposition are modulated by macromolecules that make up the extracellular matrix (Sherbet, 1982, 1987; Sherbet and Lakshmi, 1997b). In the first place, there is much evidence that implicates integrins in the modulation of cell adhesion by calreticulin. Embryonic stem (ES) cells from calreticulin knock-out mice, which are deficient in calreticulin, and fibroblasts derived from calretinin mutant mice have been reported to show severe reductions in integrinmediated cell adhesion. Transfecting ES cells with calreticulin cDNA restored this faculty (Coppolino et al. 1997). The cytoplasmic domain of the α subunit of integrins contains the amino acid sequence KXGFFKR to which calreticulin is known to bind (Shago et al. 1997). Cell adhesion is modulated by a mechanism in which the expression of the linking proteins is regulated. Overexpression of calreticulin in Lfibroblasts is accompanied by increased cell–substratum and cell–cell adhesion, and this overexpression also enhances cell spreading and decreases motility. Fibroblast cells overexpressing calreticulin also overexpress vinculin protein as well as its mRNA, and reduced levels of vinculin correlate with a down-regulation of calreticulin expression (Opas et al. 1996a). It is intriguing to note that another link in the system, namely talin, is unaffected by calreticulin overexpression. Nevertheless, calreticulin does not seem to affect the expression of α-catenin. The latter reputedly links other CBPs such as cadherin to the cytoskeleton (Sherbet and Lakshmi, 1997a, 1997b). Shago et al. (1997) have shown that calreticulin can interfere with signal transduction mediated by retinoic acid receptors that contain an amino acid sequence similar to that found in the α subunit of integrins. Retinoids are known to be able to inhibit invasive ability (Fazely and Ledinko, 1990). Furthermore, they can influence the flow of information from ligand binding to integrins. Therefore, this possible complication of the perceived events should be borne in mind when interpreting the effects of calreticulin overexpression on cell adhesion.

CALRETICULIN

IN

NEOPLASIA

Although the above discussion indicates a significant involvement of calreticulin in cell adhesion and possibly also in cell proliferation, only a few studies have dealt with its occurrence in tumours. Of the multiple functions attributed to calreticulin, its perceived effects on cell proliferation are the most relevant in relation to cancer. Calreticulin is one of several peptides of human breast cancers studied by Franzen et al. (1997). They state that it is expressed at higher levels in carcinomas than in

52

Calcium Signalling in Cancer

nonmalignant conditions of the breast, confirming an earlier study in which calreticulin expression in ductal carcinomas of the breast was compared with normal breast tissue (Bini et al. 1997). In the latter study, the enhancement of expression of calreticulin, and a number of other peptides, appeared to be specific for epithelial neoplasms. It would be premature to speculate on the nature of these correlative observations, but it has been suggested that calreticulin might control tumour progression by initiating a loss of apoptotic potential in tumours (Bruchovsky et al. 1996). Calreticulin levels increased in NG 108-15 neuroblastoma/glioma hybrid cells when induced to differentiate by treatment with db-cAMP (Johnson et al. 1998). Interestingly, however, antisense oligonucleotides for calreticulin do induce cell death in undifferentiated cells but not in differentiated cells. When the apoptotic pathway is experimentally induced in HL-69 cells by exposing them to geranylgeraniol, the expression of calreticulin is inhibited (Nakajo et al. 1996). Similarly, antisense calreticulin oligonucleotides seem to protect prostate cancer cell lines from calcium ionophore A23187-induced apoptosis. In this experimental system both androgen and calreticulin seem to be involved in the control of apoptotic events. A23187 induces apoptosis in both LNCaP and PC-3 cells. However, androgen is able to block apoptosis only in the androgen-sensitive LNCaP cells and not in the androgeninsensitive PC-3 cells. Furthermore, the inhibition of apoptosis of LNCaP cells by androgen is reversible by antisense calreticulin oligonucleotides (Zhu and Wang, 1999). One should recall here that in experimentally induced apoptosis of HL-60 cells, changes were also found in another protein species together with a downregulation of calreticulin expression (Nakajo et al. 1996). There was a decrease in the phosphorylation of a 36-kDa protein species. Thus it might be premature to link loss of calreticulin expression exclusively with apoptosis. Nevertheless, these observations suggest that calreticulin might protect cells against apoptosis. It is possible that deregulation of apoptosis is involved in both the growth of primary tumours as well as their metastatic deposits. Deregulation of growth control as well as inhibition of apoptosis can lead to cell population expansion in tumours. The phenomenon of metastatic dormancy may imply that cell mass is maintained by apoptosis-mediated regulation. Protection of cells from apoptotic death may lead to the appearance of overt metastasis. Most of these arguments are speculative. It would be interesting to see how the apoptosis regulating genes are affected by calreticulin expression.

IMMUNOLOGICAL IMPLICATIONS

OF

CALRETICULIN FUNCTION

Calreticulin has been implicated in the pathogenesis of some autoimmune disease conditions. In systemic lupus erythematosus (SLE), calreticulin might support the formation of the Ro/SS-A autoantigen complex (reviewed by Eggleton et al. 1997), and this could be related to its response to heat shock and stress factors. An interesting aspect of calreticulin in autoimmune diseases that has recently come to light is the amino acid sequence identity between calreticulin and the C1q receptor (Malhotra et al. 1993). C1q is a subunit of the first component of complement C1. C1q recognises and binds to circulating immune complexes (N.R. Cooper, 1985). A consequence of this binding is that C1q is released from the complex (Sim

Non-EF-Hand Calcium Binding Proteins

53

et al. 1979). It can now interact with the surface receptors on the various types of blood cells and endothelial cells (Tenner and Cooper, 1981; Malhotra and Sim, 1989; Peerschke and Ghebrehiwet, 1987; Peerschke et al. 1993). The binding of C1q to the receptor elicits a variety of immunological responses. The binding site of C1q to the C1q receptor or calreticulin has been identified (Stuart et al. 1996). Calreticulin occurs predominantly in association with immune complexes and C1q in sera of patients with SLE (Kishore et al. 1997). Kishore et al. (1997) suggest that inflammatory episodes occurring in autoimmune conditions could be attributed to the release of calreticulin from leukocytes, leading to an antigenic reaction, and further by virtue of its identity with C1q receptor, to an interference with C1q-mediated inflammatory effects. It is unclear at present why calreticulin should function as an autoantigen, but one reason could be that some parasitic organisms contain proteins that possess partial sequence homology with calreticulin. The inhibition of expression of steroid hormone-regulated genes is one of the postulated functions of calreticulin. As Coppolino and Dedhar (1998) have stated, it is truly remarkable that a single protein can have such a diversity of function. Clearly more work needs to be done on the multiple functions attributed to this CBP. Calreticulin has been highly conserved in the evolutionary process. Its conservation in a structural sense is probably less stringent than its functional conservation in signal transduction involving Ca2+ as a second messenger. Also it should be pointed out that calreticulin may not be the only CBP with multiple functions. The S100 family protein S100A4 has been described to participate in a wide spectrum of functions (Sherbet and Lakshmi, 1997b, 1998).

CALSEQUESTRIN AND INTRACELLULAR CALCIUM STORAGE Calsequestrin is a high-capacity, low-affinity CBP and is the muscle equivalent of calreticulin. Like calreticulin, calsequestrin is associated with specialised regions of the SR and participates in intracellular calcium homeostasis. Its major function seems to be intraluminal calcium storage. When intracellular calcium channels are activated, calcium is released from the intracellular pools leading to increases in cytosolic calcium levels, and this is an important ingredient of sustained muscle contraction. The release of calcium requires the activation of ryanodine receptor/Ca2+ channels of SR (Berridge, 1993). These ion channels are formed by tetrameric complexes consisting of 565-kDa monomers of the ryanodine receptor (Sutko and Airey, 1996). Three isoforms of ryanodine receptor are known. The type I isoform is restricted to skeletal muscle and is involved in voltage-gated Ca2+ release. The distribution of types II and III is less restricted. Type II is the main mediator of Ca2+induced Ca2+ release in cardiac cells (Lai et al. 1988; Gianini et al. 1992). Ryanodine receptors resemble IP3 receptors, which are another mediator of Ca2+ release, both in respect of their structure and function (Sutko and Airey, 1996). The calsequestrin molecule binds to and releases 4 to 50 calcium ions. Calsequestrin must be linked with the ryanodine receptor/calcium release channel, and indeed a close physical relationship does exist between them (B.E. Murray and

54

Calcium Signalling in Cancer

Ohlendieck, 1998). The anchoring of calsequestrin to the SR has been found to involve a family of transmembrane proteins. One such transmembrane protein is triadin. Triadin seems to anchor calsequestrin to junctional regions of SR and couples it with the ryanodine receptor/Ca2+ release channel (W. Guo and Campbell, 1995). A 26-kDa protein identified by Mitchell et al. (1988) as a calsequestrin binding protein was purified and characterised by L.R. Jones et al. (1995). This protein, now called junctin, bears significant sequence homology to triadin and aspartyl β-hydroxylase. Junctin contains a transmembrane domain and a short N-terminal portion extending into the cytoplasm. But a major portion of this molecule extends into the lumen of the SR (L.R. Jones et al. 1995). Junctin is distributed with ryanodine receptors and calsequestrin in the SR, which suggests that it may also be involved in calcium release from intracellular stores. The binding interactions are attributed to specific amino acid motifs that occur in the luminal portion of both triadin and junctin. L. Zhang et al. (1997) have suggested that calsequestrin, junctin, and triadin might form a quaternary complex with the ryanodine receptor to achieve calcium release. It seems possible that calsequestrin might regulate the calcium release mechanism. This is suggested by the finding that in transgenic mice overexpressing calsequestrin, the expression of junctin, triadin, and ryanodine receptor is downregulated (L.R. Jones et al. 1998). Very little is known about the involvement of calsequestrin in pathogenesis. In a recent report the sera of some patients with ocular myesthenia gravis showed the presence of antibodies that react against calsequestrin (Gunji et al. 1998). This suggests that an autoimmune mechanism might be operating in the pathogenesis of this disease.

OSTEOCALCIN IN BONE METABOLISM AND OSTEOTROPISM OF CANCER THE BIOLOGY

OF

OSTEOCALCIN

Osteocalcin is a noncollagenous protein. It is the most abundant matrix protein of bone and dentine (Price, 1992). It is regarded as a marker of bone turnover and metabolism, where bone resorption and formation are coupled. Osteocalcin is synthesised exclusively by osteoblasts and secreted into the extracellular matrix (ECM) during bone mineralisation. It is released also during osteoclastic degradation. Therefore, it is considered to be a marker of bone formation when formation and resorption of bone are uncoupled (J.P. Brown et al. 1984; Delmas et al. 1985, 1986; R.H. Christenson, 1997). Osteocalcin expression in developing chick and rat embryos coincides with the onset of mineralisation of the bone and hence it is considered to play a role in this process (Hauschka et al. 1989). Osteocalcin functions as a chemoattractant for osteoblast progenitor cells (Mundy and Poser, 1983). Also, bone resorption is poor under conditions of osteocalcin deficiency (Lian et al. 1984). These observations have supported a role for osteocalcin in bone resorption. Metastatic spread to the bone might activate bone metabolism, and therefore osteocalcin has been investigated as a potential marker of metastatic progression in certain forms of cancer, such as breast cancer and carcinoma of the prostate, which tend to show osteotropic secondary spread. Osteocalcin has turned out to be a good marker for

Non-EF-Hand Calcium Binding Proteins

55

bone turnover in osteoporosis (Delmas et al. 1983; Eastell et al. 1993). Mutations of the gla protein, a member of the osteocalcin family of proteins, have been suggested as a causal factor in the autosomal recessive Keutel syndrome, which is characterised by abnormal calcification of the cartilage (Munroe et al. 1999).

CALCIUM-BINDING PROPERTIES

OF

OSTEOCALCIN

Osteocalcin is a non-EF-hand CBP and binds 10 Ca2+ per mole protein at pH 7.5. The binding is virtually halved when pH is lowered to 5.0 (Kobayashi et al. 1998). Kobayashi et al. (1998) produced, by proteolysis, peptides of less than 8 amino acids from the N- and C-terminal ends of osteocalcin. The middle fragment bound 4 to 5 Ca2+ per mole protein. A proline-rich segment and three γ-carboxyglutamic acid residues are believed to participate in calcium binding (Heizmann, 1996).

OSTEOCALCIN GENE STRUCTURE

AND

FUNCTION

The osteocalcin gene has been mapped to human chromosome 1q25–q31 (Barille et al. 1996). The gene is regulated developmentally and in a tissue-specific manner. The tissue-specific expression seems to be due to the fact that the osteocalcin promoter functions only in cells of the osteoblastic lineage. This is suggested by the work of Ko et al. (1996), who constructed a virus vector carrying the thymidine kinase (TK) gene under the control of osteocalcin promoter. When this vector was introduced into osteoblastic cells TK gene was expressed, but no expression was detected in NIH3T3 or in a cell line derived from a transitional cell carcinoma of the bladder. The regulation of the osteocalcin gene is mediated by several transcription factors. Sequences that can bind the general transcriptional promoter AP1 have been identified in the promoter region of the osteocalcin gene (Goldberg et al. 1996; Lian et al. 1996). Lian et al. (1996) have also described two highly conserved regulatory motifs, which are, somewhat surprisingly, reported to relate to the cell type-specific expression of osteocalcin, albeit being functional in osteoblastic as well as nonosteoblastic cell lines. The modulation of expression of osteocalcin from the proliferative state to the differentiated state accompanying mineralisation of the ECM involves the function of a silencer element. An osteonectin silencer element (OSE) has been identified in the human osteocalcin gene, constituting a 7-bp (+29 to +35) sequence TGGCCT of the first exon of the osteocalcin gene (Y.P. Li et al. 1995). In proliferating cells, OSE is activated by the binding of an OSE-binding protein (OSE-bp) and consequently osteocalcin is either not expressed or is found in very low levels. When OSE-bp expression is downregulated OSE is inactivated, which leads to enhanced osteocalcin expression and the onset of ECM mineralisation. A silencer element also occurs in the first intron of the rat osteocalcin gene. Mutation of this suppressor element seems to inactivate the suppressor function (Goto et al. 1996). Other genes such as Dix-5 and MsX-2 have been shown to regulate the expression of osteocalcin during osteoblast differentiation. Dix-5 is believed to repress osteocalcin gene transcription (Ryoo et al. 1997). Interestingly, there is an increase in the expression of Dix-5 with osteoblast differentiation, which suggests an important regulatory role for this gene in the control of ECM mineralisation.

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Calcium Signalling in Cancer

Among other regulatory elements that have been identified is a short motif called the OSCARE-2. OSCARE-2 is reported to bind a number of proteins including AP1. Furthermore, this element appears to be able to bind also nuclear proteins that are induced by vitamin D3 (VD3; 1α, 25-dihydroxyvitamin D3) (Goldberg et al. 1996). As described below, VD3 receptor response elements have also been identified in the osteocalcin promoter region. This provides a molecular basis for the cooperation of VD3 and osteocalcin in the regulation of several processes such as calcium homeostasis, osteoblast differentiation, and cell proliferation and apoptosis.

REGULATION

OF

OSTEOCALCIN

BY

VITAMIN D3

Vitamin D3 (VD3) is an important regulator of calcium homeostasis. It plays a major role in bone mineralisation of type I collagen matrix, the transport of calcium and phosphate. VD3 influences several important biological processes such as cell differentiation, apoptosis, inhibition of cell proliferation, and cell signalling. It is known to suppress proliferation of T cells and their ability to produce cytokines. The function of VD3 is mediated by the VD3 receptor (VDR). VDR is a phosphoprotein transcription factor that functions in the form of a heterodimer with the retinoid X receptor (RXR). VD3 induces the formation of a heterodimeric complex between VDR and RXR (Carlsberg and Polly, 1998). VDR has a number of identifiable domains that participate in DNA binding, binding of the ligand, receptor dimerisation, and gene transactivation, as well as the C-terminal activation function (AF-2) domain required for co-factor interaction (reviewed by Issa et al. 1998). RXR seems to enhance the DNA-binding affinity of VDR as well as its specificity. VDR/RXR transcription factor regulates transcription of target genes by zinc finger-mediated DNA binding and protein–protein interaction. However, the RXR ligand 9-cis retinoic acid (RA) prevents RXR from forming heterodimers with VDR and promotes the formation of RXR–RXR homodimers. This effectively negates the transcription of VDR responsive genes. On the other hand, once VDR–RXR is generated, these seem to block the formation of RXR homodimers (Haussler et al. 1997). The genes that encode the major bone matrix proteins, namely, osteocalcin, osteopontin, and β3-integrin, are among the target genes activated by VDR. A VD response element (VDRE), which consists of hexanucleotide repeats with intervening trinucleotide spacers, has been identified in the promoter regions of these genes. VD3 induces the synthesis of osteocalcin by osteoblast cells in both in vitro and in vivo conditions (Price and Baukol, 1980). VD3 not only increases the transcription of the osteocalcin gene but also seems to increase the half-life of osteocalcin mRNA (Mosavin and Mellon, 1996). However, species differences might exist in VD3-induced osteocalcin expression. Transgenic mice carrying human osteocalcin gene do respond to VD3 and show increased serum levels of human osteocalcin, but there is no increase in the endogenous mouse osteocalcin. These species-specific differences are believed to be due to possible differences in the regulatory sequences of the mouse and human osteocalcin genes (Clemens et al. 1997; Sims et al. 1997).

57

Non-EF-Hand Calcium Binding Proteins

OSTEOCALCIN

IN

CELL PROLIFERATION

AND

DIFFERENTIATION

The osteocalcin gene is expressed in consonance with the inhibition of cell proliferation and the onset of cell differentiation and ECM mineralisation (Y.P. Li et al. 1995). VD3, as discussed above, induces osteocalcin expression, but on the other hand, it inhibits cell proliferation, apparently with the mediation of cdk inhibitors. M.J. Campbell et al. (1997) synthesised a number of VD3 analogues and demonstrated that these were capable of inhibiting proliferation of the prostate cancer cell lines LNCaP, PC-3, and DU145. The inhibition of cell proliferation was accompanied by an up-regulation of the expression of the cdk inhibitors p21waf1 and p27kip1. An up-regulation of p21waf1 expression has also been encountered in VD3-induced differentiation of human MG-63 osteosarcoma cells, and this has been shown to be independent of p53 function (Matsumoto et al. 1998). Therefore, these observations seem to define a direct and novel pathway of inhibition of cell cycle progression by VD3. Other hormones, such as thyroid hormones, that regulate the differentiation of osteoblasts also seem to function through the mediation of osteocalcin. Triiodothyronine (T3) has been found to inhibit the proliferation of the osteoblast-type MC3T3E1 cells and, in parallel, induce the expression of osteocalcin mRNA and protein and alkaline phosphatase activity (Varga et al. 1997; Luegmayr et al. 1998). Oestrogen has been reported to increase cell proliferation in the early stages of in vitro culture of osteoblasts derived from mouse bone marrow. The effects of oestrogen seem to involve the osteoblast-specific transcription factor osf2 (cbfa1) (SasakiIwaoka et al. 1999). Oestrogen increases the expression of osteocalcin, alkaline phosphatase, osteopontin, and transforming growth factor (TGF)β-1 as well as collagen type 1. Furthermore, exposure to oestrogen also increased the formation of bone nodules. Anti-oestrogens (Qu et al. 1998) blocked all these effects. Postmenopausal breast cancer patients treated with the anti-oestrogen tamoxifen have reduced (22%) levels of osteocalcin in serum (Marttunen et al. 1998). Fibronectin (FN) is a component of the ECM that has been implicated in several biological activities. Thus FN influences cell adhesion to substratum, spreading, and modulation of cell shape. It influences membrane ruffling and cell motility and also is associated with cell differentiation (Sherbet, 1987). The pattern of FN expression in osteoblast differentiation has attracted some attention. Although there is very little information about its role in vivo, there seems to be some correlation between the expression of FN and osteocalcin, in relation to the state of proliferation and differentiation in vitro. In the initial period of growth of osteoblasts, derived from foetal rat calvaria, on a collagen-coated substratum, a 50 to 70% reduction of FN occurs, but the expression of osteocalcin, osteonectin, and osteopontin is up-regulated severalfold (Lynch et al. 1995). Similarly, in chicken osteoblast cell cultures at 6 to 18 days, FN is associated with the cell membrane, but subsequently it remains associated with the ECM in a fibrillary form. Overall, FN increases in the early periods of cell culture and its levels are then maintained. In contrast, the major bone matrix ECM markers show increased expression with the onset of differentiation (Winnard et al. 1995). There is no suggestion, however, that these events are necessarily related,

58

Calcium Signalling in Cancer

beyond the recognition that collagen type I could be involved in the signal transduction pathway. Possibly, in the initial stages where it is bound to the membrane, FN could be mediating extracellular signals through the occupancy of its specific integrin receptors. The subsequent association with ECM in a fibrillary form is clearly a postsignalling event. This suggestion seems to be upheld by the experiments described by Moursi et al. (1996). These authors found that anti-FN antibodies inhibited the formation of bone-like nodules and the expression of osteocalcin and alkaline phosphatase genes. Generally compatible with this is the ability of VD3 to regulate FN expression at the transcriptional level. A VDRE has been identified in the FN gene (Polly et al. 1996). Interestingly, osteoblast differentiation requires more than the RGD domain of FN that is functional in cell adhesion. Certainly, the experiments of Moursi et al. (1996) indicate that FN regulates the differentiation of osteoblasts. There is now general acceptance that integrin receptors form an important link in the transduction of signals generated by ECM components (Sherbet and Lakshmi, 1997a). Integrin receptors are actively involved in the recognition of and interaction with ECM ligands occurring in the process of osteoblast differentiation (Uemura et al. 1997). The integrin α5β1 has been identified as the critical component in FN interaction with osteoblasts (Moursi et al. 1997). The murine MC 3T3 cell line responds to ascorbic acid treatment by synthesising a collagen matrix, and collagen type 1 ligand seems to be essential for the subsequent expression of osteoblast markers as well as the activation of the osteocalcin promoter element, OSE2. OSE2 is recognised by the osteoblast-specific transcription factor osf2 (also known as cbfa1, AML3, PEBP2, and alpha A). The latter is expressed only in osteoblastic cell lines, such as MG63, ROS 17/2.8, and MC3T3-E1, but not in cell lines of nonosteoblastic origin (Sasaki-Iwaoka et al. 1999). When the collagen type 1 receptor α2subunit is blocked, the binding of osf2 to OSE2 is also blocked, with consequent inhibition of transcription of the osteocalcin gene (Xiao et al. 1998). These experiments indicate the importance of the interaction between collagen type 1 and its integrin receptor for transduction of the signal that can elicit osteocalcin gene activation. Much effort needs to be directed toward a dissection of the pathway of signal transduction in order to provide the crucial evidence that can link these events in a coherent fashion. Intracellular adhesion as well as cell–substratum adhesion is determined by the components of the ECM. Their temporal and spatial expression is invariably associated with the alterations in the adhesive interactions, as well as changes in cell motility or migration on a substratum or invasive behaviour of cancers. The faculty of migration could be an important feature in bone resorption. Osteoclast precursors, for example, need to target sites of bone resorption and they do possess the ability of diapedesis across capillary endothelia. The modulation of certain ECM components such as FN, in conjunction with osteoblast differentiation and osteocalcin expression, has inevitably led to investigations of a potential association of osteocalcin with cell migration. Stringa et al. (1995) set up osteoblast cultures from 7day-old rat tibia fragments. They found that when the cells were exposed to parathyroid hormone they synthesised and secreted large amounts of osteocalcin

Non-EF-Hand Calcium Binding Proteins

59

together with collagen type III. The conditioned medium from these cultures stimulated the migration of EA HY-926 endothelial cells in vitro. Osteoblast cells obtained from rat calvaria adhere and show migratory behaviour upon plating on three-dimensional matrices. These migrating cells expressed osteocalcin as indicated by immunohistochemical staining (Attawia et al. 1995). TGFβ-1 mRNA expression in regenerating tissue in distraction osteogenesis is said to coincide with osteoblast migration and ECM mineralisation (Mehrara et al. 1999). However, there is also much evidence that TGFβ-1 can inhibit osteonectin expression. Hence, it is conceivable that, in this experimental model, this promotion of migration by TGFβ-1 could be an effect mediated by means other than involving osteocalcin. TGFβ-1 is a highly versatile cytokine that can elicit a wide-ranging cellular response. Not least among these is that it can induce the expression of S100A4 protein (Okada et al. 1997), which has itself been implicated in the induction of cell motility. The identity of the ECM component that might be instrumental in osteoblast migration is uncertain at present. The two major adhesion mediating glycoproteins FN and laminin both regulate adhesive interactions involving osteoblasts. As seen earlier, FN expression does vary with the state of cell proliferation and differentiation, but it has not been directly implicated in the motile behaviour of osteoblasts. Laminin, on the other hand, does seem to mediate the adhesion to substratum as well as migration of osteoclasts. There is also a suggestion that there might be some form of cooperative functioning of laminin and FN in osteoblast migration. It has been suggested, for instance, that FN might be secreted in response to laminin-2mediated adhesion (Colucci et al. 1996). This postulate needs to be tested further. There are a number of possibilities that can be tested, e.g., whether there is de novo synthesis of FN, whether there is a deletion of FN into the medium, whether there is a modulation in the expression of FN receptors, etc. But Colucci et al. (1996) have shown that osteocalcin induces the migration of osteoclasts on laminin-2- but not collagen-coated substratum. One could concede easily that osteocalcin can promote migration involving ECM components and their particular integrin receptors. However, it is unclear at present how one can envisage a physical mechanism that transduces the osteocalcin signal to the cytoskeletal machinery to bring about cell locomotion. Also relevant in this context is the question of whether other biological macromolecules, such as the cadherins, might be involved too. Cadherin has been studied extensively for its ability to suppress invasion by cancer cells and, indeed, it has often been described as an invasion suppressor gene. Now there is evidence that VD3 analogues up-regulate the expression of E-cadherin in prostate cancer cell lines (H.D. Campbell et al.1997). In osteoblastic cell lines, VD3 analogues up-regulate osteocalcin expression. It would be of much interest to examine the status of cadherin expression in osteoblasts and determine what effects VD3 exerts on cadherin. Besides its effects on cell motility, TGFβ is also a powerful modulator of growth and differentiation. TGFβ peptides are known to inhibit proliferation of a number of cell types. More than coincidental is that the mechanism by which TGFβ brings about growth inhibition involves cdk inhibitors, e.g., p27kip1 (see Sherbet and Lakshmi, 1997b for references). TGFβ isoforms subserve many functions, including bone turnover. Banerjee et al. (1996) found that TGFβ-1 down-regulated osteocalcin

60

Calcium Signalling in Cancer

expression in ROS 17/2.8 osteosarcoma cells. Similar effects have also been described in foetal mouse long bone cultures (Staal et al. 1998). Thus, although it would appear that TGFβ could be regulating bone metabolism by inhibiting osteocalcin expression, its effects on cell proliferation are achieved by a different route involving cyclin/cdks (Figure 9). This conclusion is supported by the suggestion made by J.H. Liu et al. (1999) that TGFβ might be inhibiting G1-S arrest partly by inactivating cyclin B/cdc2 kinase. TGFβ treatment results in the phosphorylation of the cdc2 component in the TGFβ receptor II–cyclin B2–cdc2 complex and downregulates its kinase activity. Despite this, it should be recognised that there is much obvious coordination in the functioning of osteocalcin, VD3, and TGFβ in the inhibition of cell proliferation and induction of cell differentiation.

FIGURE 9 A graphical illustration of how some of the functions of osteocalcin, VD3, and TGFβ overlap, and of the putative mechanisms involved. AP1, jun/fos transcription factor; cdk, cyclin-dependent protein kinase; FN; fibronectin; RA, retinoic acid; RXR, retinoid X receptor; TGFβ; transforming growth factor β; VD3; vitamin D3; VDR; VD3 receptor; VDRE; VD response element. (Based on references cited in the text.)

OSTEOTROPISM

OF

METASTATIC DISSEMINATION

Metastatic spread of cancer is a nonrandom process. The perceived organ specificity of metastasis can be attributed to a variety of factors intrinsic to the cancer cell as well as to the target organs (see Sherbet, 1982 for review). Metastatic spread to the bone is a common occurrence in certain forms of cancer. It has been argued often that bone metastases can activate the processes of bone metabolism. For that reason

Non-EF-Hand Calcium Binding Proteins

61

osteocalcin has been regarded as a potential surrogate method for detecting metastatic spread as well as for the purpose of monitoring the outcome of therapy on metastatic bone lesions. Several markers of bone metabolism have been employed in studies of this kind. Among those employed are osteocalcin, C-terminal peptide of type I procollagen (PICP), N-terminal peptide of type III procollagen (PIIINP), pyridinoline crosslinked C-terminal peptide of procollagen I (ICTP), and bone-specific alkaline phosphatase (BA-1p). Bloomqvist et al. (1996) found that ICTP and PICP levels correlated with that of osteocalcin, but not with urinary or serum calcium. All three markers correlated with the number of metastases detected by bone scans. The aminobisphosphonate ibandronate has marked osteoclast inhibitory activity and has been investigated as a treatment modality for metastatic bone disease and cancer-induced hypercalcemia. In combination with taxol/taxotere, ibandronate appears to be able to inhibit invasion of the bone by the human breast cancer cells MDA-MB231 (Magnetto et al. 1999) and the development of bone lesions in animals injected with myeloma cells (Dallas et al. 1999). Ibandronate markedly affects bone resorption in metastatic bone disease (Coleman et al. 1999). Osteocalcin, PICP, and BA-1p have been found to be reliable dose-dependent markers in a phase II clinical trial with ibandronate treatment of metastatic breast cancer. They were also found to be suitable for monitoring the effects of treatment of osteoporosis (Schlosser and Scigalla, 1997). However, Bombardieri et al. (1997) seem to disagree that any of these markers can replace bone scans. So far as prostate cancer is concerned, ICTP has been reported to reflect bone metastasis more accurately than other markers, including PSA (prostate-specific antigen). Osteocalcin showed no correlation with metastatic spread (Maeda et al. 1997). In another study, PICP and BA-1p were found to increase with progression as indicated by bone scans. A slight increase in osteocalcin was also reported in patients with remission of metastatic bone lesions, but not related to progression (Koizumi et al. 1995). Obviously more clinical trials are needed to arrive at any firm conclusions. This need is underlined by laboratory work on the differentiation of osteoblastic cells, using conditioned media of the human prostatic carcinoma PC-3 cells and cell extracts. The effects of the conditioned medium have been studied on two osteoblastic cell lines, namely, a primary cell line derived from foetal rat calvaria and a rat osteosarcoma cell line ROS 17/2.8. The conditioned medium inhibited bone nodule formation in both cell lines without affecting cell proliferation. The conditioned medium also inhibited osteocalcin mRNA but not that of osteopontin (Kido et al. 1997). These data suggest there might be other factors involved in the formation of bone metastasis. A development with much promise is the use of toxic gene therapy for cancer, with osteocalcin promoter for targeting the expression of the toxic gene in the treatment of tumours of osteoblastic lineage and metastatic tumours. Ko (1996) made an adenovirus construct with the TK gene under the control of an osteocalcin promoter (Ad-OC/TK). When this viral construct was introduced into cells of osteoblastic lineage, e.g., murine ROS cells and human MG-63 cells, the TK gene was expressed. In contrast, no TK expression occurred in nonosteoblastic cell lines. The addition of acyclovir (ACV) caused cell death in vitro. Similar growth inhibition

62

Calcium Signalling in Cancer

and cytotoxicity were encountered in vivo when the adenovirus construct was injected into murine ROS osteosarcoma followed by intraperitoneal injection of ACV. (Cheon et al. 1997) has extended its findings and has reported that the administration of the Ad-OC/TK construct coupled with methotrexate was highly efficacious in the treatment of osteosarcoma. Shirakawa et al. (1998) adopted this strategy in another experimental tumour model. They introduced ROS rat osteosarcoma cells into nude mice by intravenous route. These cells formed tumour nodules in the lung. They then injected the Ad-OC/TK construct into the tail vein with subsequent intraperitoneal ACV treatment. This treatment markedly reduced the number of tumour nodules in the lungs and significantly enhanced survival. Furthermore, the cell-type specificity of the functioning of this construct was also demonstrated by Shirakawa et al. (1998). They constructed the adenovirus vector with RSV (Rous sarcoma virus) promoter, rather than by osteocalcin promoter, and placed the E. coli β-galactosidase gene under its control. When this construct was used, there was no osteoblast-specific expression of the β-galactosidase gene, but it was expressed nonspecifically in lung parenchyma. This constitutes a rather elegant demonstration of cell-type-specific targeting of gene therapy coupled with cytotoxic drugs and certainly deserves further testing using experimental models of spontaneous metastasis.

5

The EF-Hand Calcium Binding Proteins

MOLECULAR ORGANISATION OF CALCIUM-BINDING EF-HAND PROTEINS The members of the EF-hand CBPs share a general structural feature of possessing variable numbers of domains that bind to Ca2+ with high specificity and affinity. The EF-hand structural motif was first reported from the crystal structure of carp parvalbumin (Kretsinger and Kockolds, 1973; Kretsinger, 1980). Specific structural features have been defined as being necessary for calcium binding. The EFhand consists of a consensus sequence of 12 amino acid residues in a helix–loop– helix configuration that can ligate Ca2+ (Linse and Forsen, 1995). The Kretsinger principle states that calcium binding is coordinated by five oxygen-containing amino acid residues and a conserved glycine residue, which causes the bending of the loop. The calcium-binding affinities of these proteins range from Kd 10–4 to 10–9 M, and this is dependent on the amino acid sequence of the EF-hand loop. EF-hand loops occur as a pair with antiparallel β-sheet interaction between the two loops. A high degree of cooperativity exists between calcium-binding loops (M. Zhang et al. 1995; Ames et al. 1995). The regulatory (sensor) and calcium buffer functions of EF-hand CBPs are closely related to the conformational reorganisation that occurs as a consequence of calcium binding. Conformational changes are essential for the regulatory function of EF-hand proteins, whereas for their calcium buffer function only a small conformational change appears to suffice (Ikura, 1996). A pair of EF-hands may form a globular domain and the domains within a protein may be functionally different, i.e., they may be either a regulatory domain or a buffer domain. Thus calmodulin has a regulatory domain at the Nand C-terminal regions of the protein. Troponin C and recoverin have an N-terminal regulatory domain and a C-terminal buffer or structural domain. In contrast, 9kDa calbindin has only one buffer domain at the N-terminal end. The regulatory domains undergo conformational changes upon binding to calcium and participate in the activation of target proteins (Ikura, 1996). The remarkable similarity in the genetic organisation of the genes coding for the EF-hand proteins has led to the formulation of an evolutionary profile of the EFhand protein family. As shown in Table 4, three classes of EF-hand proteins can be identified. Type I proteins include calmodulin, myosin light chains (MLCs), and spec I. The coding sequences of these are interrupted by five introns, of which four occur at identical sites. The type II proteins, the S100 and related proteins typically possess two EF-hands and the corresponding genes have two introns, one of them

63

64

Calcium Signalling in Cancer

TABLE 4 EF-Hand Calcium Binding Proteins and Their Genetic Characteristics Protein Class

Name

Number of EF Domains

Type I

Parvalbumin

3

Type II

Calmodulin Myosin light chains Centrin (caltractin) Spec I (sea urchin) S100 proteins

4 4 4 4 2

Type III

Calcyclin S100A8 (MRP-8, myeloidrelated protein) S100A9 (MRP-14) Calbindin, 9 kDa

2

Calbindin, 28 kDa

6

Calretinin

6

Genetic Organisation 5 introns interrupting EF-hand coding sequences

2 introns: one in the 5′ untranslated region and the other between the two EF-hand domains In S100A8/A9, one EF-hand occurs at each terminal region 2.5 kbp; 3 exons and 2 introns; exons 2 and 3 coding for EF-hand domains 10 introns; 7 within the EF-hand coding domains 8 introns; distribution coinciding with their distribution found in calbindin

Source: Based on Heizmann and Hunziker (1990) and Ikura (1996) and other references cited in the text.

in the 5′ untranslated region and the second separating the two coding domains. Type III proteins, such as the 28-kDa calbindin and calretinin, have six EF-hands. The corresponding genes have ten introns that occur with the EF-hand coding domain and they occur at identical sites in the 28-kDa calbindin and calretinin genes (see Heizmann and Hunziker, 1990 for a full review) (see Table 4). Kawasaki et al. (1998) have proposed a more elaborate classification of EF-hand CBPs based on the congruence of EF-hand domains. They have identified as many as 43 subfamilies on this basis. Of these, a group composed of 13 subfamilies is thought to have evolved and diversified, by gene duplication and fusion, from a progenitor domain. The remaining subfamilies do not show this probable evolutionary relationship and, indeed, some EF-hand CBPs do not appear to lend themselves to forming natural groups of subfamilies. The evolutionary relationship of EF-hand CBPs is also highlighted by the similarity that odd and even numbered domains of one protein show to the corresponding odd-and even-domains of another protein. Nonetheless, the framework for discussion adopted in the present work is based on the functional, rather than structural, homology of the CBPs.

The EF-Hand Calcium Binding Proteins

65

CALCIUM BINDING AND THE MOLECULAR CONFIGURATION OF CALCIUM-BINDING PROTEINS A characteristic feature of the binding of Ca2+ to CBPs is the conformational changes that these proteins undergo as a result. These changes in their molecular configuration have a significant impact on their function. The calcium-induced switch between active and inactive forms of target proteins is often a consequence of changes in molecular conformation. Ca2+ can induce conformational changes in both non-EFhand and EF-hand CBPs. Calcium-mediated modulation of conformational changes in actin figures prominently in actin dynamics. For instance, following calcium binding, actin seems to take a conformation that is conducive to strong binding with MLC (Avrova et al. 1998). The gelsolin family proteins are regulated by calcium, interact with actin, and induce conformational changes. These conformational changes seem to be essential in actin dynamics. Cross-linking of actin is mediated by actin-binding proteins such as fimbrin. The function of fimbrin itself is regulated by conformational changes induced by the binding of calcium ions to fimbrin. The EF-hand CBPs contain one to six EF-hand calcium-binding domains. The EF domain consists of a loop of 12 amino acid residues flanked by α-helical domains. Calcium binding to these domains brings about changes in the conformation of these domains, the major changes being the relative orientation of EF-hands. The EFhands open upon Ca2+ binding and assume a closed conformation state in the absence of Ca2+ (M.R. Nelson and Chazin, 1998). The open configuration is conducive to interaction with target proteins. However, CBPs might differ in the assumption of the closed position of EF-hands in the absence of Ca2+. Thus the C-terminal EFhand of CaM shows a closed position, whereas the C-terminal EF-hand of MLC would show a semi-open position. This would in effect mean a differential response of these proteins to the presence of calcium. In other words, the interaction of some CBPs with their target molecules might not be calcium sensitive. Sastry et al. (1998) have studied the three-dimensional structure of calcyclin and other EF-hand proteins such as calbindin D-9K (CBD9K) and reported the occurrence of major differences in the calcium-induced changes between these proteins. The flexibility and dynamics of the EF-hand are very much dependent on the amino acid sequence of the entire EF-hand (Malmendal et al. 1998). The degree as well as the nature of the various changes are probably characteristic of individual EF-hand proteins, as in the case of S100 proteins. Furthermore, a combination of changes might endow individual S100 proteins with specific functional properties. Conformational changes can affect protein function in many ways. For instance, the activation of calpains, which possess protease activity, seems to be a consequence of conformational changes in the molecule that result in the re-orientation of the protease domain to produce a functional active site of the enzyme (Hosfield et al. 1999). A classical example of the effects of another facet of molecular configuration is provided by CaM. The changes undergone by CaM seem to enable the protein to bind to a wide spectrum of target proteins and bring about their activation. Guanylate cyclase-activating protein (GCAP), on the other hand, seems to be inactivated by calcium binding to EF-hands 3 and 4. Mutations of EF-hand 3 suffice to inactivate

66

Calcium Signalling in Cancer

GCAP. Possibly, this suggests that calcium binding to EF-hand 4 is a secondary, albeit important, event. An intriguing example would be a CBP that at least putatively contains domains that are involved in diametrically opposed functions. Osteonectin is a prime example of this. Two subdomains have been identified in the osteonectin molecule, of which one is involved in the inhibition of endothelial cell proliferation and the other stimulates endothelial cell proliferation in vitro. In a similar fashion, osteonectin is able to exert differential effects on cell adhesion and spreading. Possibly, all these effects are regulated in a tissue-specific manner. The process of regulation could involve a mechanism in which molecular conformation might conceivably play a prominent part. One could postulate that changes in molecular configuration might change the relative orientation of one functional domain to the other. One can cite another example taken from the signal transduction system. As suggested in relation to osteonectin, conformational changes occurring upon Ca2+ binding might be linked closely with function. Only nonmuscle and cytoskeletal isoforms of α-actinin are capable of binding Ca2+, and only these isoforms are functional in the interaction with the cytoskeleton. This interaction is essential for successful signal transduction. Posttranslational changes of CBPs can influence calcium binding and in this way also influence molecular configuration and function. Several S100 proteins are modified at the posttranslational level. S100A8 and S100A9 are often found in the phosphorylated form. Phosphorylation can affect the functions of these proteins and can be a positive or negative regulator. It can influence the binding of Ca2+ and other ions and the configuration that molecules undergo as a consequence. These changes in molecular configuration are an essential feature of the process of target protein recognition. Phosphorylation can also target S100 proteins to specific subcompartments of the cell. For instance, S100A8 and S100A9 translocate to the cell membrane, following their phosphorylation (Guignard et al. 1996). Recoverin is myristoylated at its N-terminus. It has been suggested that this modification induces cooperative calcium binding. More precisely, calcium binding to EF-hand 3 of RCN is believed to produce reorientation of the molecule to enable EF-hand 2 to bind calcium. The myristoylation of recoverin might be involved functionally in the targeting of the molecule to the appropriate cellular compartment. Some CBPs might influence molecular changes in other CBPs merely by altering intracellular calcium homeostasis. It has been shown, for instance, that calretinin deficiency markedly alters intracellular calcium levels in calretinin-null Purkinje cells. As a result, calbindin D-28K (CBD) is saturated with calcium and undergoes conformational changes. Obviously, in this case calretinin functions as a calcium buffer and thereby influences or controls the molecular configuration and function of another CBP. Calcium binding also alters the quaternary structure and consequent oligomerisation of these molecules. Oligomerisation is essential for the binding of heat shock proteins to their targets and their oligomerisation is also known to be regulated by calcium. Such changes could alter the pattern of target recognition and in this way confer on S100 proteins the ability to interact with diverse target molecules and thus define the specificity of their function. Besides calcium, the biological functions of CBPs might be regulated by other divalent cations such as Zn2+ and Cu2+ (Heizmann

The EF-Hand Calcium Binding Proteins

67

and Cox, 1998). EF-hand proteins can bind other divalent ions such as Mg2+ and Zn2+. Indeed, a subgroup of S100 proteins that binds Zn2+ with high affinity has been identified. These bind two to four Zn2+ ions per protein monomer (Fohr et al. 1995; Fritz et al. 1998). As with Ca2+ binding, Zn2+ ion binding also occurs with much cooperativity. In S100A3, for instance, the binding of the first Zn2+ ion facilitates the binding of the second Zn2+ ion. This cooperative binding appears to be associated with and, indeed, requires structural reorganisation of the molecule (Fritz et al. 1998). Whether these conformational changes also have functional significance is still unclear. S100 proteins are known to form dimers readily in both intracellular and extracellular environments. Both homo- and heterodimers are formed by means of disulphide linkage, or the monomers may be held together by ionic bonds, depending on the environment and conditions. The relevance of the formation of dimers or tetrameric complexes to their function is yet uncertain, except probably in the case of S100A8 and S100A9. These are translocated from the cytosol to the plasma membrane. They seem to be able to form tetrameric complexes (A82/A92). Some recent work has brought to light the possibility that, at least in some CBPs, both Ca2+ and Zn2+ ions might be involved in the regulation of their function. For instance, the heterodimeric complexes of S100A8 and S100A9 bind arachidonic acid and transport it to its target site where it is metabolised. Kerkhoff et al. (1999c) have found that Ca2+ induces the binding of arachidonic acid by the S100A8/A9 complex, but this is reversed by Zn2+. This is an interesting joint functional involvement of these divalent cations, which contrasts with the demonstrations hitherto that they act on their own in bringing about changes in molecular configuration in the context of their function. Precise knowledge of the changes in conformation occurring here is still lacking. It would appear that S100A8 and S100A9 can form heterodimers in the absence of Ca2+. Possibly, Ca2+ induces a primary change in the molecular configuration of the constituent monomer to expose Zn2+-binding sites. In the wake of binding of the zinc ions a secondary molecular reorganisation could be occurring, which is incompatible, by reason of spatial constraint, with the continued association of arachidonic acid with the S100A8/A9 complex. It would be essential, therefore, to know the disposition of Zn2+-binding sites relative to the terminal calcium-binding EF-hand domains. Fritz et al. (1998) pointed out that Zn2+-binding cysteine residues are clustered in the C-terminal part of the S100A3 molecule that is said to be involved in the recognition of target proteins. Transcription factors recognise and bind to specific sequences in the promoter regions of genes, which initiates gene transcription. The binding of transcription factors to the DNA has been attributed to specific structural motifs that are present in the transcription factors. Many transcription factors possess Zn2+-binding domains in the form of protrusions and these are described as zinc fingers. The zinc finger motif binds to specific elements in the promoter. Zinc fingers, first identified in the transcription factor TFIIIA derived from Xenopus, are known to be present in a large family of proteins that includes the thyroid hormone receptor, vitamin D3 receptor, and retinoic acid receptors, among others. Four types of zinc finger motifs have been identified. The TFIIIA zinc finger is regarded as the classical type or the prototype, which binds one Zn2+ atom via two cysteine and two histidine

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conserved residues and to DNA as a monomer with high affinity. The zinc atoms seem to stabilise the structure of the zinc finger (Miller et al. 1985; Berg, 1990). The TFIIIA shows dual specificity in that it binds to DNA (5S RNA gene) as well as to 5S RNA (Pelham and Brown, 1980). The second type of zinc finger motif occurs in the steroid/nuclear receptor. Here the zinc finger binds two Zn2+ atoms binding to four cysteine residues each, and these receptors bind to the DNA as a dimer (Hard et al. 1990; Schwabe and Rhodes, 1991). The third type of motif is found in retroviral proteins and is characterised by 2 cysteine, a histidine, and a cysteine residues (Summers et al. 1990). The GAL4-type zinc finger from yeast binds two Zn2+ atoms, with six cysteine ligands to each Zn2+ (Keegan et al. 1986; Tao and Coleman, 1990; Pan and Coleman, 1991). Fritz et al. (1998) found that S100A3 binds two Zn2+ atoms. Of these, one zinc atom bound four cysteines and the second one bound four cysteines and one histidine residue. There is therefore some similarity between S100A3 and the zinc finger organisation of GAL4 transcription factor. These observations underline the potential importance of Zn2+ binding and the related changes in the molecular configuration in the function of S100A3. Gribenko and Makhatadze (1998) have proposed an attractive model in which molecular properties are postulated to change dependent upon relative the concentrations of Ca2+ and Mg2+. Calcium binding may not produce conformational changes in some proteins. S100A7 (psoriasin) has been reported to show very little change in its conformation upon calcium binding (Brodersen et al. 1998), but there were earlier reports of significant changes in conformation of S100A7 upon binding of Ca2+, Zn2+, and Mg2+ (Vorum et al. 1996). It is possible that in this particular case Ca2+-mediated changes may not be functionally relevant.

THE STRUCTURE AND ORGANISATION OF S100 FAMILY GENES The members of the S100 gene family have a shared intron–exon organisation. S100β has three exons and two introns. The first exon codes for the 5′ untranslated region and the second and third exons each code for an EF-hand (Allore et al. 1990). Similarly, the S100A4 gene (h-mts1) has three exons interspersed with two introns. The first exon is a noncoding exon and exons 2 and 3 contain the coding sequences for the mts1 protein (Ambartsumian et al. 1995).

ALTERNATIVELY SPLICED VARIANTS OF S100A4 Ambartsumian et al. (1995) also reported the occurrence of a splice variant of the h-mts1 cDNA. In the generation of this variant, alternative splicing seems to have occurred within the 5′ untranslated region of the original cDNA transcript, and a second noncoding exon is inserted in the variant form. This insertion, nonetheless, retains the main open reading frame (ORF), without creating a new longer ORF. In a recent study of the expression of S100A4 (h-mts1) in carcinomas of the breast, Albertazzi et al. (1998c) discovered the presence of another variant form, which has been designated as h-mts1v. This variant is a shorter version of the original h-mts1

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message. They have deduced the molecular organisation of the h-mts1v by reverse transcriptase polymerase chain reaction (RT-PCR). These studies have suggested that a loss of exon 1 (1a as well as 1b) sequences corresponding to one or more primers used for PCR may have occurred in the variant cDNA. The organisation of the h-mts1 gene and that of the variant cDNA is presented in Figure 10. The only and striking difference is the loss of exon 1 (1a as well as 1b) in the shorter splice variant, which is present in the larger variant reported by Ambartsumian et al. (1995).

FIGURE 10 Molecular organisation of the h-mts1 (S100A4) and that of the variant h-mts1v cDNA. Exon 1 (1a and 1b) of h-mts1 is not translated. This is spliced out in h-mts1v. Exons 2 and 3 encode the protein. (From Albertazzi et al. 1998c.) Reprinted by permission of the publisher Mary Ann Liebert Inc.

FUNCTIONAL SIGNIFICANCE OF ALTERNATIVELY SPLICED ISOFORMS Alternative splicing of pre-mRNAs is a ubiquitous phenomenon in higher eukaryotes. However, the significance of the occurrence of splice variants is poorly understood. Alternative splicing has been regarded as a mechanism of regulation of gene function. Splice variants may have different functions and different intracellular locations, and may indeed subserve opposing functions (Sherbet and Lakshmi, 1997b). Alternative splicing of pre-mRNA can generate isoforms of biologically active molecules and of membrane receptors for extracellular ligands functioning as biological response modifiers (Gunthert et al. 1991; Kim and Yamada, 1997; Shi et al. 1994). This results in changes in the mode of function. Splicing out of the transmembrane domain of the IL-6 receptor appears to cause its deletion from the cell membrane (Horiuchi et al. 1994). The generation of splice variants of the kinases and phosphatases might be a common mechanism associated with the regulation of cell cycle progression. The PITSLRE protein kinases are members of the p34 (cdc2) kinase family, and are so named on the basis of the amino acid sequence of an important regulatory region. Several splice variant isoforms of the PITSLRE protein kinases have been described

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(Xiang et al. 1994). They may be expressed differentially in different cell types and subserve different functions (Lahti et al. 1995). Similarly, the phosphatase cdc25B, which is involved with the function of the cyclin/cdk complex in cell cycle regulation, occurs in the form of several splice variants, which may be involved together with the wild-type phosphatase in the regulation of the cell cycle (Forrest et al. 1999). Another striking example is provided by the splice variants of annexin VI. Annexin VI has marked effects on cell proliferation. In A431 cells, it inhibits the mobilisation of Ca2+ induced by EGF. But this effect is produced only by the larger isoform. The shorter isoform does not affect Ca2+ mobilisation nor does it influence cell growth (Fleet et al. 1999). Alternative splicing could also generate protein isoforms that may be unable to interact with target proteins or, as in alternatively spliced p53, to bind to DNA (Bayle et al. 1995). Possibly, the 5′ region is most prone to splice modification, and the stability and translation efficiency could be affected in these splice variants (N. Roy et al. 1992; Bingham et al. 1988). There is the further possibility that the 5′ untranslated region may have a role in the regulation of the mRNA translation, as demonstrated for the alternatively spliced variants of the muscle-specific enolase gene (Oliva et al. 1995). On similar lines, the noncoding exons 1 and 2 of the angiotensin II receptor gene have been shown to be able to regulate the translation of the transcripts (Curnow et al. 1995). The 3′-untranslated region seems to inhibit the expression of the cell membrane receptor of luteinising hormone. The inhibition has been attributed to a decrease in the half-life of the receptor mRNA (Nair and Menon, 2000). As stated before, Ambartsumian et al. (1995) reported that the occurrence of the larger splice variant of h-mts1 that contained an additional noncoding exon. It has been argued that splice variants may subserve different, often opposing, functions. If this were the case, one would expect to find a differential expression of splice variants in tissues (Sherbet and Lakshmi, 1997b), which appears to be the case with respect to the h-mts1 splice variants (Ambartsumian et al. 1995). Ambartsumian et al. (1995) have stated that this splice variant showed marked variations in the level of expression in different human tissues. This large isoform was not found in the breast cancers, which Albertazzi et al. (1998b) investigated. However, there are indications that the expression of the h-mts1v isoform described here may have some bearing upon nodal spread of the disease (Albertazzi et al. 1998c), although h-mts1v did not appear to influence nodal spread as decisively as h-mts. It would be premature to speculate on the significance or relevance of h-mts1v expression to the state of the disease, because the deleted exon 1a/1b is not transcribed and exons 2 and 3 that encode the functional protein are intact. It may be argued that the loss of the 5′ untranslated exon could be affecting the secondary structure of the transcript and its stability and translatability as a consequence. Possibly, the integrity of the S100A4 protein and its molecular properties might be affected by the splicing process, and this might impair the ability of S100A4 to promote metastatic spread.

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REGULATION OF EXPRESSION OF S100 FAMILY GENES The widespread involvement of S100 genes in diverse physiological function, and the demonstration that abnormal expression of many of these genes is associated with aberrant physiological events and the pathogenesis of several diseases, has inevitably led to the investigation of how the expression of S100 genes is regulated. Two modes of gene regulation have been investigated: transcriptional regulation and epigenetic regulation.

TRANSCRIPTIONAL REGULATION

OF

S100 GENES

The promoter regions of several S100 genes have now been characterised. The functional promoter of S100β has been localised to a region –168 to +697 and contains 168 bp upstream of the transcription start site. The activity of this promoter has been identified in several cell types. Furthermore, there are both positive and negative regulatory elements in the promoter region. There are positive regulatory elements in –788 to –391 and –1012 to –788. Further upstream, in the region –4437 to –1012 and in –1012 to –788, occur negative regulatory elements. The regulatory element in –4437 to –1012 has been found to suppress promoter activity in many cell types (Castets et al. 1997). Several potential regulatory elements, such as the CRE and AP2, have been identified in the promoter region of S100β (Allore et al. 1990). AP1-like binding sites are found also in the promoter region of the calcyclin gene, but the latter shows no binding by transcription factors such as AP-2, AP-3, or NF1 (Bottini et al. 1994). The murine homologue of S100A4 has been investigated by Tulchinsky et al. (1990, 1992), who have found homology between the 5′ region of S100A4 and the promoter regions of rat fibrinogen and human prothrombin, and also with certain enhancer elements of SV40. Tulchinsky et al. (1992) found no cis-acting control elements in the 5′ region of S100A4. Subsequently, a 16-nucleotide cis-acting transcription regulatory element has been identified in the first intron of the gene, and this element has been found to bear a high degree of sequence homology to the enhancer element of the CD delta gene. The S100A4 promoter region also has been reported to contain motifs that bear a high degree of homology to p53 binding negative regulatory elements and AP1-like enhancer elements in the 3′ region (Parker et al. 1994a). Okada et al. (1998) have identified a cis-acting element stretching from –187 to –88, upstream of the first intron, that they believe specifies the transcription of the S100A4 (FSP1) gene in fibroblasts. They have also reported the occurrence of an enhancer element in the first intron. This does not show cell type specificity. Okada et al. (1998) have reported further that a 5-bp domain TTGAT at –177 to –173 interacts specifically with nuclear extracts derived from fibroblasts. This fibroblast-specific expression is not a typical feature of S100A4, which is indeed expressed to a variable degree in a variety of normal and neoplastic tissues. Thus, there are clear suggestions that the function of S100 genes may be controlled by a variety of transcription factors.

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GENE EXPRESSION

BY

DNA METHYLATION

DNA methylation is an epigenetic mechanism that regulates gene expression and gene imprinting. The CpG dinucleotide sites or islands that occur within and around genes are the targets of methylation (Ng and Bird, 1999). The process of methylation alters the conformational state of the DNA and it has been suggested therefore that methylation may play a role also in genomic stability. The association of the state of DNA methylation with gene expression has been known for many years (Kass et al. 1997). Hypomethylation is associated with constitutive gene expression. Demethylation of promoter regions of a gene, e.g., by treatment of cells with 5-azacytidine, increases the level of its expression (S. Lu and Davies, 1997). The regulation of tissue-specific expression of a tyrosine hydroxylase gene is dependent on the methylation status of the cytosine bases occurring around the CRE of the promoter (Okuse et al. 1997). Similarly, the expression of the manganese superoxide dismutase gene is suppressed when certain cytosines present in the 5′ flanking region are methylated (Y.H. Huang et al. 1997). Methylation of regulatory elements may interfere with the binding of transcription factors, as shown by Ryhanen et al. (1997). These authors found that AP-1 transcription factor bound to unmethylated response elements with far greater affinity than to methylated ones. The pattern of DNA methylation has been studied extensively. Methylation patterns may be somatically inherited, but probably not through the germ line. Epigenetic DNA methylation may be linked with genetic changes. However, in contrast with the generally held view that DNA methylation is a heritable and stable phenomenon, Ramchandani et al. (1999) have postulated that it is indeed a reversible signal. This postulate makes it easier to appreciate how DNA methylation might be involved in developmental processes as well as in neoplastic progression, both inherently associated with changes in the patterns of gene expression. Wachsman (1997) has argued that DNA damage, e.g., by alkylation of bases, oxidative lesions, etc., can interfere with the methylation of CpG dinucleotides by DNA methyltransferases (DNA-MTases) and alter the distribution of 5-methylcytosine (5-MC). The presence of 5-MC results in an increased risk of mutagenesis. CpG islands are hot spots for mutations with the presence of 5-MC and DNA-MTases. Furthermore, the levels of DNA-MTases correlate with the state of DNA methylation. Quite clearly, epigenetic changes of DNA methylation and mutations are interrelated and also are associated with carcinogenesis.

DNA METHYLATION

IN

CANCER

It has been known for many years that changes in methylation patterns are associated with normal developmental processes (E. Li et al. 1992; Neumann and Barlow, 1996) as well as human cancers (P.A. Jones, 1986; P.A. Jones and Chandler, 1986). Whether abnormal methylation status is associated with neoplastic transformation has been the subject of many investigations. Tumorigenic and non-tumorigenic cell lines differ markedly in their DNA-MTase levels (Kautiainen and Jones, 1986), and human tumours show a range of 5-MC levels (Gamasosa et al. 1983). The expression of DNA-MTase is said to be deregulated in colonic carcinomas, when compared with

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normal colonic epithelia and adenomatous lesions. The carcinomas have also been reported to be highly heterogeneous with regard to DNA-MTase expression (De Marzo et al. 1999). However, because cancers tend to be intrinsically heterogeneous, the heterogeneity of DNA-MTase might not be sufficient grounds for postulating an association between possible deregulation of its expression and the process of carcinogenesis. The overexpression of certain oncogenes in animal tumour models has been associated with their demethylation (Wainfan and Poirier, 1992). Indeed, the oncogene ras was reported many years ago to be hypomethylated in human cancers (A.P. Feinberg and Vogelstein, 1983). Using an experimental tumour model, Counts et al. (1997) have demonstrated that hypomethylation of Ha-ras and raf oncogenes is related to tumour promotion, and further that carcinomas were more hypomethylated than adenomas. In breast carcinomas, marked hypomethylation has been reported in the vicinity of the promoter region of the calcitonin gene (Hakkarainen et al. 1996). pS2 is a protein associated with oestrogen-induced breast cancers. The expression of this protein has been found to be associated with hypomethylation of a CCGG site within the promoter/enhancer region of the pS2 gene (V. Martin et al. 1997). In contrast with oncogenes, tumour suppressor genes appear to be hypermethylated and inactivated in association with tumorigenesis (Zingg and Jones, 1997; Denissenko et al. 1997). The suppressor gene p53 is mutated in a large number of human tumours. Not only does a large number of somatic mutations occur at methylated CpG dinucleotide sites, but also these might be preferred targets for mutagenic agents (Denissenko et al. 1997; M.S. Tang et al. 1999). There is a generalised DNA hypermethylation in chronic lymphocytic leukaemia. More specifically, two tumour suppressor genes — cyclin-dependent kinase inhibitor genes CDKN2A and CDKN2B — have been reported to be methylated. Apparently their expression is suppressed thereby in B-cell chronic lymphocytic leukaemia (Martel et al. 1997), and also in many other forms of human cancer, such as carcinoma of the breast, prostate, and kidney (Herman et al. 1995). The candidate suppressor gene HIC1 is relatively unmethylated or hypomethylated in peripheral blood or in acute myelogenous leukaemia as compared with recurrent acute lymphocytic leukaemia and blast crisis chronic myelogenous leukaemia (Issa et al. 1998). The inactivation or silencing of other tumour suppressor genes has also been reported, e.g., the MUC2 gene in colorectal cancer (Hanski et al. 1977) and the Wilms tumour gene (Kleymenova et al. 1998). The promoter region of the E-cadherin gene, which is regarded as a putative tumour and invasion suppressor, often has been found to be hypermethylated in gastric carcinomas. This has been associated with the reduced expression of E-cadherin in these tumours, and it has been suggested that hypermethylation of the promoter might occur as an early event in gastric neoplasia (G. Tamura et al. 2000). In prostate cancer, there is a decreased expression of endothelin receptor as compared with normal prostate epithelium. This appears to be due to methylation of a 5′ CpG island covering the transcriptional regulatory region of the endothelin receptor gene (J.B. Nelson et al. 1997).

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S100 GENE TRANSCRIPTION

BY

METHYLATION

The expression of S100 genes may also be regulated by methylation. This has been suggested by Tulchinsky et al. (1995) for S100A4. They have implicated the methylation of the first exon and the first intron in the repression of transcription of S100A4. Subsequently, Tulchinsky et al. (1996) showed that the first intron of this gene contains a methylation-dependent AP1 binding site. They further stated that the transcription of the gene is related to hypomethylation of the first intron in murine adenocarcinoma cells. In human colonic adenocarcinoma cell lines, the expression of S100A4 closely correlates with hypomethylation of the second intron of the gene. Also, where S100A4 expression was low, this could be increased by treating the cells with 5-Aza-2′-deoxycytidine (N. Nakamura and Takenaga, 1998). Indeed, the pattern of methylation of the gene could be a reason why S100A4 often shows a differential pattern of expression (D.S. Chen et al. 1999). S100A2, on the other hand, may be regarded as a tumour suppressor because it is expressed in normal breast tissue but it is down-regulated in the progression of breast cancer. Wicki et al. (1997) demonstrated the presence of an enhancer element 1.2 kb upstream of the transcription start site. Here they identified two AP1-like binding sites. The same enhancer element regulates the expression of S100A2 in normal as well as neoplastic breast epithelia suggesting the involvement of an epigenetic mechanism of methylation. Although their observations are not conclusive, it would appear that an element proximal to the transcription start site shows differential states of methylation in normal cells, tumorigenic cells, and cells derived from a breast tumour biopsy. Nonetheless, they have demonstrated that site-specific in vitro methylation of S100A4 gene in normal cells leads to a down-regulation of its expression. Perhaps it is unhelpful to overemphasise the significance of the regulation of gene expression at the transcriptional level. Ambartsumian et al. (1999) have reported that, in S100A4 transgenic mice, S100A4 mRNA was expressed in all organs. The mRNA was expressed at a higher level in some organs. However, they have made the crucial observation that the S100A4 protein was down-regulated in the organs that did not express the gene in the wild-type animal. It is obvious from this that there must exist a complex mechanism that regulates the expression of the protein at the translational level or regulates the decay of the protein. This is not unique to S100A4, and the expression of other S100 proteins in common with a wide variety of cellular proteins might also be controlled at the posttranscriptional level. For instance, S100α1 protein and its mRNA are differentially expressed in differentiating neuronal and skeletal muscle cells (Zimmer and Landar, 1995).

6

The Calmodulin Family of Calcium Binding Proteins

A number of calcium regulatory proteins may be treated as belonging to the calmodulin family of CBPs. In addition to calmodulin, troponins, calbindins, recoverin, and calretinin can be assigned to this family. The regulatory function of these CBPs in normal as well as aberrant physiology will be discussed in the following pages.

CALMODULIN AND ITS PHYSIOLOGICAL FUNCTION STRUCTURE

AND

MODE

OF

ACTION

OF

CALMODULIN

Calmodulin (CaM) is a 17-kDa CBP. Structurally it may be described as a dumbbellshaped molecule with two lobes each containing two Ca2+-binding domains. The two lobes are joined by an α-helix comprising 28 amino acid residues. A conformational change occurs in the CaM molecule in the presence of calcium and as result CaM can bind to a large number of target molecules. The conformational change involves the exposure of two methionine-rich hydrophobic areas, one in each globular domain, and these take part in the interaction with target proteins and in their activation. CaM can activate a large number of proteins, and this ability has been attributed to the highly conserved methionine residues (Kincaid et al. 1987; Perrino et al. 1992; Milan et al. 1994), the mutation of which might affect activation of target proteins. The α-helix linker also plays a crucial role in this process of activation. Tabernaro et al. (1997) studied a mutant form of CaM in which the amino acid residues Thr 79 and Asp 80 are deleted from the α-helix. This deletion appears to alter the relative orientation of the globular domains, which results in the hydrophobic patches coming closer together and consequently becoming less accessible to interaction with target proteins. The CaM binding domains of target proteins occur in short stretches of 15 to 25 amino acid residues (O’Neill and De Grado, 1990; Ikura et al. 1992; Crivici and Ikura, 1995). CaM appears to recognise certain conserved structural features, which might differ from one target protein to another with respect to their topographical arrangement. CaM orientates its globular domains by virtue of the flexible nature of the linker. An interaction with CaM is essential for the activation of several of the target proteins (Kretsinger, 1992). Specificity of activation of the target enzymes seems to be achieved by the phosphorylation of CaM (Williams et al. 1994; Sacks et al. 1995; Quadroni et al. 1994; Saville and Houslay, 1994). Several kinases phosphorylate 75

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CaM, among them the myosin light chain kinase (MLCK) (Davis et al. 1996); casein kinase II (Sacks et al. 1992), and receptor tyrosine kinases (Graves et al. 1986; San Jose et al. 1992). Ubiquitination of calmodulin also has been suggested as a possible regulatory mechanism (Laub and Jennissen, 1991). The occurrence of numerous target proteins indicates the broad spectrum of physiological function in which CaM participates. CaM mediates the function of enzymes that are involved in cyclic nucleotide metabolism, phosphorylation and dephosphorylation, and smooth muscle contraction (Means, 1988; Harrison et al. 1988; MacNeil et al. 1985; G. Li et al. 1989).

CALMODULIN-MEDIATED SIGNAL TRANSDUCTION There is currently a large body of evidence demonstrating the important role calmodulin plays in the transduction of signals of cell proliferation and growth control, and in cell locomotion and invasion. The signalling pathways that are initiated by the activation of cell membrane receptors, as alluded to earlier, involve the raising of intracellular calcium levels and the subsequent phosphorylation of target proteins. Because Ca2+/CaM interacts with and activates target enzymes, CaM has been deemed as an important component of the signalling pathway by mediating protein phosphorylation via CAMKs. CaM kinases subserve a variety of functions. Several examples can be cited for the purposes of discussion and illustration. Thus T-cell activation, cell migration, and proliferation will serve amply to underline the significance of CaM as a signalling molecule. The activation of the T-cell receptor not only increases intracellular calcium but also activates CaM-dependent kinases, which results in the regulation of phosphatases (Ostergard and Trowbridge, 1991). CAMKs are also involved in cell proliferation. CAMK II has been shown to be involved in the induction of S-phase delay in fibroblasts exposed to γ-irradiation. The exposure to radiation also induces CAMK II activity. This induction does not occur if the cells are pretreated with CAMK II antagonists (Famulski and Paterson, 1999). An outstanding example of CaM-mediated signal transduction is the transduction of angiotensin signals. The hormone angiotensin II, derived from the decapeptide angiotensin I, generates many cellular responses. Angiotensin II possesses cytokinelike activity. It functions as a cardiovascular growth factor and is mitogenic to cardiac fibroblasts. It has also been attributed with the ability to induce angiogenesis. Not surprisingly, therefore, angiotensin signal transduction has many similarities to that of growth factors. Some of the effects of angiotensin are indeed mediated by growth factors such as PDGF, EGF, basic fibroblast growth factor (bFGF), and TGFβ. The transduction of the angiotensin signal involves two types of angiotensin receptors: type 1 (AT-1) and type 2 (AT-2). The binding of angiotensin to AT-1 triggers a cascade of signalling events and the activation of transduction pathways (Thomas, 1999). The C-terminal domain of the AT-1, close to the inner membrane, interacts with CaM in a Ca2+-dependent fashion. This interaction seems to be essential for the transduction of angiotensin binding to AT-1A (Thomas et al. 1999). In cardiac fibroblasts, AT-1-induced signals appear to transactivate epidermal growth

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factor receptors (EGFr) and eventually result in DNA synthesis. This effect is inhibited by CAMK inhibitors (Murasawa et al. 1998). Angiotensin is able to induce the synthesis of TGFβ and the type II TGFβ receptor (Wolf et al. 1999). TGFβ and its receptor activity have potentially profound effects on the composition and properties of the ECM. Furthermore, angiotensin and TGFβ can both induce cytoskeletal changes, such as the formation of actin stress fibres and the phosphorylation of focal adhesion proteins (Riedy et al. 1999). There can be little doubt that the cytoskeletal modulations and the remodelling of the ECM will influence cell migration as well as cell proliferation and differentiation. Cell migration is another cellular activity that demonstrably involves CaM in signal transduction. Cell migration is believed by some to involve activation of the pathway that includes members of a family of serine/threonine kinases called extracellular signal-regulated receptor kinases (ERK), which are also known as the MAPK pathway. In PC12 cell, calcium influx activates ERK. This activation is inhibited by the CaM inhibitor W13, which indicates that CaM is involved in this pathway (Egea et al. 1998). Egea et al. (1998) also showed that other receptors, e.g., trk A and EGFr, are not involved in the activation of the ERK pathway. In gastric epithelial cultures also CaM appears to be involved in the transduction of the cell migration signal (Ranta-Knuuttila et al. 1998). Of late, much evidence has emerged that links CaM with G-protein-mediated signal transduction. There is a marked parallelism between angiotensin signal transduction and the transduction of growth factor signals. It has been recognised, therefore, that G-proteins might be involved in angiotensin signal transduction. Angiotensin II has been known to bind to a high-affinity G-protein receptor (T.J. Murphy et al. 1991; Sasaki et al. 1991; Sandberg et al. 1992). Recently, CaM has been reported to enhance the Ca2+-dependent binding of guanosine triphosphate (GTP) to the ras-related protein Ral-A (Wang and Roufogalis, 1999). Fischer et al. (1998) found that the PI3K, an enzyme that functions downstream of G-proteins, possesses high-affinity binding sites for CaM. The association of CaM with G-proteins is not restricted to animal cell systems. CaM appears to be closely involved with G-proteins in pollen germination and the growth of pollen tubes (Ma et al. 1999).

CALMODULIN

AND

CELL PROLIFERATION

As stated earlier, among the targets of CaM are the cytoskeletal proteins MAP-2 and the tau protein. The conclusion is inescapable, therefore, that CaM might be involved in cytoskeletal reorganisation. Both cell proliferation and cell motility have been regarded as the natural foci of CaM involvement. CaM seems to be actively associated with cell proliferation, as amply demonstrated by several investigators. Indeed, it is regarded as an essential ingredient of cell proliferation and the progression of the cell cycle (Means, 1994). The exit of cells from the cell cycle has been reported to be accompanied by a decrease in CaM levels (Christenson and Means, 1993). CaM levels increase as cells progress into the mitotic phase, and there is an elegant demonstration that both Ca2+ and CaM are essential for this process. When either of these is reduced cells undergo G2 arrest (K.P. Lu et al. 1993).

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Some investigators have examined the levels of CaM expression in relation to growth responses. Exaggerated growth responses have been recorded in cardiomyocytes resulting from an overexpression of CaM (Gruver et al. 1993). Recently, however, Prostko et al. (1997) found no effects on growth responses arising from an overexpression of CaM in C6 glioma cells in culture, but reduction of CaM expression was found to inhibit their growth. The antiproliferative effects exerted by CaM inhibitors have also provided a substantial body of evidence that suggests an association of CaM with growth responses. Several CaM inhibitors have been tested to date. Schuller et al. (1991) found that B859-35, which is a dihydropyridine derivative, markedly inhibited proliferation of three human lung cancer cell lines. Hait et al. (1994) reported that several phenothiazine antipsychotic drugs inhibit CaM and also the proliferation of C6 glioma cells. They also found that the antiproliferative effects corresponded with the inhibition of CaM-sensitive phosphodiesterases. Further work from the same laboratory has shown that KS-501 and KS-502 similarly affect cell proliferation, not by direct action on the enzymes but by interfering with their function of activating CaM (Hait et al. 1995). In other words, the failure to activate CaM appears to lead to an inhibition of cell proliferation. Glass-Marmor et al. (1996) and Glass-Marmor and Beitner (1997) have investigated the effects of another class of CaM inhibitors, which reduce intracellular levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, and ATP and also detach glycolytic enzymes bound to the cytoskeleton. Four antagonists tested — thioridazine, CGS 9343B, clotrimazole, and bifonazole — brought about a marked reduction in cell viability (Glass-Marmor et al. 1996). The detachment of glycolytic enzymes associated with the cytoskeleton can also affect the function of the latter in cytokinesis. CaM cDNA has been transfected in sense as well as in the antisense orientation into C6 glioma cells. Transfection with sense-cDNA has been found to produce more clones than transfection with antisense constructs. The DNA content of cells has been reported to correlate with CaM levels (Liu G.X. et al. 1996). This is compatible with the finding that cells experience a delay in DNA synthesis in the presence of CaM inhibitors W7 and W13 and the CaM-dependent protein kinase inhibitor KN-62 (Mirzayans et al. 1995). CaM can influence the transduction of growth factor signals via the receptor kinases by modulating their phosphorylation by means of CaM-dependent protein kinases. Thus one can visualise several ways in which CaM could regulate physiological processes.

CALMODULIN

IN

NEOPLASIA

An enhancement of CaM has been recognised as a feature of cell transformation and of malignant cells. CaM levels of human lung cancer cells are higher than that of benign tumours of the lung or normal lung tissue (Liu G.X. et al. 1996). These authors also describe a correlation between tumour grade and TNM stage and levels of CaM. However, Edelman et al. (1994) have described a CaM-like protein that is apparently restricted to epithelial cells. This CaM-like protein was identical in size and largely homologous to CaM, but, unlike CaM, its expression seems to be significantly lower in malignant cells.

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79

The CaM antagonist J8 inhibits the invasive behaviour of the cutaneous melanoma cell line A-375SM and uveal melanoma cells (Dewhurst et al. 1997). Tamoxifen, its metabolites N-desmethyltamoxifen, and 17β-oestradiol also inhibit invasion in the absence of oestrogen receptors, which suggests that the inhibition produced by these anti-oestrogens was mediated by mechanisms other than receptor binding, e.g., CaM inhibition. A deregulation of intracellular calcium resulting in cell death is produced by tamoxifen at high concentrations where its effects are believed to be not mediated by oestrogen receptors (Jain and Trump, 1997). CaM antagonists could inhibit invasion by starving the cytoskeletal machinery of local ATP generation. Glass-Marmor and Beitner (1997) found that the CaM inhibitors, which they had previously shown to reduce the levels of certain glycolytic enzymes and ATP and cause loss of viability (Glass-Marmor et al. 1996), also detach these enzymes from their association with the cytoskeleton. It is possible that CaM could be influencing cell invasion by altering the expression of ECM-associated enzymes. Some years ago A. Ito et al. (1991) suggested that CaM could be differentially modulating the expression of TIMP and prometalloproteinases 1 and 3 in fibroblasts derived from human cervical carcinoma. The expression of these enzymes is known to markedly alter the invasive behaviour of cancers (Sherbet and Lakshmi, 1997b). Possibly, therefore, modification of the expression of ECM-associated enzymes and the properties of the ECM as a means of modulating cell behaviour, ought to be seriously considered. S100A4 does seem to operate through such a mechanism (Merzak et al. 1994b; Lakshmi et al. 1997). It would seem, therefore, that CaM might involve more than one target enzyme in the modulation of invasive behaviour.

RECOVERIN SUBFAMILY OF NEURAL CALCIUM BINDING PROTEINS AND THEIR FUNCTION Recoverin (RCN) and related proteins belong to a four-EF-hand recoverin family of neural calcium-binding proteins (NCBPs) (Table 5). Two types of NCBP can be distinguished. Type A proteins, e.g., recoverin, have two canonical EF-hands. Type B proteins, e.g., VILIP (visinin-like protein) and NCS-1, possess three regular EFhands. Flaherty et al. (1993) have elucidated the three-dimensional structure of RCN. According to them, the four EF-hands are arranged in a compact array. The tertiary structure and arrangement of the EF-hand may control calcium binding. Although generally described as NCBPs, individual NCBPs do show characteristic differences in their localisation within the neural tissue and the retinal component itself.

THE G-PROTEIN SIGNALLING

PATHWAY

Many extracellular modifiers of biological response transduce their signals via heterotrimeric guanine nucleotide regulatory proteins (G-proteins), which consist of α, β, and γ subunits (Koelle, 1997; Dohlman and Thorner, 1997; Berman and Gilman, 1998). These G-proteins are coupled to cell surface receptors. Several agents, such as epinephrine, norepinephrine, cytokines, and nonsteroid hormones, activate Gprotein receptors (Hein and Kobilka, 1997; Watson et al. 1996). Upon activation

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Calcium Signalling in Cancer

TABLE 5 Recoverin Family of Neural Calcium Binding Proteins Protein Designation Recoverin (S-modulin) Rem-1 Visinin s26 VILIP NCS-1

Occurrence

Ref.

Photoreceptor cells; mammalian bipolar cells Retinal cells; haematopoietic cells; gut cells Photoreceptor cells Photoreceptor cells (frog retina) Inner retina Photoreceptor inner segments; inner plexiform layer; ganglion cells

De Raad et al. (1995) Kraut et al. (1995)

Kawamura et al. (1996) De Raad et al. (1995) De Raad et al. (1995)

these receptors promote guanine nucleotide exchange of guanosine diphosphate (GDP) to GTP on the Gα. This in turn leads to the dissociation of Gβγ complex from Gα. These then regulate the activity of the target protein, leading eventually to the mobilisation of second messengers. A family of proteins called RGS (regulators of G-protein signalling) can preferentially bind to activated Gα. RGS proteins appear to function as guanosine triphosphatase (GTPase) activating proteins (GAP) (Berman et al. 1996; Watson et al. 1996; Hunt et al. 1996) and attenuate or block the signalling pathway (Popov et al. 1997; Tesmer et al. 1997; Hepler et al. 1997). The G-protein-dependent and calcium-signalling pathways are often closely allied. For example, neurotransmitters inhibit calcium channel current, which seems to be regulated by the photoreceptor G-protein called transducin (Jeong et al. 1999; also, see below). Similarly, dopamine-coupled receptors inhibit voltage-activated calcium channels (Wolfe and Morris, 1999). Dolphin et al. (1999) have identified the calcium channel-protein domains that might be involved in the modulation of calcium channel currents by G-proteins. In the signalling events associated with the acrosome reaction in spermatozoa, G-proteins require calcium together with PLA2 to induce acrosomal reaction (Dominguez et al. 1999).

RECOVERIN

AND ITS

FUNCTION

The transduction of the extracellular sensory signal involves the interaction of activated rhodopsin with the photoreceptor G-protein, transducin. The rhodopsin intermediate meta II activates transducin by catalysing the exchange of GDP to GTP. The RGS protein down-regulates this signalling pathway by promoting Gα GTPase activity and additionally by down-regulating cGMP phosphodiesterase, which effectively enhances GTPase activity. RCN (a homologue of this from the frog is known as S-modulin) is a 23-kDa protein that plays a specific role of phototransduction in mammalian retinal photoreceptors. The transduction of visual signals requires rhodopsin to be activated by phosphorylation. Upon illumination rhodopsin is phosphorylated by rhodopsin kinase at several serine and threonine residues. The activated rhodopsin is dephosphorylated by phosphatases to return it to the basal state.

The Calmodulin Family of Calcium Binding Proteins

81

RCN is involved in the process of light and dark adaptation by rod cells by regulating rhodopsin phosphorylation, thereby controlling photoreceptor light sensitivity, which is Ca2+ dependent. Several mutant forms of RCN, with mutations in EF-hands 2 and 3, and others with mutation of EF-hand 4, have been isolated recently. Of these, EFhand 4 RCN mutants were found to be able to inhibit rhodopsin kinase more effectively than could wild-type RCN (Alekseev et al. 1998). Apparently, a decrease in cytoplasmic calcium levels is necessary for light adaptation (Figure 11). Besides RCN, other calcium-binding proteins are involved in this process. Among them are the photoreceptor-specific GCAP and calmodulin, together with their targets, namely rhodopsin kinase, guanylate cyclase, cGMP-gated channel, and nitric oxide synthase (Koch, 1995; Gorczyca et al. 1995).

FIGURE 11 The pathway of sensory signal transduction involving recoverin and RGS (regulator of G-protein signalling) protein.

The RCN gene has been mapped to human chromosome 17p13.1 (J.F. McGinnis et al. 1995). Interestingly, the autosomal-dominant progressive cone dystrophy (CORD5) gene maps to chromosome 17p12–p13. The genes, coding for retinal guanylyl cyclase and pigment epithelium-derived factor, and the retinitis pigmentosa (RP) genes also occur in the RCN region (Balciuniene et al. 1995). Recoverin occurs predominantly in mammalian photoreceptor cells. It may be found in other retinal cell types and may therefore subserve other functions besides phototransduction (McGinnis et al. 1997). RCN appears to be a highly conserved protein, as suggested by its occurrence in the photoreceptor cells of the lamprey (Dalil-Thiney et al. 1998). Its expression may be developmentally regulated (Yan and Wiechmann, 1997). It is found to be transiently expressed in developing follicular and parafollicular pinealocytes in the developing chick embryo (Bastianelli and Pochet, 1994). Four isoforms of RCN occur and each of these shows N-terminal myristoylation. RCN not only participates in phototransduction, a role suggested by its localisation in photoreceptor cells, but it is also associated with the pathogenesis

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Calcium Signalling in Cancer

of the autoimmune state of cancer-associated retinopathy and uveoretinitis. However, it is uncertain whether RCN is involved with autosomal recessive RP.

MODE

OF

ACTION

OF

RECOVERIN

Recoverin regulates photoreceptor response by inhibiting rhodopsin phosphorylation. Rhodopsin kinase is active in the absence of RCN (Gorodovikova et al. 1994a). Rhodopsin phosphorylation and consequent cGMP hydrolysis are Ca2+- and ATPdependent processes. When the free Ca2+ level is raised, phosphorylation of rhodopsin is reduced and there is an increase in the lifetime of phosphodiesterase. This Ca2+ effect is negated by anti-RCN antibodies, which has been interpreted as suggesting that the calcium effects observed are a result of the inhibition of rhodopsin kinase (Gorodovikova et al. 1994b). Upon addition of RCN, rhodopsin kinase becomes sensitive to free Ca2+. Calcium-dependent interaction between RCN and rhodopsin kinase is indeed necessary for the inhibition of rhodopsin phosphorylation by RCN (C.K. Chen et al. 1995). All four isoforms of RCN inhibit rhodopsin phosphorylation in the same free calcium range (0.3 to 0.8 µM), but they differ with respect to the magnitude of inhibition achieved, which appears to be related to their hydrophobicity (Sanada et al. 1995).

POST-TRANSLATIONAL MODIFICATION

OF

RECOVERIN

Four isoforms of RCN have been identified; all appear to be posttranslationally modified at the N-terminal glycine residue with myristic acid or related lipids. Myristoylation is believed to be essential for the function of RCN. Senin et al. (1995) compared the inhibitory effect on rhodopsin phosphorylation of myristoylated and nonmyristoylated forms of recombinant RCN. They found that both forms of RCN inhibit rhodopsin kinase in the presence of Ca2+, but myristoylated RCN was more efficient in inhibiting the kinase. However, others believe that myristoylation is not necessary for the kinase inhibitory effect of RCN and that it only induces a cooperative Ca2+ dependence of the process (Kawamura et al. 1994; Calvert et al. 1995). Ames et al. (1994) have suggested, on the basis of the flexibility of the N-terminal helix in myristoylated calcium-free and nonmyristoylated calcium-bound form, that calcium binding to the EF-hand 3 domain induces EF-hand 2 to adopt a conformation that promotes calcium binding to RCN. Covalent attachment of a myristoyl or related N-acyl group to the N-terminal glycine appears to promote the binding of RCN to the optic disc membrane when free Ca2+ is raised. The so-called calcium–myristoylation switch is believed to play an essential role in the targeting of RCN to the cell membrane. The addition of the myristoyl group has been found to reduce the calcium affinity of RCN and induce cooperative calcium binding. Two conformational states have been recognised viz. the T and R states. In the T state the myristoyl group is sequestered inside the protein, whereas in the R state it is exposed, and furthermore, calcium binds to the R state several thousand-fold more strongly than to the T state (Ames et al. 1995). Calcium binding to the myristoylated RCN induces its translocation to the membrane. The

The Calmodulin Family of Calcium Binding Proteins

83

nonmyristoylated RCN is not translocated in this way (Tanaka et al. 1995). It would appear, therefore, that posttranslational modification of RCN is required for targeting to and interaction of RCN with the membrane. The RCN family protein VILIP also shows calcium-dependent targeting to the cell membrane. It has been found to interact with the actin component of the cytoskeleton in its recruitment to the cell membrane (Lenz et al. 1996). Braunewell et al. (1997) have confirmed that myristoylated VILIP can be shown to be associated with membranes, whereas nonmyristoylated VILIP is not. Membrane association may stabilise the RCN–rhodopsin kinase complex, as suggested by experimental demonstration of a preferential association of RCN with cell membrane with concomitant increase in rhodopsin kinase inhibition (Sanada et al. 1996). However, Johnson et al. (1997) found that conformational changes and distribution of RCN in the cell were not influenced greatly by calcium concentration and suggest, therefore, that the calcium–myristoylation switch may not be the only mechanism involved in the targeting of RCN to the cell membrane.

RECOVERIN

AND

CANCER-ASSOCIATED RETINOPATHY

Recoverin and Cancer-Associated Retinopathy in Small Cell Lung Cancer A degeneration of the retina is infrequently associated as a paraneoplastic condition with some forms of cancer. This condition, often described as cancer-associated retinopathy (CAR), is an autoimmune syndrome involving the degeneration of the photoreceptor cells of the eyes. Injection of RCN had previously been shown to induce degeneration of photoreceptor cells of the eyes of Lewis rats, and this was correlated with high titres of RCN antibodies in the circulation (Adamus et al. 1994). There are several reports of incidence of CAR in patients with small cell lung carcinoma (SCLC) (Polans et al. 1995; Matsubara et al. 1996). Polans et al. (1995) showed RCN is expressed in lung tumour biopsies of patients who had CAR but not in tumour samples of patients without CAR. They further identified two regions of RCN that were associated with its immunogenicity. Amino acid residues 64–70 and 48–52 were the major determinants. Immunisation of Lewis rats with peptide 64–70 caused photoreceptor degeneration in the animals. This 64–70 region is in close proximity to the EF-hand 2 calcium-binding domain. The autoimmune reactivity has been found to depend on changes in the conformation of the 64–70 stretch of amino acid residues induced by calcium binding to the EF-hand 2 present in the neighbourhood (Adamus and Amundson, 1996). Murphy et al. (1997) have encountered high titres of antibodies to a 60-kDa retinal protein, as well as high titres of RCN antibodies, in association with SCLC. The titres of both declined with treatment and Murphy et al. (1997) have therefore suggested that the detection of RCN antibodies alone should not be used as the sole criterion for diagnosing CAR. A recent report described a patient with non-SCLC with recoverin antibodies in the serum (Salgia et al. 1998).

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Retinopathy Associated with Other Forms of Human Cancer Retinopathy is associated also with other neoplasms such as breast and cervical tumours (Holz et al. 1997) and metastatic melanoma (Kirati et al. 1997). Again, this has been attributed to autoimmune responses to retinal proteins. RCN is expressed in cell cultures derived from retinoblastoma. Using antibodies raised against recombinant RCN, Weichman (1996) demonstrated that RCN is expressed in the cytoplasm of retinoblastoma cell line Y79. The expression was greatly increased by treating cells with 2 mM butyrate, and to a lesser degree by db-cAMP, together with the formation of neurite-like cellular processes indicating a state of induced differentiation. This possible link-up between RCN expression and differentiation is worthy of further investigation. In the sera of patients with CAR, heat shock protein (HSP) 70 has been detected in addition to recoverin (Ohguro et al. 1999). The involvement of bacterial HSPs in the induction of autoimmunity has been recognised with the demonstration that HSP65 from the tubercle bacillus mycobacterium can induce autoimmune disease in certain animal tumour models. Certain peptides derived from mycobacterial HSP65 and their homologous peptide obtained from patients with Behcet’s uveitis or iridocyclitis induce uveitis in Lewis rats. High antibody titres against these peptides are found in rats that developed uveitis as compared with those that had not (Uchio et al. 1998). This suggests that HSPs do function as autoantigens. Therefore, the detection of HSP70, together with recoverin, might suggest that both these antigens stimulate humoral autoimmunity in the pathogenesis of CAR. This view would be in line with the implication of HSPs in other autoimmune diseases such as rheumatoid arthritis (RA) (Winfield, 1989) and SLE (Minota et al. 1988; Conroy et al. 1994). Antibodies against human HSP60 and E. coli HSP60 have been found in patients with RA and SLE and in Reiter’s syndrome which combines arthritis with conjunctivitis, but the antibody titres are far higher than the titres of antibodies against mycobacterium HSP65 (Handley et al. 1996). This seems to suggest that the immune system recognises certain epitopes of these HSPs (Van der Zee et al. 1998). Although these observations suggest that HSPs might play a role in autoimmune conditions, they do not explain the coexpression of RCN with HSPs. It may be that some other HSP function, such as protein folding, may be involved here. As we have seen above, certain critical conformational changes are required in the proper functioning of RCN. Although, in the realms of speculation, the possibility that HSPs influence RCN conformation in such a way that sequestered autoimmune epitopes might be exposed cannot be excluded. It is unclear whether RCN forms complexes with HSPs. This is an interesting avenue to approach; one can envisage a situation where HSPs regulate RCN function by forming a complex. HSPs are known to form complexes with important biological macromolecules such as p53, rb proteins, and S100A4, and possibly influence their function as regulators of the cell cycle (Sherbet and Lakshmi, 1997b).

The Calmodulin Family of Calcium Binding Proteins

85

Is Recoverin Involved in Retinitis Pigmentosa? Retinitis pigmentosa (RP) is an autosomal-recessive disease involving degeneration of photoreceptor cells. RP has been attributed to mutations in the genes coding for rhodopsin and for the rod cGMP-gated channel. Naturally, therefore, the possibility of RCN implication in RP has received some attention. However, mutations in the RCN gene have been ruled out in 42 Spanish families with autosomal-recessive RP. Furthermore, the study by Parminder et al. (1997) did not encounter any mutations in the RCN gene in RP or allied heritable retinal diseases.

GUANYLATE CYCLASE-ACTIVATING PROTEINS The mammalian retina contains two GCAPs: GCAP1 and GCAP2. They are photoreceptor-specific calcium binding protein. GCAP1 is expressed at a high level in the outer segments of rods and cones. GCAP2 occurs mainly in the cone inner segments and is less intensely expressed in inner retinal neurons. Therefore, whereas GCAP1 has a phototransduction function, GCAP2 may not (Otto-Bruc et al. 1997). Furthermore, the expression of GCAP1 and 2 gene transcripts is substantially reduced in retinal degeneration (rd/rd) mutant chicken. Here, GCAP2 protein seemed to be normal, but the expression of GCAP1 proteins was down-regulated by more than 90% and this seems to be consistent with the loss of phototransduction (SempleRowland et al. 1996). GCAP1 has four EF-hand motifs. When not bound to calcium, GCAP is able to activate GC. Calcium binding inactivates GCAP. This seems to be a consequence of Ca2+ binding by EF-hands 3 and 4 and the conformational changes in the molecule (Rudnicka-Nawrot et al. 1998). The involvement of EF-hand 3 is also indicated by an inactivating mutation A → G in codon 99, exon 2 of the GCAP1 gene (Payne et al. 1998). This mutation is reported to occur in the GCAP1 gene in a family with autosomal-dominant cone dystrophy.

7

The Structure of Contractile Proteins

THE ACTIN COMPONENT OF CONTRACTILE MACHINERY OF THE CELL The physical alterations of the shape of the cell, its structure, motility and contractile ability are an attribute of the structure and organisation of the cytoskeletal machinery, of which the intermediate filaments (IF) (10 nm diameter), actin microfilaments (7 nm diameter), and microtubules are the important components. Actin microfilaments are polymers of F- and G-actin. These filaments are flexible, double-helical polymeric structures made up of F-actin strands. The G-actin subunits participate in binding to the myosin globular heads. G-actin also possesses ATP- or ADP-binding domains. Actin filaments have a defined polarity, which flows from the fact that the monomers themselves show a distinct polarity. In association with actin-binding proteins, the actin filaments form specialised structures. To these organised structures the cell owes its faculty of modulation of shape, motility, and other adhesionmediated phenomena. The reorganisation of actin cytoskeleton is also involved with plant cell division, differentiation, and light-induced plastid migration, among other cellular features (Staiger et al. 1997).

ACTIN ISOFORMS Four actin isoforms have been identified, of which two, the α and γ isoforms, are smooth muscle specific and the other two are the cytoplasmic β isoforms. The transfection of mutated β-actin genes in normal diploid cells alters their morphology and growth characteristics, and at the same time mutated β-actin protein is seen to be incorporated into the cytoskeleton (Leavitt et al. 1987a). Leavitt et al. (1987b) have further shown that the transfectant cells that expressed mutant βactin possessed greater tumorigenic potential than those with lower levels of mutant actin. Cells derived from these tumours had a shorter latency period for tumour formation as compared with the original transfectant cells. None of these interesting studies seem to have been followed up to examine motility, invasiveness, and metastatic potential, which, in retrospect, seems highly desirable in light of the changes in cell morphology and growth features engendered by the expression of mutant actin. A natural extension would be to investigate whether wildtype β-actin possesses tumour suppressor function. The suppressor function of αactin has been demonstrated recently. The rat fibroblast cell line 3Y1 expresses α-actin, but this is down-regulated when the cells are transformed by RSV. In

87

88

Calcium Signalling in Cancer

contrast, transfection of α-actin into RSV-transformed fibroblasts resulted in the reduction of tumour growth and invasiveness (Okamoto-Inoue et al. 1999).

REGULATION

OF

ACTIN DYNAMICS

Actin polymerisation and depolymerisation occurring in the dense actin cytoskeleton at the leading edge of a cell is instrumental in achieving locomotion (Tilney et al. 1983; Zigmon, 1993). The polymerisation of actin provides the protrusive power that allows the formation of lamellipodia or filopodia in the direction of cell movement. There is net polymerisation at the front edge while net depolymerisation occurs at the rear of the lamella. The relative rates of polymerisation and depolymerisation are at the basis of cell motility regulation. Actin dynamics are affected and may, indeed, be regulated by many cellular proteins. The actin-binding proteins regulate cell motility by influencing the rates of actin polymerisation and depolymerisation. Actin-binding proteins such as gelsolin thymosin, and profilin are cytosolic proteins that inhibit the process of actin polymerisation and profoundly influence cell morphology and motility. It follows, therefore, that these proteins could be indirect determinants of the altered cell motility and metastasis that often accompany neoplastic transformation. Among other notable cellular proteins that affect cytoskeletal dynamics is the S100A4 calcium-binding protein. S100A4 has been shown to be closely associated with the invasive and metastatic behaviour of cancer cells, and also to promote depolymerisation of the cytoskeletal structures (Lakshmi et al. 1993, 1997). It would be worthwhile, therefore, to discuss the role played by thymosin, cofilin, profilin, and fimbrin in altering cytoskeletal dynamics and assess their impact on cell behaviour. The currently held view is that the actin-binding proteins alter the ratio of monomeric G-actin and F-actin in cells. G-actin-binding proteins such as thymosin β4 (Tβ4) and profilin sequester G-actin from the cell pool and thereby prevent the process of polymerisation, whereas F-actin-stabilising factors such as myosin subfragment I and phalloidin can promote repolymerisation from the Tβ4/G-actin complex (Ballweber et al. 1994) (Figure 12). In continuous cellular locomotion or inherent invasive ability, the cell has to maintain a net high level of actin polymerisation and a constant F-actin content. Actin subunits derived from depolymerisation occur at the rear of the lamella, are assembled into filaments at the front of the lamella (Y.L. Wang, 1985; Small 1995). It would be of considerable interest to review how the expression of these actin-binding proteins affects cell behaviour and especially if any one of them shows any correlation with the aberrant behavioural properties manifested by neoplastic cells.

COFILIN

IN THE

REGULATION

OF

ACTIN DYNAMICS

Recently it has been shown that the phosphoprotein cofilin, also known as the actin depolymerisation factor, is actively involved with actin depolymerisation and in cellular processes that require cyclical changes in the actin cytoskeleton (Abe et al. 1996; Lappalainen and Drubin, 1997; Theriot, 1995). Cofilin is a 20-kDa protein found in muscle as well as nonmuscle cells. Two isoforms of this protein occur in

89

The Structure of Contractile Proteins

Profilin

Tβ4

G-actin ≈ F-actin

Sequestration of G-actin

Tβ4/G-actin complex

Profilin/G-actin

Myosin Fragment I Phalloidin

Prevention of polymerisation

Repolymerisation FIGURE 12 Schema of the participation of actin-binding proteins in the regulation of actin dynamics Tβ4, thymosin β4.

mammals: the muscle (M) type and nonmuscle (NM) type. M-type cofilin expression is up-regulated in the myogenesis of C2 cells in vitro (Ono et al. 1994). Goode et al. (1998) identified a 37-kDa protein in budding yeast, and they named it twinfilin because it contains two cofilin-like regions. Twinfilin is able to sequester actin monomers, but it does not inhibit actin polymerisation. Homologues of this protein may occur in man, mouse, and Caenorhabditis elegans, as sequence data searches have revealed, and Goode et al. (1998) suggest twinfilin may be highly conserved in evolution. Cofilin regulates actin polymerisation in a pH-dependent manner. Rapid cycles of assembly and disassembly of actin depend on this protein (Lappalainen and Drubin, 1997). Overexpression of cofilin leads to disorganisation of actin filaments (Ono et al. 1996). Its intracellular distribution as well as its interaction with actin are regulated by phosphorylation (Obinata et al. 1997). Cofilin is phosphorylated by LIM kinases (LIMK-1 and -2) (Arber et al. 1998; Yang et al. 1998; Sumi et al. 1999). Phosphorylated cofilin loses its normal function of depolymerising actin filaments. Cellular response in the form of alterations in cellular morphology, membrane ruffling, and neuronal outgrowths results from the growth factors being able to activate cofilin. In the case of neurite extension, the dephosphorylation of cofilin

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Calcium Signalling in Cancer

has been found to correlate with its translocation to the membrane (Meberg et al. 1998). Actin-binding peptide derived from cofilin has been reported to compete with gelsolin segments 2 and 3 for actin-binding. These binding sites of cofilin and gelsolin have structural homologies and therefore they possibly share the same binding domain on the actin filament (Van Troys et al. 1997). How this structural relationship between cofilin and gelsolin translates in functional terms is yet uncertain. However, Van Troys et al. (1999) seem to have made a beginning by providing a framework based on the structure of the actin-binding modules that occur in actinbinding proteins, the similarities and differences between these binding domains, and how, overall, they might impinge on actin dynamics.

PROFILIN

IN THE

REGULATION

OF

ACTIN DYNAMICS

Profilin is another protein that participates in and regulates actin dynamics in cellular responses to external as well as internal signals (Staiger et al. 1997). It is a 14 to 15-kDa protein that has been found to sequester monomeric actin, to promote nucleotide exchange occurring on actin monomers, and to be capable of inducing actin polymerisation when barbed ends of actin filaments are free. Profilin also binds to poly-l-proline and phosphoinositol lipids. In mammalian cells two isoforms of profilin have been described and these also may be differentially expressed in different tissues (Witke et al. 1998). Profilin forms a high-affinity complex with actin (Di Nubile and Huang, 1997). The importance of this complex formation in the cellular function of actin filaments is demonstrated by the effects of deleting residue proline 96 and threonine 97 that occur near the major actin-binding site. Deletion of these amino acids reduces the ability of profilin to bind to actin and its ability to promote nucleotide exchange on actin monomers. When injected into Swiss 3T3 fibroblasts, the mutant profilin failed to affect microfilament organisation, which the wild-type was capable of achieving (Hajkova et al. 1997). A diminution of profilin expression has been shown to result in abnormalities of cytokinesis and the formation of multinucleate cells (Cao et al. 1997). Profilin is also a strong competitor against Tβ4 in binding to actin (Ballweber et al. 1998; also, see below). It would be reasonable to accept that profilin is directly involved in actin polymerisation and in this way influences morphological features and biological behaviour of cells.

RHO GTPASES

IN

ACTIN DYNAMICS

AND

SIGNAL TRANSDUCTION

The Rho family of GTPases are ras-related GTPases. In the yeast Saccharomyces cerevisiae, certain members of the family are known to control the process of budding and the determination of cell polarity (Johnson and Pringle, 1990). The mammalian Rho proteins have been implicated in cytoskeletal remodelling, and cofilin could be the terminal target that brings this about. Rho proteins also have been associated with the control of microfilament assembly in vitro and the formation of cytoskeletal structures such as filopodia, lamellipodia, adhesion plaques, and intercellular junctions (Ridley and Hall, 1992; Nobes and Hall, 1995, 1999; Peterson et al. 1990; Van Aelst and D’Souza-Schory, 1997). Intercellular adhesion is another cellular faculty in which Rho proteins are involved (Morii et al. 1992; Tominaga et al. 1993). The

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91

processes of cell spreading and alterations in cell morphology and the transduction of growth factor signals also require the participation of and are regulated by Rho proteins (G.A. Murphy et al. 1999). At the fundamental level in all these cell functions is the contractile ability of the actin cytoskeleton, which has been attributed to the activation of Rho GTPases in response to microtubule depolymerisation (B.P. Liu et al. 1998). The Rho GTPases seem to activate a host of signalling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. These kinases phosphorylate downstream target proteins, directly or indirectly via the phosphorylation of other kinases. Rho and Cdc42 induce the reorganisation of the actin cytoskeleton and promote the formation of stress fibres. This process appears to be related to the activation of LIMK-2. LIMK-2 phosphorylates cofilin and inhibits its normal function of cytoskeleton depolymerisation (Sumi et al. 1999). The Rho-activated serine/threonine kinases (ROCK) seem to be important elements in cancer invasion. Itoh et al. (1999) transfected a dominant active mutant of ROCK cDNA into rat MMI hepatoma cells. These transfectant cells were found to possess greatly enhanced invasive ability, in comparison with cells that had been transfected with a dominant negative ROCK cDNA. The MLCK of endothelial cells is another kinase that seems to be regulated by Rho (Garcia et al. 1999). The possibility that Rho proteins might regulate the contractile apparatus of endothelial cells and alter the permeability of endothelia as a consequence has serious implications for cancer cell dissemination, in which the permeation of the endothelial barrier is an essential event. The induction of membrane ruffles has been attributed to Rac, a GTPase that belongs to the Rho family. Rho GTPases have been implicated in Rho kinasemediated phosphorylation of transmembrane adhesion proteins, such as the CD44 splice variants, and their interaction with the cytoskeletal protein, ankyrin (Bourguignon et al. 1999). This interaction between CD44 and ankyrin is manifested as changes in membrane activity in the form of ruffling. In this particular instance, Rho A GTPase stimulates ROCK, which phosphorylates CD44. This leads to an enhancement of CD44–ankyrin interaction. Bourguignon et al. (1999) also demonstrated that the induction of CD44-associated membrane ruffling can be achieved by injecting the catalytic domain of ROCK and, furthermore, that the membrane activity is inhibited by anti-CD44 antibodies. A more indirect route might be adopted in the Rho-mediated reorganisation of the cytoskeleton involving cofilin. Here ROCK phosphorylates and activates LIMK, which then phosphorylates cofilin (Maekawa et al. 1999). Some members of the S100 protein family are known to be able to modulate and possibly regulate actin dynamics. Among them is S100B, which has recently been shown to bind and activate the serine/threonine kinase Ndr (Millward et al. 1998). The Ndr kinase has structural and possibly also functional similarities with ROCK. It is suggested, therefore, that there might exist a general and common mechanism by which S100 proteins could regulate actin dynamics and the cellular processes and functions upon which the modulation of actin dynamics and cytoskeletal organisation might strongly impinge. A family of proteins called WASP [Wiskott-Aldrich syndrome (WAS) protein] is regarded as the regulators of actin reorganisation. WAS is an X-linked condition

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that affects male children. The syndrome is generally described as including immunodeficiency, thrombocytopenia, and eczema, but the actual phenotype may be variable. The WAS gene is mutated in patients and the type of mutation seems to relate to the phenotype of the disease. WASPs are cytoplasmic proteins. One WASP isoform is found only in the lymphocytes. Another isoform called the N-WASP, which was isolated from neural cells, shows a more ubiquitous distribution. These proteins are known to bind to several cellular proteins and kinases and also to Rho GTPases, such as Cdc42 and Rac. They interact directly or indirectly with the actin cytoskeleton. The haemopoietic cells derived from patients with WAS also have been reported to show abnormalities of the actin cytoskeleton. These observations suggest that WASPs play an important part in the transduction of extracellular signals to the cytoskeleton (O’Sullivan et al. 1999). They are believed to function downstream of Rac. The association of WASP with the induction of membrane activity has been demonstrated recently. Castellano et al. (1999) showed that experimentally induced recruitment of activated Cdc42, or its downstream effector WASP, to the cell surface results in actin polymerisation and the formation of filopodia. Other actin-binding proteins, such as profilin, that are associated with actin dynamics may therefore be expected to enter into this picture of WASP- and Rhomediated regulation and organisation of the actin cytoskeleton. Suetsugu et al. (1998) generated a mutant profilin, H119E, that is defective in actin-binding. This mutant profilin suppressed actin polymerisation induced by N-WASP. Under normal circumstances profilin associates with N-WASP, and is essential for a rapidly polymerising actin by N-WASP; this cannot happen with mutated profilin.

INTERACTION

OF

FORMIN

WITH

PROFILIN

AND

RHO GTPASES

The formin family of proteins constitutes another component of the actin regulatory system. Formin and related proteins, which are expressed ubiquitously in several organisms, seem to knit together profilin and Rho GTPases in their function of regulating cytoskeletal dynamics. Members of the formin family share many structural features such as the coiled-coil molecular organisation, the occurrence of homology sequences known as formin homology (FH) 1 and FH2 domains, the collagen-like domain, nuclear localisation signals, and phosphorylation sites. The formin gene was identified by mutations in the gene associated with recessive limb bud deformity (Maas et al. 1990, 1991; Chan et al. 1995). The gene has 24 exons encompassing 400 kb of genomic DNA (C.C. Wang et al. 1997). The formins are predominantly nuclear proteins participating in cytokinesis and cell polarisation (Zeller et al. 1999). They are essential for the correct orientation and alignment of the cell division spindle (L. Lee et al. 1999). Formin also participates in morphogenesis, e.g., in the yeast budding process. The importance of their role in morphogenesis is also indicated by the fact that mutation in the formin gene leads to disruption of epithelial–mesenchymal interactions, which in turn leads to abnormal limb development. This has suggested the possibility that it may participate in the transduction of morphogenetic signals (Zeller et al. 1999). Formin has several ligands. Prominent among them is profilin (Mittermann et al. 1998). Rho GTPase is a downstream target of formin (Watanabe et al. 1997). It seems, therefore, that

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formin is an important element in profilin/Rho-dependent actin polymerisation of cytoskeletal dynamics and in this way might regulate cytokinesis, cell morphology, and adhesion-dependent processes such as cell motility and invasion.

THE ROLE OF THYMOSIN FAMILY ACTIN-BINDING PROTEINS IN ACTIN DYNAMICS SEQUESTRATION

OF

ACTIN

BY

THYMOSINS

The beta thymosins constitute an important family of actin-binding proteins, which are recognised as major participants in the regulation of actin dynamics. Among its members are Tβ4, Tβ10, and Tβ15, which are the most intensively investigated forms of thymosin. Tβ4 is a 4.5-kDa protein consisting of 43 amino acid residues. Tβ14 is another protein isolated from the sea urchin, that has 40 amino acid residues and a molecular size of 4.53 kDa and bears a high degree of sequence homology to Tβ4 (Stoeva et al. 1997). Tβ15 is a 5.3-kDa protein. Thymosins seem to occur ubiquitously. Actin-binding proteins showing a high degree of homology to mammalian thymosins have been isolated from invertebrates. The thymosins from invertebrates have 40 amino acid residues, but they appear to differ with respect to their affinity for binding to rabbit muscle actin (Safer and Chowrashi, 1997). The thymosins function by sequestering monomeric actin and thereby diminishing actin polymerisation (Safer, 1992). Tβ4 binds to actin monomers by means of its N-terminal 5 to 20 amino acid residue sequence (Van Compernolle et al. 1992; Czisch et al. 1993; Van Troys et al. 1996). In vitro this peptide adopts a folded conformation for achieving high-affinity interactions with actin (Feinberg et al. 1996a). Feinberg et al. (1996b) have discovered homologous short sequences in Tβ4 and gelsolin that bind to the C-terminal region of actin. They have also commented on the ability of these short sequences to form secondary structures and its relationship to their biological function. However, Safer et al. (1997) found that Tβ4 crosslinks to both barbed and pointed ends of actin, and this requires that the C-terminal domain, which participates in the binding, is in an extended conformation.

EFFECTS

OF

THYMOSINS

ON

CELL PROLIFERATION

The ability of Tβ4 to inhibit actin polymerisation is well documented. Also adequately demonstrated is the involvement of thymosins in proliferation, motility, cell differentiation, and angiogenesis, all of them important features of embryonic development and cancer development and progression. Recently, Sanger et al. (1995) experimentally enhanced the intracellular levels of Tβ4 and studied the structural integrity of actin bundles of stress fibres and cytokinetic furrows. They microinjected a viral vector carrying Tβ4 cDNA, transfected the vector into PTK2 cells, or injected purified Tβ4 protein into these cells. Both microinjection and transfection of cDNA produced a disassembly of stress fibres. The effects of injection of the pure protein were even more marked; a disassembly of stress fibres occurred within 10 min after injection, resulting in delayed cytokinesis. However, although Tβ4 reduces actin

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polymerisation by sequestering monomeric G-protein, at higher concentrations its ability to depolymerise F-actin appears to decrease (Carlier et al. 1996). The effects of thymosins on cell proliferation are therefore inevitable. Sanger et al. (1995) demonstrated that an overexpression of Tβ4 delays cell division. The N-terminal part of the protein contains an acetylated tetrapeptide sequence of Ac–N–Ser–Asp–Lys–Pro that is known to inhibit haemopoiesis. This peptide has been shown to inhibit the growth of normal bone marrow progenitor cells. It markedly reduces the growth of granulomacrophagic and erythroid progenitor cells and the size of their S-phase fraction (Bonnet et al. 1996). However, Bonnet et al. (1996) also found that although the inhibitory effect of the Tβ4 is similar to that of the tetrapeptide, Tβ4 from which this sequence is deleted retains the inhibitory effect.

THYMOSINS

AND

CELL MOTILITY

AND

DIFFERENTIATION

Needless to say, the amply confirmed effects of thymosins on the structural integrity of the cytoskeleton are bound to find expression in altered cell motility. Tβ4 has been shown to enhance strongly the migratory behaviour of an established cell line of endothelial cells derived from human umbilical vein (Malinda et al. 1997). Of the primary human cell lines that Malinda et al. (1997) examined, the migration of only human coronary artery cells seemed to respond to Tβ4. They also reported an increase in the production of metalloproteinases upon treatment with the thymosin. These enzymes can remodel the extracellular matrix and aid migration by altering cell adhesion to the substratum. NIH3T3 cells induced to overexpress Tβ4 have been reported to be more adherent than corresponding control cells (Golla et al. 1997). This enhanced adhesion appears to be due to adhesion-mediating proteins such as α-5 integrin as well as the transmembrane proteins such as vinculin that link cell membrane proteins to the cytoskeleton. There are also important implications of thymosin overexpression for cell migration in the context of cancer invasion, because thymosins have been reported to be overexpressed in neoplastic cells (see below). Especially significant are the reported specificity of response by endothelial cells to Tβ4 and the associated overexpression of metalloproteinases. Metalloproteinases have been closely associated with breaching of the endothelium and aiding the diapedesis of cancer cells into the vascular compartment (Sherbet and Lakshmi, 1997b). Tβ4 is reported to be highly expressed in the intrinsically highly motile embryonic mesenchymal cells (Carpintero et al. 1996). The expression of Tβ15 seems to be up-regulated in the highly motile Dunning rat prostate cancer cell lines. Besides, the transfection of antisense constructs of Tβ15 gene apparently alters the motility of these cells (Bao et al. 1996).

EXPRESSION

OF

THYMOSINS

IN

EMBRYONIC DEVELOPMENT

Differential cell adhesion and migration patterns are an integral feature of cell differentiation and morphogenesis. The expression of Tβ4 and Tβ10 shows some relationship to specific developmental stages (Carpintero et al. 1996), although there are as yet no indications that it is developmentally regulated. Carpintero et al. (1996) found a marked increase in Tβ10 mRNA in early postimplantation mouse embryos.

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This mRNA is also found in certain tissues, including mesenchymal tissues and the spinal cord, in 9.5 to 12.5-day old embryos. These authors have also encountered differences in the distribution of Tβ4 and Tβ10 mRNAs, which could conceivably suggest differences in their function in embryonic development. Tβ4 has been detected in early chick embryo cells but not in adult skeletal muscle cells. Cofilin also is associated with muscle development, but its relative contribution may change during muscle development (Nagaoka et al. 1996). These observations are consistent with the notion of development-related expression of thymosins. Gomez-Marquez et al. (1996) showed that Tβ4 mRNA occurs in mouse embryonic stem cells, and further that Tβ4 is able to induce embryonal P19 cells to differentiate into neuronal- and glial-type cells or into cardiac and skeletal muscletype cells. Furthermore, Tβ4 mRNA was demonstrable around blood vessels and in heart tissues.

POTENTIAL ROLE

OF

THYMOSINS

IN

CANCER PROGRESSION

As stated before, the putative association of overexpression of Tβ4 with increased production of metalloproteinases in normal cells together with the relationship between Tβ4 and high invasive ability of the Dunning rat prostate cancer cell lines point to the relevance of thymosins in cancer invasion and metastasis. Both Tβ10 and Tβ15 have been investigated for their possible relationship with cancer progression. Tβ10 protein was reported to occur at high levels in the malignant cell rather than the normal tissue component of human breast cancer, and the level of expression increased with tumour grade (Verghese-Nikolakaki et al. 1996). The expression of Tβ10 gene was higher in five thyroid carcinoma cell lines as compared with normal thyroid-derived primary cells. Expression of the gene was higher in anaplastic tumour tissue (Califano et al. 1998). Tβ15 levels have been examined in several tumour cell lines of various histological origins. An up-regulation of the gene has been found in highly metastatic mouse lung and human breast cancer cell lines. This study also involved an examination of the levels of Tβ15 protein in human breast and prostate cancer tissues. Higher expression of Tβ15 has been correlated with the Gleeson grade in prostate carcinomas (Bao et al. 1996). In breast cancer, higher levels of the protein correlated with greater metastatic potential (Bao et al. 1998). This is in contrast with their earlier studies (Gold et al. 1997). Gold et al. (1997) had encountered higher Tβ15 expression in nonmetastatic breast cancer, which suggested the involvement of thymosins in the early stages of breast cancer development rather than in the late stages of the disease. Although much attention has been focused on β-thymosins, Tsitsilonis et al. (1998) found α-thymosins to be of prognostic value in breast cancer. They determined levels of prothymosin α and parathymosin α in breast carcinoma tissue as well as in tissues from benign breast disease, and found that levels of both are much higher in carcinomas than in benign tumour tissue and normal breast tissue. They also reported that these levels correlated with overall survival of patients. As stated before, Tβ4 expression has been found to be very high in Dunning rat prostate carcinoma cells, which possess high motility. When Tβ4 antisense constructs were transfected into these cells the invasive ability was suppressed,

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suggesting that the high levels of Tβ4 expression might be associated with high invasive ability. Contrary to this, transfection of adenovirus E1A and E1B genes has been carried out in a highly metastatic human melanoma cell line by Van Groningen et al. (1996) who reported that a number of markers for tumour progression were down-regulated as a consequence. Among them was Tβ10. Admittedly, E1A gene products can inhibit oncogene-mediated cell transformation as well as invasion and metastasis in certain animal models. E1A protein is known to be able to induce and stabilise p53 phosphoprotein, which controls the G1-S checkpoint transition of cells, and this is accompanied by apoptic loss of cells. On the other hand, E1B protein can bring p53 levels to normality (Sherbet and Lakshmi, 1997b). However, the raft of changes produced by the transfection of the adenovirus genes makes it a rather inappropriate model for the study of tumour progression. Although the transfected cells may have shown a reduction in tumorigenicity upon implantation into compatible hosts, under these circumstances it would be difficult, even unacceptable, to try to extrapolate as to the significance of the suppression of individual markers in relation to tumorigenicity. Some of the difficulties are inherent in the interpretation of data obtained from experimental studies such as gene transfer in vitro and on tumour tissues from patients. Therefore, it is imperative that further studies be undertaken to assess the potential value of thymosins in determining the state of tumour progression.

THE FIMBRIN FAMILY OF ACTIN-BINDING PROTEINS MOLECULAR FEATURES

OF

FIMBRIN

Fimbrins are EF-hand calcium-binding proteins that actively participate in binding to and bundling of actin. In actin filaments, one molecule of fimbrin might bind eight actin monomers under optimal conditions (Namba et al. 1992). Fimbrins are highly conserved in evolution, with regard to both their structure and function. Fimbrin from diverse origins, e.g., from Dictyostelium discoideum to humans, share structural and biochemical properties. Fimbrin-like proteins have also been isolated from plants, such as wheat (Triticum aestivum) and Arabidopsis thaliana (CruzOrtega et al. 1997). The Dictyostelium fimbrin is a 67-kDa protein containing two EF-hands that are followed by two actin-binding sites (Prassler et al. 1997). The Nterminal actin-binding domain is a highly conserved domain that fimbrin shares with other actin-binding proteins. This N-terminal domain contains two tandem calponin homology (CH) domains which are implicated in the binding of F-actin (Goldsmith et al. 1997). Fimbrin-mediated cross-linking and bundling of actin is regulated by free calcium, which it binds with high affinity and selectivity. However, the optimal concentration for cross-linking of actin was determined to be below 0.15 µM. This process was progressively inhibited at higher free calcium concentrations and halfmaximal inhibition of cross-linking occurred at 1.6 µM Ca2+ concentration (Pacaud and Derancourt, 1993). Pacaud and Derancourt (1993) also noted that calciumbinding of fimbrin, in the concentration range of 0.15 to 1.5 µM, produced conformational changes in the molecule, to which they attribute the calcium-mediated regulation of fimbrin function. It may be that the conformational alterations actuated

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by calcium at different concentrations could render actin cross-linking reversible. A further possibility has arisen from the studies of Hanein et al. (1997a) that fimbrin might induce conformational changes in actin itself. Both types of change will inevitably contribute to cell behaviour. Fimbrins form a large family of proteins. Four genes encode fimbrin in Salmonella (Collinson et al. 1996). Three isoforms of fimbrin have been identified and designated as I-fimbrin, L-fimbrin, and T-fimbrin. I- and L-fimbrin are characteristically associated with intestinal and kidney epithelia, leukocytes, and tumours, whereas T-fimbrin shows a more general distribution in a variety of cells and tissues (Chafel, 1995).

FUNCTION

OF

FIMBRIN

IN

CYTOSKELETAL ORGANISATION

A major function of fimbrin appears to be in the assembly of actin filaments. Fimbrin as well as actin capping protein (CP) are required for proper assembly of these filaments in the yeast Saccharomyces cerevisiae. There is a reduced filament assembly and fimbrin in the CP mutants of the yeast. Actin obtained from CP mutants shows defects in polymerisation as well as in its binding to fimbrin (Karpova et al. 1995). In mammalian cells, Rho GTPases may mediate actin filament assembly and bundling. A constitutive expression of the Rho GTPase Cdc42Hs causes impairment of cytokinesis of HeLa cells. This seems to be a consequence of a reorganisation of F-actin with which, among other actin-binding proteins, T-fimbrin is associated (Dutartre et al. 1996). Functional conservation of fimbrin isoforms has been amply demonstrated by Adams et al. (1995), who found that human T- and L-fimbrin can substitute for the yeast fimbrin called Sac6p. T- and L-fimbrin were both able to complement the temperature-sensitive growth defect that is seen in sac6 null mutants, and they could also restore normal cytoskeletal organisation and cell shape in these mutants. The null mutants show defective sporulation, which is restored by human T- and L-fimbrin (Adams et al. 1995). Fimbrin isoforms show not only a tissue-specific distribution, but the specificity appears also to extend to differentiation and morphogenesis. Fimbrin shows a definable temporal and spatial pattern of expression in the course of the development of the cochlea and may be involved in the formation of the inner and outer stereocilia of the hair cell (Zine et al. 1995). The differentiation of intestinal epithelium is associated with the expression of T-fimbrin in the apical area of the cell and Lfimbrin in the basal area, until 14.5 days of development. Both isoforms are said to be totally down-regulated in expression by day 16.5, but instead, I-fimbrin appears on day 14.5 of development to give the epithelium a brush border-like localisation. These findings, reported by Chafel et al. (1995), suggest possible differences in their function in the course of differentiation. A number of the events occurring in morphogenesis and differentiation require changing patterns of cell adhesion, for which fimbrin seems to be ideally placed. Babb et al. (1997) studied the localisation of fimbrin in mature as well as differentiating osteoclasts. Fimbrin is a component of the osteoclast adhesion complexes called podosomes. During migration podosomes are found at the cell periphery. Microfilament organisation and podosome assembly is important in the rapid modulation of adhesion to the substratum, and

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in the motility of cells. T- and L-fimbrin, but not I-fimbrin occurred as an integral component of the core of the podosomes throughout the process of monocyte-derived osteoclast differentiation. The levels of T-fimbrin increased with and appeared to be related to the formation of podosomes. The involvement of fimbrin in podosomes is compatible with the association of fimbrin with the adhesive organelles called fimbriae that have been described in bacteria. Fimbriae are fibre-like structures that mediate the attachment of bacteria to host cells. These are assembled from fimbrin subunits (Smyth et al. 1996). Frederick et al. (1996) investigated the adhesive interactions between lymphokineactivated killer (LAK) cells and came to the conclusion that fimbrin might be an important factor in intercellular adhesive contacts. They also showed that contact between LAK cells and target tumour cells, namely SK-Mel-1 human melanoma cells and Raji lymphoma cells, leads to the phosphorylation of L-fimbrin of the LAK cells. S.L. Jones et al. (1998) have also suggested that induction of fimbrin phosphorylation is an important step in fimbrin function. However, as discussed below, the question of whether phosphorylation leads to fimbrin activation must be regarded as sub judice at present. Podosomes that participate in cell adhesion and locomotion have been found to contain other cytoskeletal linking proteins such as talin and vinculin, besides fimbrin. L-fimbrin has been shown to regulate integrin-mediated adhesion of leukocytes (S.L. Jones et al. 1998). It is possible that fimbrin is instrumental in the organisation of physical pathways, such as fimbriae and podosomes, by which extracellular signals for intercellular and cell–substratum adhesion are transduced to the cell. Another membrane organelle with which fimbrin might be associated in organising a signal transduction machinery is the caveola. Caveolae are plasma membrane invaginations, of 50 to 100 nm dimension, that occur in many cell types. Caveolae have been attributed with many functions, notably transport of molecules across endothelia and signal transduction (Lisanti et al. 1995). A major component of caveolae is a 21- to 24-kDa protein called caveolin. Caveolin is said to function as a scaffolding protein that organises the signalling molecules in the caveolae. That caveolae contain essential components of the signal transduction machinery may be deemed to be firmly established. Thus caveolin has been shown to interact directly with signal transduction molecules such G-protein α subunits and the ras protein (Lisanti et al. 1995; Song et al. 1996). The receptors for PDGF and EGF are located in caveolin-rich microdomains, and caveolin has been shown to interact directly with the EGF receptor (G.X. Liu et al. 1996; Couet et al. 1997). The interaction of caveolin negatively regulates RTK activities associated with activated EGFr and cerbB2 (Couet et al. 1997), and in mammary tumours expressing c-erbB2, caveolin expression is down-regulated (Engelman et al. 1998). Therefore, this interaction between growth factor receptors and caveolin may deregulate the transduction of the growth factor signals. Compatible with this view are recent reports that overexpression of caveolin results in growth inhibition of tumour cells (S.W. Lee et al. 1998; T. Suzuki et al. 1998). On the other hand, caveolin has been associated with cancer progression. It is reported to be overexpressed in infiltrating ductal carcinoma of the breast and prostate. In prostate cancer, caveolin is expressed in both primary and metastatic tumours (N. Yang et al. 1998). These two sets of data may appear to

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99

contradict each other, but it is possible to envisage caveolin functioning as a growth inhibitor in the early stages of tumour development. Caveolin might function in later stages of tumour progression merely by virtue of its ability to promote cell–cell and cell–substratum adhesion, which are essential requisites for a successful transition of the cancer cell along the metastatic cascade. In this connection, it would be worthwhile to note that T-cadherin, an adhesion-mediating molecule, also occurs in caveolin-rich plasma membrane microdomains (Philippova et al. 1998). After a seemingly long digression, it should also be noted that fimbrin is a component of the caveolae. Fimbrin occurs together with other proteins, e.g., src and ezrin, in cell membranes that are rich in caveolin (Mirre et al. 1996). The available evidence, albeit circumstantial in nature, may be interpreted as implicating fimbrin in tumorigenesis and tumour progression by deregulating signal transduction pathways and cell adhesion mechanisms.

REGULATION

OF

FIMBRIN EXPRESSION

The apparent tissue-specific distribution of fimbrin isoforms has led inevitably to investigations directed toward understanding the mechanisms of fimbrin expression and whether there is any tissue-specific regulation of its expression. C.S. Lin et al. (1997) have characterised the promoters of human and murine L-fimbrin. They described considerable similarity in the organisation and found that both promoters functioned with equal efficiency in most cell types. Therefore, the apparent tissuespecific and differentiation-related expression of fimbrin could be due to posttranscriptional modification of fimbrin rather than to its regulation at the transcriptional level. Phosphorylation could be a mechanism of regulation of fimbrin function, as demonstrated for L-fimbrin. The serine residues of the head-piece region of L-fimbrin are phosphorylated (Messier et al. 1993), although earlier, Namba et al. (1992) found unphosphorylated L-fimbrin of human T cells to be quite effective in F-actin bundling. Shinomiya et al. (1995) reported that LPS stimulated the phosphorylation of L-fimbrin in macrophages. Phosphorylation occurred on amino acid residues at the N-terminal region close to the first Ca2+-binding domain. Shinomiya et al. (1995) also noticed that the phosphorylated region contained motifs that are phosphorylated by CK II, protein kinase A (PKA), and PKC, but not motifs specific for MAPK. Frederick et al. (1996) also have recently implicated PKC in fimbrin phosphorylation and have confirmed further that only serine, not tyrosine, residue is phosphorylated. The possible linkage of fimbrin function with phosphorylation is indicated by the ability of cytokines and phorbol esters, among other agents, to induce fimbrin phosphorylation. Polymorphonuclear leukocytes (PMN) stimulated by IL-8, IL-1, neutrophil-activating proteins, monocyte-derived neutrophil chemotactic factor, and TNF have been reported to stimulate the phosphorylation of I-fimbrin. Phosphorylation was also influenced by phorbol 12-myristate 13-acetate (PMA) (Shiroo et al. 1988; Shibata et al. 1993a, 1993b). Fimbrin of T cells is phosphorylated in response to IL-2 (Zu et al. 1990). The adhesion of neutrophils to immune complexes induces L-fimbrin phosphorylation. Furthermore, it appears that phosphorylation-mediated regulation may be distinct from calcium-mediated regulation of fimbrin (Jones and Brown, 1996). It seems likely that the transduction of extracellular signals involves

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Calcium Signalling in Cancer

this important component of the membrane cytoskeleton. However, despite the clear demonstration of a fimbrin phosphorylation response to extracellular signals, there is little direct evidence that these features are functionally related. Messier et al. (1993) provide circumstantial evidence for this. They found phosphorylated fimbrin was mainly associated with the insoluble cytoskeleton. When this is read with their observation that the serine residues of the head piece were specifically phosphorylated, one might be justified in concluding that phosphorylation could regulate the process of actin-binding and bundling by fimbrin. Whether the isoforms have different functions in the regulation of cell morphology is a question that has been addressed, albeit superficially, by Arpin et al. (1994). In CV1 fibroblast-like cells, both T- and L-fimbrin cause change of cell shape and reorganisation of the actin stress fibres, and only L-fimbrin is associated with microfilaments. In epithelial cells such as LLC-PK1 cells, T-fimbrin remains associated with actin filaments of microvilli and produces shape changes in them. Lfimbrin has no effect on these structures. These observations might suggest functional differences between the isoforms. The invasion of Shigella flexneri, a bacterium that causes dysentery in humans, involves the formation of heavy actin polymerisation and actin bundling near the site of host cell contact with the bacterium. These bundles form protrusions with which the bacterium coalesces. Adam et al. (1995) experimentally overexpressed T- and L-fimbrin in HeLa cells and demonstrated that Tfimbrin might be preferentially recruited to the zone of bacterial entry.

IS FIMBRIN INVOLVED

IN

CANCER?

The potential of fimbrin to modulate cell behaviour is obviously quite considerable. There are references in the literature since the early 1980s that fimbrin expression increases with cell transformation, especially in fibroblast cells. However, little incisive investigation seems to have been carried out. More recent investigations have pursued the worthwhile aim of establishing a role for fimbrin in neoplastic transformation and tumour progression. The expression of L-fimbrin has been investigated in cell lines derived from carcinoma of the prostate. L-fimbrin has been found in many carcinoma cell lines, but not in normal epithelial cell lines derived from the prostate. L-fimbrin occurs at higher levels in prostate cancer tissue as compared with normal prostate tissue. Immunohistochemical staining has suggested that the increase of fimbrin occurs predominantly in the glandular epithelial cells of the carcinoma, whereas in normal prostate tissue, it is found mainly in the fibromuscular stroma. It would have been interesting to explore the fimbrin expression pattern of benign prostatic hyperplasia (BPH). Nonetheless, the differences shown to exist between normal prostatic epithelium and carcinomatous glandular epithelium are persuasive enough to accept that fimbrin expression may be related to the carcinomatous changes. Interestingly, fimbrin expression was also reported to be rapidly up-regulated in LNCaP cells by dihydrotestosterone and oestradiol (J.P. Zheng et al. 1997). This observation does not per se contribute much to the argument that fimbrin is involved in the transformation of normal epithelium to carcinoma. Testicular hormones are the major factors that regulate the growth and development of the prostate. It is

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The Structure of Contractile Proteins

possible that changes in hormonal milieu are responsible for the development of BPH and onward to the development of prostatic carcinoma. Androgen receptors (ARs), oestrogen receptors (ERs), and progesterone receptors (PgR) occur in BPH and in prostate cancer (Srinivasan et al.1995). Indeed, AR occurs in all histological types and all clinical stages of prostate gland cancer. Because androgens are involved in the development of the prostate itself, it would be reasonable to suppose that AR occurs in the developing prostate as well. Therefore, one would expect that the androgen-sensitive cell line LNCaP would respond by an altered level of fimbrin. What is crucially important is to find out whether normal epithelial cells respond differently from carcinomas. Testicular hormones bind to the appropriate receptors and stimulate the transcription of androgen-responsive genes, including those that regulate the growth of prostate cells. It should be borne in mind, further, that the progression of prostate cancer is associated with a change from androgen-dependent to androgen-independent state. The fimbrin gene may be a steroid-regulated gene, and it is important to determine whether this is the case, and whether androgen differentially stimulates fimbrin transcription in normal prostatic epithelium and prostatic carcinoma cells. Another isolated observation, which may, nonetheless, be significant in the context of drug resistance of tumours, is the relationship discovered by Hisano et al. (1996) between cisplatin resistance and fimbrin expression. They observed that cisplatin-resistant cells possessed severalfold greater levels of T-fimbrin compared with sensitive cells. They transfected T-fimbrin antisense cDNA into cisplatin-resistant cells and showed that reduction of fimbrin expression resulted in increased sensitivity to the drug.

MODULATION OF ACTIN DYNAMICS DISSEMINATION

AND

CANCER CELL

Conceptually there can be no difficulties in accepting the proposition that the modulation of actin dynamics should be involved in some way with tumour dissemination. A reasonable body of experimental evidence, in the form of changes in the expression of gelsolin, thymosins, and fimbrin in cancer progression, has been adduced in support of this concept. In this context of actin-interacting proteins, the vasodilator-stimulated phosphoprotein (VASP) deserves a mention. VASP is approximately 45 to 50 kDa in size and occurs in domains of the plasma membrane that are involved in the formation of lamellipodia (Reinhard et al. 1992, 1995b). Together with the Drosophila- and murine-enabled (Ena) proteins, VASP belongs to a family of actin-binding proteins that are actively engaged in the regulation of the actin cytoskeleton. VASP has been shown to interact with actin stress fibres via its Cterminal region (Huttelmaier et al. 1999). VASP as well as Ena can interact with the actin CP profilin (see below) (Reinhard et al. 1995a) and with focal adhesion proteins (Reinhard et al. 1995b, Brindle et al. 1996; Lanier et al. 1999; AhernDjamali et al. 1998). This suggests that the VASP family members might be involved in signal transduction (Huttelmaier et al. 1998). Besides, during in vitro morphogenesis of human umbilical endothelial cells, VASP, profilin, and gelsolin are markedly up-regulated (Salazar et al. 1999). In view of the close association of all three

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proteins in actin dynamics, Salazar et al. (1999) have postulated that reorganisation of the actin cytoskeleton is required for the alignment of the endothelial cells during capillary morphogenesis. The expression of profilin or VASP in neoplastic transformation has not been studied in great detail. NIH3T3 fibroblasts that have been made VASP deficient by experimental manipulation have been reported to be able to form tumours in nude mice. In vitro, these cells show loss of contact inhibition and a deregulation of cell proliferation (Liu K.Y. et al. 1999). With the demonstration that these proteins are associated with capillary morphogenesis, it would be of much interest to see whether VASP and/or profilin are down-regulated in neoplastic transformation. One can envisage that perturbation of the actin cytoskeleton could make the cell membrane malleable and the cell more deformable and in this way aid invasion. Mechanistic perturbations in endothelial cell alignment could generate abnormal fenestration in tumour-associated microvasculature that might serve as ports of entry into the vascular compartment for tumour dissemination. These observations highlight the potential importance of CBPs in cancer invasion, and also provide a fertile ground for extracting information that might be highly relevant in assessing the degree of malignancy of cancers and in cancer management.

α-ACTININ The actin cytoskeleton is regulated by several actin-binding and cross-linking proteins that are themselves regulated by free calcium. α-actinin is an EF-hand protein approximately 110 kDa in size and it forms a major component of the cytoskeleton in many cell types. It forms antiparallel, highly stable homodimers. Actinin isoforms can also form heterodimers, both in vivo and in vitro (Y.M. Chan et al. 1998). Y.M. Chan et al. (1998) suggest the possibility that heterodimers formed by different isoforms could potentially have new functional characteristics. The dimerisation of smooth muscle actinin is believed to be mediated by a segment of the C-terminal region of the molecule (Baron et al. 1987; Imamura et al. 1988; Kahana and Gratzer, 1991; A.P. Gilmore et al. 1994). This segment has two cross-linking sites (A and B), and the actinin molecules form cross-links in an antiparallel fashion by the binding of an A site of one molecule with the B site of the second molecule (Imamura et al. 1988). Several isoforms of α-actinin have been identified from vertebrate cytoskeletal, skeletal, and smooth muscle sources (Duhaiman and Bamburg, 1984; Imamura and Masaki, 1992; Landon et al. 1985; J.P. Bennett et al. 1984). Isoforms also have been isolated from invertebrate sources such as Dictyostelium discoideum (Noegel et al. 1987) and Drosophila melanogaster (Fyrberg et al. 1990). A cDNA has been cloned from the nematode Caenorhabditis elegans. The sequence of this clone predicts that it codes for an α-actinin, which possesses a high degree of sequence homology to actinin from other sources (Barstead et al. 1991).

MOLECULAR STRUCTURE

OF

α-ACTININ

α-actinin is an EF-hand calcium-binding protein. Two EF-hands are located at the Cterminal region of the molecule. An actin-binding domain occurs at the N-terminal

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end, followed by the rod domain, which consists of four spectrin-like repeat elements, followed by the EF-hand to the C-terminus (A. Blanchard et al. 1989; Flood et al. 1995). The rod domain is required for stable dimerisation of the molecule, and dimerisation is reduced markedly if either of terminal repeats 1 and 4 is deleted (Flood et al. 1995). Although all the isoforms possess EF-hands, these have been found to be in a functional state only in the nonmuscle or cytoskeletal isoforms of α-actinin (Burridge and Feramisco, 1981; Duhaiman and Bamburg, 1984). Their significance has been elucidated by introducing point mutations to make them nonfunctional. The first EF-hand seems to regulate the Ca2+-dependent cross-linking activity of α-actinin (Janssen et al. 1996). Nonmuscle isoforms might themselves differ in calcium-binding ability (Imamura et al. 1994). The EF-hands of α-actinin isoforms 2 and 3 of human skeletal muscle are not capable of binding calcium, and therefore the binding of these isoforms to actin might not be calcium sensitive (Beggs et al. 1992). Calcium sensitivity or the lack of it can be viewed from a functional viewpoint. The EF-hands appear to undergo conformational changes from a closed position in the absence of calcium, to an open position when calcium is present (Trave et al. 1995). The open conformation may be conducive to protein–protein interactions, which are required for the transduction of signals via the actin cytoskeleton.

α-ACTININ ISOFORMS Two skeletal isoforms of α-actinin have been designated as α-actinin-2 and -3 and two nonmuscle isoforms as α-actinin-1 and -4 (Millake et al. 1989; Beggs et al. 1992, 1994; Honda et al. 1998). The skeletal muscle isoform α-actinin-2 is found in both human skeletal and cardiac muscle, but α-actinin-3 occurs only in limb skeletal muscle (Beggs et al. 1992). The isoforms α-actinin-1 and -4 occur in the actin microfilament bundles and at the adherens junctions. In skeletal, cardiac, and smooth muscle, α-actinin is localised to the Z-discs and dense bodies, and it participates in the anchoring of the actin thin filaments and giant titin molecules to the Z-disc (Endo and Masaki 1984; Geiger et al. 1990). α-actinin has been shown to interact with an N-terminal domain of titin in vitro (Ohtsuka et al. 1997). In the Zdisc, the assembly of the actinin–titin complex seems to involve two types of interaction between titin and α-actinin. In the other regions of the Z-disc, titin binds by means of a single binding site with the outermost pair of α-actinin molecules, but in the middle of the Z-disc the titin binds several α-actinin molecules through binding sites at their C-terminal region (Young et al. 1998). Thus α-actinin seems to play a major part in anchoring actin thin filaments of the two halves of the sarcomere at the Z-disc (see Figure 15). Abnormal expression of α-actinin and other thin filament-associated proteins are known to interfere with the assembly of Zdiscs, which could lead to the formation of so-called nemaline bodies. Two loci are known to be involved in nemaline myopathy: the tropomyosin-3 locus and the nebulin locus on 2q21.2–q22. Mutations of both genes have been implicated in this form of myopathy (Laing et al. 1995; Pelin et al. 1999). The formation of the complex of tropomyosin, titin, nebulin, α-actinin, and actin can conceivably be affected by these mutations, leading to abnormalities in Z-disc assembly and the formation of nemaline bodies.

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FUNCTION

Calcium Signalling in Cancer OF

α-ACTININ

α-actinin functions predominantly in F-actin bundling and anchoring of the filaments to specific sites within the cell as well as to the cell membrane and in the linking up of the cytoskeletal machinery to the ECM. α-actinin might form a bridge between the actin cytoskeleton and integrin receptors then function as a receptor for components of the ECM (Burridge et al. 1990; Geiger et al. 1990). The cytoplasmic tail of βintegrins and intercellular adhesion molecule-I (ICAM-I) interact with α-actinin via binding sites occurring in the rod domain. Furthermore, α-actinin appears to link the cytoskeleton to the cell membrane (Baron et al. 1987). Kahana and Gratzer (1991) have identified binding sites for long-chain fatty acids in the spectrin-like repeats of the α-actinin domain rod. Han et al. (1997) recently have shown that bundling of actin filaments occurs if diacylglycerol is present in the membrane. It follows, therefore, that α-actinin would be an important component of the signal transduction pathway as well as an important link in the cytoskeleton-mediated changes in cell shape and motility. Miyamoto et al. (1995) have delineated the pathway of integrin-mediated signal transduction. They reported that integrin aggregation induced the accumulation of several signal transduction molecules, such as Rho A, Rac1, ras, raf, and others, of cytoskeletal components, such as vinculin, talin, and α-actinin. The tyrosine kinase inhibitor genistein inhibits accumulation of both the signal transduction molecules and the cytoskeletal signal transduction components.

ACTININS IN CELL ADHESION, MOTILITY, AND SIGNAL TRANSDUCTION The cross-linking of actin filaments increases filament elasticity and viscosity and may be expected to affect the structural properties of actin filaments. It follows from this that actinin might, in this fashion, change the properties of the cell membrane, such as intercellular and cell–substratum adhesion, which in turn would be reflected in alterations in cell shape and motility. These changes will have serious implications for cancer invasion and secondary spread. Furthermore, there are strong indications that actin cross-linking in endothelial cells might result in metabolite transport and permeability. Although yet to be demonstrated, one would expect that alterations in the organisation of adherens junctions might effectively alter endothelial integrity and lead to enhanced tumour cell diapedesis across the endothelium. Changes in the structural properties of the actin cytoskeleton would impugn the link-up between it and the ECM. Such changes would affect seriously the physical pathway of signal transduction, and effectively lead to its deregulation, and eventually to the deregulation of intercellular adhesion and communication and of cell proliferation and growth.

THE CADHERIN–CATENIN COMPLEX CELL ADHESION

IN

SIGNAL TRANSDUCTION

AND

A signal transduction complex that occurs in the cell membrane, consisting of the transmembrane glycoprotein cadherin and other components such as α-actinin, βcatenin, and the adenomatous polyposis coli (APC) protein, has assumed considerable significance by virtue of its apparent dual function, namely in signal transduction

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and cellular adhesion. Intercellular adhesion mediated by cadherin is dependent on the integrity of the link-up with the actin cytoskeleton (Sherbet and Lakshmi, 1997b) (Figure 13). β-catenin subserves two functions, viz. as a signal transduction molecule and in the formation of adherens-type junctions. Its participation in signal transduction has become apparent with the demonstration that plakoglobin and β-catenin are homologues of the armadillo protein of Drosophila that is involved in segment polarity. N-terminus

2+

Extracellular Ca

binding domains

Plasma membrane β-catenin α-catenin α-actinin Actin filaments

Adherens junction protein APC Protein

FIGURE 13 Schematic representation of the interaction of the transmembrane adhesion protein cadherin with the actin cytoskeleton, via β-catenin, α-catenin, and α-actinin. (Based on van Roy [1992] and Sherbet and Lakshmi [1997b]). Reprinted by permission of the publisher Academic Press, from The Genetics of Cancer, (Sherbet and Lakshmi, 1997b).

The armadillo (arm) protein is a component of the signal transduction pathway of the wg (wingless) molecule (Riggleman et al. 1990). The arm family of proteins, e.g., β-catenin, plakoglobin, and the p120ctn protein, are characterised by a central arm repeat domain (Riggleman et al. 1989). The p120ctn gene potentially can code for a large number of isoforms that are generated by alternative splicing, and these have been regarded as constituting a subfamily of signalling proteins (Keirsebilck et al. 1998b). Jou et al. (1995) have shown that β-catenin binds to a C-terminal 25 amino acid region in the cytoplasmic domain of E-cadherin and to the N-terminal domain of α-catenin. The p120ctn protein, on the other hand, seems to bind to a juxtamembrane domain of the cadherin cytoplasmic tail (Yap et al. 1998). The armadillo and wg proteins exert similar effects on embryonic development (Peifer et al. 1991). A family of wnt proteins has been identified. The wnt protein is a secreted glycoprotein signalling factor. It is a vertebrate homologue of wg and has been shown to be a regulator of morphogenesis of Xenopus (Gumbiner, 1996; Miller and Moon, 1996). In a similar vein, β-catenin and plakoglobin are components of the signal transduction pathway of the wnt gene as well as being involved in the formation of adherens junctions and Ca2+-mediated cell–cell adhesion (Hinck et al. 1994; Peifer, 1995). Cadherin, as a part of this signalling complex, not only regulates intercellular adhesion, but also seems to negatively regulate the signalling function of β-catenin. These two functions can be dissociated. Fagotto et al. (1996) noticed that β-catenin deletion mutants affect cadherin-mediated adhesion but not its signalling function. Sanson et al. (1996) showed that a full length E-cadherin and a

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truncated form of cadherin that have opposite effects on cadherin-dependent adhesion, nonetheless function effectively in wg signalling. Two further elements are involved in the signal transduction complex involving E-cadherin and catenins. One is the APC protein and the other component is axin or conductin. β-Catenin forms complexes with both axin and APC. The APC protein is known to compete with cadherin in its binding to the internal repeats of β-catenin. The β-catenin/APC complex is phosphorylated by the glycogen synthase kinase GSK3β. A consequence of this phosphorylation is that the degradation of β-catenin is greatly enhanced (Munemitsu et al. 1995; Hayashi et al. 1997; Hart et al. 1998). Axin also induces β-catenin degradation and is suggested to function downstream of APC (Behrens et al. 1998). The GSK3β-mediated phosphorylation is inhibited when the wnt signal transduction pathway is active. This results in the accumulation of β-catenin in the cytoplasm (Hinck et al. 1994; Giarre et al. 1998; Papkoff and Aikawa, 1998) and leads to the formation of a complex with the T-cell factor/lymphoid enhancer factor (Tcf/Lef). The binding between them occurs via the arm repeats. This complex functions as a transcription factor in the wnt/wg signalling pathway (Behrens et al. 1996; Van de Wetering et al. 1997). The pathway might be deregulated by inhibition of β-catenin degradation, leading to a constitutive activation of the transcription complex (Figure 14).

Axin/conductin Wnt/Wg

APC/β-catenin

APC-P/β-catenin-P

GSK3b

β-catenin Cell adhesion

β-catenin degradation

β-catenin/Tcf-Lef Transcription complex

FIGURE 14 The wnt signal transduction pathway and cell adhesion signal involving βcatenin, APC, and cadherin. (Based on references cited in the text.)

The deregulation that accompanies neoplastic changes seems to be more frequently due to mutation of β-catenin or APC protein than to inactivating mutations of E-cadherin (Morin et al. 1997; Efstathiou et al. 1999). These mutations seem to stabilise β-catenin. They occur at the phosphorylation sites that are essential in the ubiquitination and degradation of the protein. β-catenin mutations occur in approximately 50% of colorectal tumours. Again, these tend to occur in the serine/threonine phosphorylation sites. They seem to occur more frequently in small adenomas than

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in larger adenomatous lesions. Furthermore, a majority of the mutations have been found in adenomas rather than in carcinomas (Samowitz et al. 1999). A high rate (61% of 31 patients) of somatic mutation of β-catenin has been encountered in human anaplastic thyroid carcinoma (Garcia-Rostan et al. 1999). In a murine hepatocarcinoma model, mutations have been reported to occur in the carcinomas but not in adenomas. These findings suggest that deregulation of signal transduction occurs as an early event in the pathogenesis and progression of tumours. The βcatenin mutations in colorectal tumours reported by Samowitz et al. (1999) were not accompanied by APC mutations. The suggestion has been made, therefore, that mutations of APC and β-catenin might be functionally different. Mutations of APC leading to the loss of its suppressor function have been identified with the familial adenomatous polyposis, and are also closely related to the progression of the disease. Furthermore, mutated APC seems to lack the ability to regulate β-catenin levels. Therefore, APC may be regarded ipso facto as functioning upstream of β-catenin. The adhesion function of β-catenin flows from its linkage to cadherin, which spans across the cell membrane, on the one hand, and to the cytoskeleton, on the other, by means of two other elements, namely α-catenin and α-actinin. In fibroblasts, this linkage has been demonstrated by Knudsen et al. (1995), who also noted that α-actinin specifically immunoprecipitates with β-catenin and cadherin. Niesset et al. (1997) have identified the binding sites involved in the interaction of α-catenin with β-catenin on the one hand and with α-actinin on the other. In normal thyroid epithelial cells, the intercellular adhesion junctions show the presence of cadherin together with the catenins. In contrast, CGTHW-2 cells derived from thyroid carcinoma show a marked alteration in the pattern of distribution of these linking proteins in the foci of intercellular adhesion. Further evidence in support of the importance of the integrity of the cadherin–catenin complex in intercellular adhesion comes from the apparent involvement of the Rho GTPase family in the regulation of cadherin-mediated cell adhesion. IQGAP1, which is an effector of two members of the family, namely Cdc42 and Rad, has been shown to be able to dissociate α-catenin from the cadherin/β-catenin/α-catenin complex. This results in the disruption of intercellular adhesion. Both Cdc42 and Rad counteract this process (Kaibuchi et al. 1999). The inhibition of Rho and Rac (another member of the Rho GTPase family) has been reported to lead to a disruption of E-cadherin localisation at keratinocyte–keratinocyte cell junctions. However, when the GTPases themselves are inhibited, the cadherin-mediated adhesive contacts are reestablished (Braga et al. 1999). Rho protein, when microinjected into four-cell blastomere-stage embryos, disrupts cortical microfilaments and reduces interblastomere adhesion. Cdc42 in the same way also disrupts the cortical cytoskeleton and interblastomere contacts (Clayton et al. 1999). But the modes of involvement of Rho and Cdc42 are clearly distinguishable in NIH3T3 cells transformed by the dbl oncogene, which codes for a supposed exchange factor for RhoA and Cdc42. The transformed cells respond to adhesion to fibronectin substratum by changing cell shape. This involves the activation of RhoA and the associated ROCK and CRIK, but not Cdc42. In nontransformed cells, however, change of cell shape seems to require Cdc42 activation (Olivo et al. 2000). Rho GTPases may also be involved in the regulation of endothelial cell adhesion involving cadherins. Rho

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GTPases are also involved in the regulation of the function of other adhesionmediating glycoproteins such as CD44. Therefore, prima facie there is a reasonable basis for investigating their role in maintaining the integrity of the vascular endothelia and possible implications for metastatic spread of cancer. Other proteins that link the plasma membrane with the actin cytoskeleton may disrupt cadherin-mediated adhesion. The proteins ezrin, radixin, moesin, and merlin subserve such a linking function. Hiscox and Jiang (1999) used antisense nucleotides to inhibit ezrin expression in colon carcinoma cells and noticed that this resulted in a loss of intercellular adhesion and acquisition of motility. They further noticed that ezrin co-precipitated with E-cadherin and β-catenin. In cells treated with antisense ezrin nucleotides, this could result in a reduced interaction between ezrin and the cadherin complex. Such a reduction indeed occurs when ezrin is phosphorylated or when the cells are treated with hepatocyte growth factor. Huang et al. (1998) found no cadherin or γ-catenin at the adhesion junctions, and β- and α-catenins were distributed diffusely in the cytoplasm of most cells. However, β-catenin, when detected at intercellular junctions, was found to colocalise with α-actinin. Huang et al. (1998) have therefore suggested that the loss of intercellular adhesion could be due to an incorrect assembly of the linking components. Implicit also in this is the suggestion that such an incorrect assembly might lead to the acquisition of invasive ability. Although this is an attractive hypothesis, it should be mentioned here that Honda et al. (1998) have reported the occurrence of a novel actinin, namely actinin-4, whose expression is said to be upregulated with enhanced cellular migration. This new isoform is said to occur in the cytoplasm. It is reported to be associated with cytoplasmic extensions and is found in peripheral migrating cells of cell clusters. Actinin-4 appears to be translocated from the cytoplasm to the nucleus, when actin is depolymerised. Honda et al. (1998) have also examined the expression of this novel actinin in breast cancers. It is expressed in infiltrating breast carcinomas, and the expression was found to correlate with poor prognosis. Mamura and Masaki (1996) identified a 115-kDa α-actinin in vascular endothelial cells. This actinin differed from other known actinins of muscle or nonmuscle origin in its sensitivity to calcium. However, this isoform of actinin did occur in heart tissue, the pectoralis muscle, and the gizzard. What could be significant about this actinin in relation to cancer progression is its apparent location in the vascular endothelium. It is well known that several inflammatory agents induce a reversible change in endothelial cell shape that results in the formation of intercellular gaps. If cancer cells can reduce the endothelial integrity in this way, that could form ports of entry into the vascular system for cancer cells. There is a view that invading cancer cells take advantage of naturally occurring fenestration of the endothelium. There is no demonstration to date that they might secrete substances that could induce the formation of ports of entry. From this point of view it would be interesting to see if cancer cells induce any changes in the expression of actinins of the endothelial barrier. A new facet that can be added to the story of actinins in invasion and metastasis is their apparent relationship to the expression of metalloproteinases. It was shown some years ago that the production of these enzymes often paralleled the invasive

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and metastatic abilities of cancers, which has suggested the possibility that metalloproteinases might reconstruct the ECM and provide cancer cells with ECM properties conducive to invasion (Sherbet and Lakshmi, 1997b). Crawford et al. (1999) have made some interesting observations on the expression of matrilysin and βcatenin. They found that β-catenin and matrilysin mRNAs were expressed in parallel in murine intestinal adenomas. A further observation of considerable importance is that β-catenin significantly up-regulated the matrilysin promoter. Because β-catenin is a structural component of signalling pathway and intercellular adhesion machinery, these experiments bring together, in a unique fashion, transduction of an extracellular signal that can putatively remodel the ECM and change the invasive behaviour of cells. Other cell adhesion molecules such as the ICAM similarly require interaction with the actin cytoskeleton. A peptide containing a cytoplasmic sequence of ICAM2 has been shown to interact with α-actinin in vitro. Indeed, interaction occurs between several sites of α-actinin and ICAM-2. Besides, ICAM-2 co-localises with α-actinin (Heiska et al. 1996). The epithelial ICAM (Ep-ICAM), an adhesion molecule regarded as specific for epithelial cell adhesion, has a cytoplasmic domain that regulates the cellular adhesion function. It appears that Ep-ICAM achieves this via α-actinin, with which Ep-ICAM appears to interact directly by means of specific binding sites (Balzar et al. 1998). Shigella flexneri, which causes bacillary dysentery, is known to induce changes in the cytoskeleton of epithelial cells. These changes result in the production of membrane protrusions that engulf the bacterium. The bacterium secretes a protein called IipaA. The cytoskeletal reorganisation, which is essential for this process of bacterial invasion, involves the recruitment by IipaA of the linking protein vinculin as well as α-actinin (Van Nhieu et al. 1997). The effects of the loss of α-actinin on cell shape, aggregation, and motility have been studied in Dictyostelium. Rivero et al. (1996) generated mutants lacking αactinin as well as the “gelation factor” and these mutants presented a rounded shape rather than the typical polarised morphology of aggregating cells. The mutants also showed considerable loss of motility. In rat bladder carcinoma cells, the experimentally induced loss of motility was associated with the reorganisation of F-actin and α-actinin (Morton and Tchao, 1994). The transmembrane integrin α2β1 functions as the receptor for the ECM components collagen and laminin. This integrin as well as α-actinin have been implicated in the invasive ability of melanoma cells in vitro. L.M. Duncan et al. (1996) reported that α-actinin was not detectable in benign melanocytic naevi and in laterally spreading lesions, but it occurred in all nodular melanomas and in metastatic melanomas. It would have been of much interest whether there was an association between α-actinin expression and the vertical growth of melanoma. EGF is known to alter cell morphology as well as promote cell invasion in vitro (Shiozaki et al. 1995). These cellular responses seem to originate from the linkage of the activated EGFr to the cytoskeletal system (Roy et al. 1989, 1991; Van Bergen en Henegouwen et al. 1989). There is some recent evidence that EGFr regulates intercellular adhesion by interfering with the formation of a complex between the invasive suppressor transmembrane protein E-cadherin and the actin cytoskeletal

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elements, including α-actinin and β-catenin (Hazan and Norton, 1998; see also Figure 13). As alluded to earlier, the proliferative signal imparted to the cell by EGF is mediated by PKC. N.R. Murray et al. (1999) generated transgenic mice that express PKC-βII at high levels. The colonic epithelium showed high cell proliferation. Besides, the epithelium was more prone to preneoplastic changes. They found that these effects were also accompanied by increased expression of β-catenin and a reduction in glycogen synthase kinase 3β activity. These studies suggest, therefore, that EGFr-mediated signal follows the wnt/APC pathway of transduction leading to cell proliferation. This pathway also may be utilised in neoplastic changes of epithelia. Overall, these data now provide a physical basis for previous observations that high EGFr expression correlated with poor prognosis, and that EGFr status is a powerful marker of clinical aggressiveness and metastatic potential of tumours (Sherbet and Lakshmi, 1997b).

MYOSIN FILAMENTS Myosin is a filament protein found in association with actin in both muscle and nonmuscle cells. Like actin, myosin is a bipolar protein. It possesses a globular head region (subfragment S1), which interacts with actin, and a fibrous region, which promotes aggregation into filaments. The filaments consist of two myosin heavy chain (MHC) subunits and four myosin light chain (MLC) subunits. The class II skeletal myosin has two heads whereas other myosins have only one head. The N-terminal globular domain comprises the motor domain. The heavy chain subunit possesses the ATPase activity that provides the energy for the contractile force. The process of muscle contraction is driven by a cyclical interaction between actin and myosins. Myosins have been described as mechanoenzymes that catalyse the hydrolysis of ATP and release of energy, which is converted into mechanical force (Hasson and Moosekar, 1995). Energy is transduced by a series of interactions between myosin heads and hydrolytic changes of ATP. This is believed to deform the protein, and this change is transmitted to actin as a mechanical effect. The mechanical displacement and force generated in this way has been measured. The force generated by double-headed myosin is nearly twice as large as that produced by single-headed myosins (Tyska et al. 1999). In the skeletal muscle, the two heads of myosin interact when it is bound to filamentous actin (Rayment et al. 1993). The smooth muscle myosin heads interact in the presence of filament actin and in the absence of ATP (Onishi et al. 1992). Such head–head interaction also is required in the phosphorylation-dependent regulation of smooth muscle myosin (Matsuura and Ikebe, 1995). The processes of muscle contraction and cell motility are usually evaluated by an in vitro technique in which actin filaments are slid over myosin and measured using an electron microscope. Actin sliding as well as actin activation of myosin-ATPase is dependent on phosphorylation when myosin is dimerised, but not while myosin is in the monomeric form. Furthermore, the sliding velocity of the dimer is twice as large as that of the monomer (Sata et al. 1996).

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MYOSIN HEAVY CHAIN (MHC) ISOFORMS A large number of MHC isoforms are known to exist (Table 6). There are preliminary reports that the pattern of their expression is modulated in embryonic development and also in association with certain disease processes. For instance, thyroid hormone is reported to alter the pattern of MHC isoform expression in embryonic development (Maruyama et al. 1995). During metamorphosis of Xenopus tadpoles, thyroid hormone has been reported to down-regulate the expression of embryonic but upregulate adult isoforms (Sachs et al. 1997). Modulations in MHC isoform expression have been reported in the progression of human atherosclerosis (Aikawa et al. 1995). Similar changes have also been described in tumour-associated fibrosis and in neoplastic transformation of breast epithelium (Chiavegato et al. 1995).

TABLE 6 Myosin Heavy Chain (MHC) Isoforms MHC Isoform Myosin I slow MHC 1β, α Myosin II fast MHC II a, b, x MCH 3 II c MHC1, MCH 9 Myosin II SM1 (204 kDa); SM2 (200 kDa) Nonmuscle MIIA, B1, B2 Unconventional myosins V, VI Myosin VIIa

Tissue Skeletal, cardiac, foetal Skeletal Foetal Skeletal, immature fibres Smooth muscle Fibroblasts, endothelial cells, macrophages Intestinal brush border, Va in a variety of cell types Cochlea, retina, testes, lung, kidney

Source: Based on several sources including Kelley and Adelstein (1994); Heintzelman et al. (1994); A. Kimura et al. (1995); Chiavegato et al. (1996); Galler et al. (1997); Hasson et al. (1997); Wu et al. (1998).

The metastasis-associated S100A4 (mts1), which is itself a CBP, has been found to bind to the myosin rod in the light meromyosin region and could be destabilising the myosin filament. Besides, S100A4 binding has been found to inhibit the actinactivated ATPase of myosin (Ford et al. 1997). These interactions could conceivably alter the faculty of motility, which has been associated with cancer invasion, and this is especially associated with high expression of S100A4. It would be of interest to note here that the unconventional myosin V could be involved in the mechanisms associated with filopodial extension of neuronal growth cones. When myosin V is inactivated, cones of dorsal root ganglia show a rapid retraction of the filopodia (F.S. Wang et al. 1996). The distribution of the nonmuscle isoforms of myosin II (A and B) shows a pattern that is compatible with their involvement in cell locomotion. These myosins occur in association with lamellipodia and around the cell nucleus. However, myosin IIA shows a preferential localisation along the leading edge, but

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myosin IIB occurs along the trailing edge of cells in locomotion (Kolega, 1998). It is significant that, when injected into the cells, these isoforms show the same pattern of localisation as the endogenous variety. This has led to the suggestion that their localisation pattern is an intrinsic property of locomoting cells (Kolega, 1998). The apparent specificity of localisation may not be a consequence of their synthesis at specified sites, but could indicate that they are being targeted to those locations. HSPs are believed to behave like molecular chaperones for S100A4. HSPs might target S100A4 to the plasma membrane to be secreted into the ECM, and in this way endow the cells with invasive ability. Therefore, further effort into the possible mechanisms involved in the perceived specificity of myosin IIA and B localisation in the locomoting cell would be eminently worthwhile. Among other myosin-driven biological phenomena that may be cited here is cytokinesis in cell division. The myo2 gene of Schizosaccharomyces pombe encodes a protein called myo2, which shows a high degree of homology to myosin type II heavy chain. A contractile ring composed of actin and myo2 is formed before cytokinesis. Disruption of myo2 reportedly leads to an inhibition of cell proliferation (Kitayama et al. 1997). In Dictyostelium the formation of the contractile ring requires phosphorylation of myosin (Sabry et al. 1997). The unconventional myosin Va has been localised to the microtubule organising centre of interphase cells and to the mitotic apparatus in dividing cells. Using specific antibodies against myosin Va, Wu et al. (1998) have been able to demonstrate its association with microtubules in a number of cell types. The specificity of this association was obvious from experiments using cells that were derived from mice with null (dilute gene –/–) phenotype, in which no association with microtubules could be detected.

ACTOMYOSIN ASSEMBLY Myosin thick filaments are composed of two MHCs of 220 kDa and four MLCs of 20 kDa molecular size. The N-terminal end of MHC forms the globular head. The tail region of the filament is an α-helical coiled coil that constitutes part of the long rigid tail, of which the part proximal to the head region is composed of the Cterminal half of MHC and the light chains form the distal end of the myosin tail. The light chains are of two classes: a phosphorylatable form found in heart muscle and the CNS, but not in skeletal muscle, and a nonphosphorylatable form. Indeed, the N-terminal section of MHC together with two light chains, one of each class, form the globular head. MLCs bind Ca2+ with great affinity and regulate myosinATPase activity. The conformation of the myosin globular heads is regulated by the phosphorylation of the MLC by MLCK, which binds to actin via residues 2–42 of the N-terminal end of the kinase (Gallagher and Stull, 1997). This phosphorylation is believed to be a Ca/CaM-dependent process. Although reversible phosphorylation of MLC is generally regarded as the regulatory event, other proteins, e.g., caldesmon and calponin, which are capable of binding to the actin–myosin assembly, could subserve important functions. The skeletal and heart muscle cells possess a characteristic striated pattern. The striations are due to individual fibrils within the muscle cells. The striations are produced because the fibrils are arranged in register, which accentuates the striation.

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FIGURE 15 A sarcomere with its bands. The pattern is a result of the arrangement of actin, myosin, and titin. (Based on Gaub and Fernandez, 1998.)

The striations form three distinct zones: the dark and birefringent A-band with a lighter zone in the middle called the H-band, which in turn is divided by the Mline; the I-band, which is lighter than the A-band; and the Z-line, which is a dark line in the middle of the I-band (Figure 15). The contractile, regulatory, and structural components of these zones are summarised in Table 7. The A-band is composed of

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TABLE 7 Contractile, Regulatory, and Structural Component Proteins of Myofibrils of Vertebrate Skeletal Muscle

Protein Contractile Myosin Actin Regulatory elements Troponin Tropomyosin M-protein C-protein α-actinin Structural component Titin Nebulin

Location Band

A I I I M-line A Z-line A–I I

Content % wt.

Molecular Weight (kDa)

43 22

ca. 500 42

5 5 2 2 2 10 5

70

ca. 110 3000 600–800

Source: The data in this table have been collated from references cited in the text.

thick myosin filaments. These are arranged in a parallel array and in register and therefore they determine the length of the A-band. Thin actin microfilaments form the lighter I bands. However, the thick myosin filaments form bridges with the microfilaments, especially in the region of overlap of A and I bands. In transverse section of the muscle, the organisation of the filaments can be described as a lattice. Each thick myosin filament has six thin actin microfilaments and each actin filament has two neighbouring myosin filaments. In the thin filaments, tropomyosin occurs in a head-to-tail orientation in one groove of actin helix by means of seven actinbinding sites. It is believed that molecular elasticity is regulated by a species of giant rod-like proteins called titin or connectin. Human titin is a ca. 3000-kDa protein consisting of variable numbers of immunoglobulin (Ig)- and FN-like repeat domains and a P (proline), E (glutamate), V (valine), and K (lysine)-rich region of variable length. Titin overlaps the A-band and the I-band, extending from the Z-line to the M-line (see Figure 15). It is believed that the part of titin that overlaps the I-band forms the elastic region of the molecule (Linke et al. 1998). Alternative splicing of the I-band titin is said to result in changes in the component Ig and FN modules and in the length of the PEVK region (K. Wang et al. 1991; Labeit and Kolmerer, 1995a). Another large protein called nebulin, of approximately 600 to 800 kDa molecular size, spans the whole length of the thin filaments of the I-band. Indeed, nebulin is regarded as the determinant of the length of the thin filaments (Kruger et al. 1991; Jin and Wang, 1991; Labeit et al. 1991; Trinick, 1994). The assembly of mature Z-discs occurs from precursors called I-Z-I bodies, which contain titin,

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nebulin, actin, and α-actinin (Holtzer et al. 1997). Tropomyosin seems to be involved in nemaline myopathy. Two loci involved in this disease are the tropomyosin-3 locus and the nebulin locus on 2q21.2–q22 (Laing et al. 1995; Pelin et al. 1999). Mutations of both genes have been implicated. The formation of the complex of tropomyosin, titin, nebulin, α-actinin, and actin can conceivably be affected by these mutations, leading to abnormalities in Z-disc assembly and the formation of nemaline. The C-terminal region of nebulin contains an SH3 domain, which may anchor the nebulin filament system to the Z-disc (Labeit and Kolmerer, 1995b). This SH3 domain has been found to interact with actin, tropomyosin, troponin and calmodulin. This could suggest that nebulin, together with tropomyosin and troponin, might form a complex that is calcium-regulated (K. Wang et al. 1996). In cardiac muscle, a nebulin-related protein called nebulette substitutes for nebulin (Millevoi et al. 1998). Nebulette is similar to skeletal muscle nebulin in its organisation and contains four domains: an N-terminal domain followed by a domain that contains nebulin-like repeats, a linker domain connecting the nebulin repeats to the C-terminal SH3 domain. The nebulin repeats possess actin-binding ability and the linker zone appears to target the protein to the Z-line. This is suggested by the binding of recombinant nebulette fragments to actin, tropomyosin, and α-actinin in vitro (Moncman and Wang, 1999).

MYOSIN LIGHT CHAIN (MLC) PHOSPHORYLATION

AND

FUNCTION

As stated earlier, there are two classes of myosin light chain (MLC), viz. a nonphosphorylatable and a phosphorylatable regulatory chain. The reversible phosphorylation of the regulatory MLC is regarded as the key event in the regulation of myosin-ATPase and the generation of the contractile force. The phosphorylation of MLC is carried out by the MLCK. MLCK is a Ca2+/CaMdependent kinase (Stull et al. 1993) and is itself subject to regulation by kinases and phosphatases (Quadroni et al. 1998). MLCK is known to be able to bind to actin and thereby regulate actin–myosin interaction (Kohama et al. 1992; see also below). Naturally this will influence the function of the ATPase. On the other hand, binding of calponin and caldesmon to actin may inhibit myosin-ATPase and this inhibition may be reversed by calcium-binding S100 proteins and calmodulin (see Fattoum, 1997). Rho kinase also seems to be able to phosphorylate MLC, together with inactivation of myosin phosphatases (Fukata et al. 1999) (Figure 16). The regulation of the contractile machinery of the cell has obvious implications for cell motility and diapedesis of cells. The ability of activated PMN to traverse the endothelium appears to be related to the state of MLCs phosphorylation of endothelial cells. Hixenbaugh et al. (1997) found that activated PMNs increase phosphorylation of serine 19 and threonine 18 of MLCs of endothelial cells. Garcia et al. (1998) have confirmed the enhanced phosphorylation of MLC in endothelial cells and have further shown that inhibitors of MLCK significantly inhibit the diapedesis of activated PMNs across the endothelium. Phosphorylation of MLCK by protein kinase C also inhibits MLC phosphorylation and causes enhanced

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Muscle contraction

Caldesmon Calponin

Actomyosin Mg-ATPase

Myosin

Actin

S100/S100A4 Caltropin Ca 2+/Calmodulin

RMLC +P PKC

+P

MLCK

Ca2+/CaM +P Casein Kinase II

-P PPIA/PPIB

FIGURE 16 Control of regulatory myosin light chain (MLC) function in muscle contraction. CaM, calmodulin; MLCK, MLC kinase; PKC, protein kinase C; PP1A/PPiB: protein phosphatases; RMLC, regulatory MLC.

epithelial resistance (Angle et al. 1998). The association with actin and the assembly of myosin II into the cytoskeleton is dependent on the phosphorylation of the regulatory chain. MLC associated with the cytoskeleton of cultured endothelial cells has been reported to show a fivefold greater phosphorylation than MLC in soluble form. The phosphorylation of serine 19 and threonine 18 appears to regulate the assembly of MLC in vitro. In contradistinction, soluble MLC seems to be phosphorylated at threonine 9 (Kolega and Kumar, 1999). It would seem that the phosphorylation of the cytoskeletal machinery could increase the intercellular gaps thus allowing cell penetration. It would be worthwhile to investigate whether, like activated PMNs, cancer cells affect the state of MLC phosphorylation of endothelial cells. Unquestionably, vascular permeability is an important determinant of metastatic spread of cancer. However, invasive ability is an intrinsic property of the cancer cell. Gillespie et al. (1999) have recently shown that the MLCK inhibitors, ML7 and KT5926, inhibit the invasive ability of glioma cells. MLC phosphorylation in both the invading tumour cell and the endothelial cell barrier might determine the degree of successful entry of the tumour cell into the vascular compartment (Figure 17). Some studies have implicated MLC phosphorylation in the regulation of certain other biological features such as cell growth and apoptosis. Mills et al. (1998) have reported that membrane blebbing associated with apoptotic cell death could be reduced by MLCK inhibitors, and, further, that there was increased phosphorylation

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Diapedesis

MLC-P +P

Inhibitors

MLCK

+P

MLCK kinases (PKC)

+P MLC-P

䊲

Cancer Cell Invasion FIGURE 17 Possible involvement of MLC phosphorylation in the migration of cells across the endothelial membrane as well as with the intrinsic invasive potential of the tumour cell. MLC, myosin light chain; MLCK, MLC kinase; PKC, protein kinase C.

of MLCs in membrane blebbing. There are also suggestions based on preliminary findings that MLC phosphorylation may also be associated with cell proliferation (Bresnick et al. 1996; Yamakita et al. 1996). MLCK may affect actin reorganisation by a mechanism that is independent of its kinase activity. As stated earlier in this section, MLCK is known to be able to bind to actin and influence actin–myosin interaction. Another aspect of MLCK function is that it can bind to and result in actin bundling into filaments (Hayakawa et al. 1994). Two sites on MLCK have been identified and characterised; one of them is Ca2+/CaM sensitive and the other is Ca2+/CaM insensitive (Ye et al. 1997). These participate in actin filament assembly in vitro (Hayakawa et al. 1999). By inference from and in common with the properties of other actin-binding and bundling proteins, it would be reasonable to suggest that MLCK might influence cell shape and flexibility and thereby affect cell migration and diapedesis, quite independently of its kinase activity.

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TROPONINS AND TROPOMYOSINS IN THE REGULATION OF MUSCLE CELL CONTRACTION THE REGULATORY ROLE MUSCLE CONTRACTION

OF

TROPONINS

AND

TROPOMYOSINS

IN

Troponins are CBPs that play a major regulatory role in cardiac and skeletal muscle contraction in consort with tropomyosins. Three troponin (Tn) molecules — TnT, TnI, and TnC — bind to each tropomyosin molecule. Many tropomyosin isoforms with a range of molecular sizes have been identified (see below). The high molecular weight isoforms bind actin more strongly than the others (Matsumura et al. 1985). There is a complex and specific form of association between the troponins and tropomyosin that produces conformational changes in actin that lead to muscle relaxation or contraction. TnT binds to the C-terminal region of tropomyosin and forms a link between TnI and TnC, which both bind to tropomyosin. The structural domain C and regulatory domain N of tropomyosin interact with specific amino acid sequence motifs of TnI (Pearlstone et al. 1997). Calcium binding to the N domain of TnC can alter the affinity of the N- and C-termini of TnI for TnC. In other words, calcium binding can provide a switch between these two sites for binding with TnC (Tripet et al. 1997). At any rate, the whole complex interacts with actin via TnI. TnI is the myosin-ATPase inhibitory protein. TnC is the calcium binding subunit. In the absence of Ca2+ binding to TnC, the conformation of actin filament is such that its interaction with myosin heads is weak and the muscle fibres are in a relaxed state. The ATP that is bound to myosin is hydrolysed by a myosin-catalysed ATPase. In the absence of calcium, the troponin/tropomyosin complex inhibits myosin-ATPase. But when intracellular calcium levels are raised, Ca2+ binds to TnC and myosinATPase inhibition is released, ATP hydrolysis occurs and muscle contraction ensues. The contracted state is maintained when Ca2+ and ATP levels are maintained at high levels (Figure 18). The SR around myofibrils stores Ca2+ as well as the membraneATPase that is required for the release of Ca2+ and raises intracellular levels. There is therefore a homeostatic mechanism at work, maintaining a 10-7 M Ca2+ level in the resting muscle, and the increase of this by an order of magnitude produces the cascade of events leading to muscle contraction. Tropomyosin

Tnl

TnT

Muscle contraction

TnC

Ca

Actin

FIGURE 18

Myosin-ATPase

2+

conformational change

Cascade of events leading to muscle contraction upon Ca2+ binding to TnC.

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The calcium–TnC trigger leads to the contraction of cardiac and skeletal muscles. However, different mechanisms may be involved in the activation of myofilaments of the heart and skeletal muscles. In heart myofilaments, Ca2+ binds to one regulatory site on TnC but to two sites in skeletal myofilaments. Besides, there are also differences with regard to Ca2+-induced conformational changes between cardiac and skeletal muscle (Spyracopoulos et al. 1997). In the smooth muscle, i.e., involuntary muscle under CNS control and muscle tissue of invertebrates, there is no SR. In these, intracellular Ca2+ alterations tend to be slower than those found in striated muscle. Furthermore, Ca2+ regulation of contraction involves MLCs, which have a high affinity for Ca2+. At low calcium levels the interaction of myosin head with actin is weak, but when the light chains bind Ca2+, the myosin head–actin interaction activates myosin-ATPase leading to muscle contraction.

TROPOMYOSIN ISOFORMS

IN

BENIGN

AND

MALIGNANT CELLS

Tropomyosin isoforms are expressed in cells of muscle as well as nonmuscle origin. They are found in many organisms from unicellular ones to mammals (Pittenger et al. 1994). In human fibroblasts, eight isoforms have been identified and characterised. They are products of four genes (Novy et al. 1993a, 1993b). The isoforms of tropomyosin that have been identified range in molecular size from 32 to 40 kDa (J.J.C. Lin et al. 1985; Matsumura et al. 1983; Novy et al. 1993; Pittenger et al. 1994; Warren et al. 1985). The isoforms seem to differ with respect to the strength of their association with actin and may also differ in their function (Matsumura et al. 1985; Novy et al. 1993a; Gunning et al. 1997). The possible functional differences between the isoforms has been emphasised by the finding that they appear to be spatially sorted out or compartmentalised in neuronal development (Gunning et al. 1997). The localisation of tropomyosin isoforms shows a distinctive pattern in the kidney epithelial cells LLC-PK1. Temm-Grove et al. (1998) found that these cells expressed both high and low molecular weight isoforms of tropomyosin. The high molecular weight isoforms were associated with stress fibres but not with adhesion belts. In contrast, the low molecular weight isoforms were found on adhesion belts. These differences in localisation were also encountered when these isoforms were introduced into cells by microinjection or transfection. These observations support the view that the different isoforms might subserve different functions. The loss of cell shape, cell adhesion to the substratum, and the loss of stress fibres in cellular transformation has focused attention on the expression of tropomyosins in benign and malignant cells and tissues. The expression of tropomyosins has been reported to be frequently down-regulated in transformed cells (Cooper et al. 1985; Hendricks and Weintraub, 1981, 1984; Leavitt et al. 1986; J.J.C. Lin et al. 1985; Matsumura et al. 1983). In chick embryo fibroblasts transformed by RSV, a differential repression of tropomyosins has been reported to occur at the transcriptional level (Hendricks and Weintraub, 1984). The normal human fibroblast KD cells express four tropomyosin mRNAs. The expression of two of these of 1.1 and 3.0 kb was found to be markedly reduced in transformed cells and in a cell line derived from pancreatic cancer (Novy et al. 1993b). Leavitt et al. (1986) have reported alterations of tropomyosin isoforms that are expressed in human fibroblasts trans-

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formed by chemical carcinogens. In these cells, the high molecular weight tropomyosins appeared to be down-regulated. However, there was a slight down-regulation of one and an up-regulation of another high molecular weight isoform in nontumorigenic immortalised cells. A change in pattern of isoform expression has also been reported to occur upon cell transformation by viruses (Matsumura et al. 1982, 1983). Oncogenic retroviral genes indeed have been shown to suppress tropomyosin synthesis (H.L. Cooper et al. 1985). Furthermore, the suppression of the tropomyosins appears to have a bearing on cellular morphology. Prasad et al. (1999) transfected v-src-transformed cells, which express the TM1 isoform at very low levels, with TM1 cDNA. This resulted in higher levels of TM1 expression in the transfected cells, which also showed a reduction in cell growth and enhanced cell spreading. These effects also were accompanied by alterations in the architecture of microfilaments. Therefore, differential expression of the isoforms could be seen as suggesting differences in their cell function. A loss of high molecular weight tropomyosin isoforms and MLCs has been reported in tumorigenic cell lines as compared with nontumorigenic cells derived from prostate epithelium transformed by SV40 or X-irradiation (Prasad et al. 1997). However, TPA, which is a tumour promoter, is able to induce a high molecular weight isoform in human melanocytes (Vogt et al. 1997). Ovarian carcinomas reportedly have lower levels of a high molecular weight isoform of tropomyosin as compared with benign tumours (Alaiya et al. 1997). Franzen et al. (1996) found that the average levels of high molecular weight isoforms were far lower in breast carcinomas and nonmalignant tumours. The level of expression of one of these isoforms seemed to relate to the presence of tumour in the axillary lymph nodes, with the primary tumours showing 1.7-fold greater expression as compared with those that showed no nodal dissemination. The same high molecular weight isoform was found to be expressed at higher levels in H-ras-transformed fibroblast cell lines that possessed high metastatic ability than in transformed cells with lower metastatic ability. It may be pointed out that this implies that the more malignant the cell the greater is the level of expression of the high molecular weight isoform. This contradicts the relationship subsisting between the normal cell and its transformed counterpart, where transformed cells show a down-regulation of tropomyosin expression. Nonetheless, such a comparison might lead to oversimplified conclusions. It should be recognised that viral transformation of cells does not always lead to metastatic spread. The criterion of tumorigenicity does not contain any element of metastasis. For instance, tumours formed by SV40-transformed 3T3 fibroblasts rarely, if at all, metastasise. Furthermore, tumours are too heterogeneous and therefore association of metastatic ability with specific isoforms requires a detailed investigation of specific subpopulations of the tumour. Also desirable would be an investigation of primary tumours and the corresponding metastatic tumours. Hashimoto et al. (1996) compared K-1735 murine melanoma cells with differing metastatic potential. They reported that the β isoform of tropomyosin was expressed only in the low metastasis variant of K-1735 melanoma. The differential expression of the high and low molecular weight isoforms in adhesion belts and stress fibres (Temm-Grove et al. 1998) underwrites these efforts at exploring differential expression of tropomyosin isoforms in the progression of tumours to the metastatic state.

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121

Cell–cell and cell–substratum adhesion are important factors in tumour cell dissemination and their adhesion to metastatic target tissues. However significant might be the association between tropomyosins and neoplasia, it would be worth recalling here that TnI, which is the inhibitory component of the troponin/tropomyosin complex, might itself be associated with metastatic progression of cancer. As shown by Moses et al. (1999), TnI can inhibit angiogenesis both in vitro and in vivo and also inhibit metastatic dissemination.

THE REGULATORY ROLE

OF

CALDESMON

The cyclical interaction of the globular head domain myosin with thin actin filaments coupled with the breakdown of ATP drives muscle contraction. Although reversible phosphorylation of MLC is regarded as an important regulatory event, there is general recognition that not all the events in muscle contraction can be explained by invoking this mechanism alone. Additional mechanisms have been sought. Caldesmon and calponin are two major proteins, both capable of binding to the major components such as actin, myosin, tropomyosin, and phospholipids (see Fattoum, 1997; Childs et al. 1992; Szymanski and Tao, 1997; Bogatcheva and Gusev, 1995), and both have been seen as offering alternative modes of regulation. Furthermore, both proteins interact with calmodulin in the presence of Ca2+ (Medvedev et al. 1996; Zhang and Vogel, 1997; Graether et al. 1997). Whether caldesmon and calponin have individual roles in the regulation of smooth muscle contraction is being debated. Some argue that the two proteins interact with each other. Graceffa et al. (1996) showed that a strong binding occurred between calponin and the carboxyl domain of caldesmon. They have suggested that this interaction could lead to the stabilisation of the complex. However, Czurylo et al. (1997) subscribe to the view that these two proteins have independent roles in muscle contraction. Caldesmon is found in both smooth muscle and nonmuscle cells. It is a regulatory protein that occurs in the groove of the actin helix. Caldesmon and calponin inhibit actin-mediated activation of myosin-ATPase in smooth muscle fibres. Several segments of the C-terminal region of the caldesmon molecule may be involved in binding to actin and in its inhibitory function (Heubach et al. 1997). Whereas the C-terminal end binds to actin, the N-terminal end appears to be dissociated from but present in the close vicinity of actin and seems to mediate the link between myosin and actin filaments (Graceffa, 1995, 1997). In the C-terminal domain encompassing residues 663-763, three regions have been identified that take part in myosinATPase inhibition: a central segment encompassing residues 747–767, a segment that is N-terminal, and another segment C-terminal to this central segment. The central segment is essential but not sufficient to produce myosin-ATPase inhibition (I.D.C. Fraser et al. 1997). Two isoforms of caldesmon arising from alternative splicing of pre-mRNA are known. The high molecular weight (89–93 kDa) isoform is restricted to adult and fully differentiated smooth muscle cells, whereas the nonmuscle low molecular weight isoform is found in de-differentiated nonmuscle cells. There are several conserved domains in the caldesmon molecule, which determine its interaction with and binding to actin, calmodulin, myosin, and phospholipids. All the isoforms are

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powerful inhibitors of myosin-ATPase, but the specific nature of their distribution suggests possible differences in function. In nonmuscle cells, caldesmon could be stabilising the microfilament network and in smooth muscle, it may be involved in the inhibition of muscle contraction (see Huber, 1997). The function of caldesmon in the regulation of cytoskeletal organisation and contractile processes is not limited only to animal cells. A caldesmon-like protein occurs in higher plants. A 107-kDa protein with properties similar and immunologically related to caldesmon has been isolated from extracts of pollen tubes of Ornithogalum virens. This protein binds to actin in a CaM/Ca2+-dependent manner (Krauze et al. 1998). This is consistent with the view that actomyosin cytoskeleton participates in the growth and elongation of pollen tubes. Pollen tube elongation is a calcium-dependent process (Masserli and Robinson, 1997). Caldesmon also can bind to S100 α and β proteins. The S100 binding site appears to be in the C-terminal region. Interestingly, the cross-linking of caldesmon via cysteine residues with S100 seems to reduce the inhibitory effect of caldesmon on myosin-ATPase activity, and S100 seems to be as effective as calmodulin in this respect (Polyakov et al. 1998). There are no implications, as yet, that the different isoforms of caldesmon differ functionally, because there is extensive sequence homology between isoforms occurring in different species (Novy et al. 1991). However, as stated above, the expression of some isoforms has been related to the state of differentiation. Furthermore, early studies by Novy et al. (1991) have shown a two- to four-fold reduction in the expression of caldesmon in certain transformed cell lines.

CALPONIN: ITS FUNCTION

AND

REGULATION

Calponin is a 33-kDa protein highly expressed in smooth muscle cells. The human smooth muscle calponin gene contains seven exons encompassing >11 kb and it has been assigned to chromosome 19p13.2 (Miano et al. 1997). Calponin is a thin filament-associated TnT-like protein. It is strongly implicated in smooth muscle contraction. As with caldesmon, calponin binds to actin, myosin, tropomyosin, and CBPs such as calmodulin. Actin possesses two calponin-binding sites (Kolakowski et al. 1997; Mino et al. 1998). The interaction between calponin and myosin occurs via defined regions of the calponin molecule (Szymanski and Tao, 1997). Three isoforms of calponin have been described: the basic h1 isoform, the neutral isoform h2, and the acidic isoform. Two distinct genes encode the isoforms h1 and h2. They are not generated by alternative splicing of pre-mRNA (Fukui et al. 1997). All three isoforms contain the calponin-homology (CH) actinbinding domain in the N-terminal region of the protein. The CH domain comprises a sequence motif containing approximately 100 amino acid residues. Some actincross-linking proteins may contain two distinct CH domains and have contested the rather generalised concept that a single CH domain is sufficient to confer actinbinding properties on these proteins (Stradal et al. 1998). CH domains also occur in other actin-interacting proteins. Fimbrin is an actin-cross-linking protein. Its association with F-actin is similar to that of calponin (Hanein et al. 1997b) and, furthermore, it contains two CH domains. The proto-oncogene vav, which is

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123

expressed in haematopoietic cells, codes for a signalling molecule that contains a CH domain (Castresana and Saraste, 1995; Romero and Fischer, 1996). Nonmuscle isoforms of calponin have been reported in brain tissue. The h2 isoform was identified by Fukui et al. (1997) from human skin tissue and human keratinocytes in culture using antibodies raised against smooth muscle calponin. Calponin generally has been attributed with the ability to inhibit myosin-ATPase. In vivo, calponin occurs in the ratio of approximately one calponin molecule to seven actin monomers (Lehman and Kaminer, 1984; Takahashi et al. 1986; Lehman, 1991), but there is a view that higher levels of calponin may be required for an effective inhibition of myosin-ATPase (Gimona and Small, 1996). Exogenous calponin has been shown to inhibit Ca2+-responsive muscle contraction (Horowitz et al. 1996; Naka et al. 1998). Calponin serves as a substrate for several kinases and phosphatases, and its function seems to be regulated by phosphorylation. Pohl et al. (1997) have shown that unphosphorylated calponin is able to inhibit actin sliding, which is demonstrable using in vitro motility assays, but when calponin is phosphorylated by PKC this effect is inhibited. In vivo, calponin purified from tracheal smooth muscle stimulated with carbachol (carbamyl choline chloride), which is a Ca2+-mediated stimulator of phosphorylation, showed two-fold greater phosphorylation than unstimulated muscle. Naka et al. (1998) also demonstrated a correlation between calponin phosphorylation and contraction of porcine coronary artery. However, as discussed below mechanisms other than phosphorylation have been adduced for calponin regulation, e.g., one involving caltropin. Aside from being regulated by phosphorylation, it has been reported that calponin co-precipitates with MAPK, but there is no obvious phosphorylation of calponin. The latter also co-precipitated with PKC-ε. A translocation of calponin to the cell membrane occurs in cells that are stimulated with phenylephrine, when MAPK and PKC-ε are also translocated to the cell membrane. On the basis of these findings, Menice et al. (1997) have implicated calponin in signal transduction. Indeed, the targeting of these various components to the cell membrane might be a consequence of the tight binding of calponin to cytoplasmic β-actin (Parker et al. 1998). Calponin seems also to be able to regulate the amount of free actin available for cytoskeletal organisation. The h2 isoform, for instance, appears to be localised in the cytoplasm of basal cells in situ, whereas in cultured keratinocytes it occurs along the cell–cell contact areas, which suggests that it might play a part in the organisation of the cytoskeleton. Tang et al. (1997) believe that calponin, being polycationic in nature, could promote the formation of F-actin bundles by reducing the polyanionic repulsive interaction between actin filaments. Calponin can also bind to tubulin, which decreases with increases in Ca2+ concentration and ionic strength (Fujii et al. 1997). These authors also state that calponin has distinct sites for binding actin and tubulin. Furthermore, calponin seems to bind strongly to desmin and is incorporated into IF and, therefore, it is possible that it might mediate the association of desmin IF with actin (Mabuchi et al. 1997). The ability of calponin to promote cytoskeletal organisation may be reflected in its ability also to inhibit cell growth (Z. Jiang et al. 1997). It also has been reported recently that the expression of calponin and smooth muscle MHC expression can distinguish between in situ and invasive carcinoma of the breast. Antibodies against

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Calcium Signalling in Cancer

MHC and calponin stain epithelial cells of ducts and acini of normal breast tissue and carcinomas in situ, but these antibodies hardly show any reactivity in invasive carcinomas (Wang et al. 1997). Calponin is prominently expressed in myofibrils of leiomyosarcomas (Meyer and Brinck, 1997). Overall, calponin seems to show a generally reduced expression in human leiomyosarcomas. Compatible with the lack of its expression in invasive breast carcinomas as compared with in situ tumours are the recent findings of Horiuchi et al. (1999). These authors transfected human calponin h1 into leiomyosarcoma cells. The transfectants showed a one third reduction in cell proliferation, together with reduction in anchorage-independent growth as well as tumorigenicity. Nonetheless, much further work is required before one can attribute a tumour suppressor function to calponin.

CALTROPIN-MEDIATED REVERSAL OF MYOSIN-ATPASE INHIBITION BY CALDESMON AND CALPONIN It is obvious from the above discussion that caldesmon and calponin both inhibit myosin-ATPase and influence muscle contraction. The binding of caldesmon and calponin to actin appears to produce this inhibition and this effect is reversible by calmodulin. Another CBP, i.e., caltropin, appears to be involved in the regulation of the inhibitory effects of caldesmon and calponin on myosin-ATPase. Caltropin is an 11-kDa protein derived from smooth muscle. It occurs as a dimer in the native form (Willis et al. 1994). Some years ago, Mani et al. (1992) demonstrated that caltropin, like CaM, was able to reverse the inhibition of myosin-ATPase by caldesmon in the presence of Ca2+. They found that caltropin directly interacted with caldesmon. This seemed to influence the interaction of caldesmon with the actin component of heavy meromyosin (Mani and Kay, 1993, 1995a). In vitro, caltropin seems to inhibit the polymerisation of G-actin (Mani and Kay, 1995b), thus providing further confirmation of interference by caltropin in caldesmon–actin interactions. Although Mani et al. (1992) state that caltropin is as effective as CaM in reversing caldesmon-mediated inhibition of myosin-ATPase, there is evidence that the binding affinity of caltropin to caldesmon is far greater than that of CaM (Zhuang et al. 1995). In consonance with these observations are the findings of Willis et al. (1994) that caltropin also is able to form a complex with calponin in a calcium-dependent fashion in the ratio of 2:1 and reverse the inhibitory action of caldesmon. Caltropin has been described as being more efficient than CaM. Thus there seems to be a reasonable body of evidence, albeit from the same laboratory, that caltropin has considerable implications in the regulation of myosin-ATPase function.

8

Structure and Biology of Calbindin

Calbindins D-28K (CBD) and D-9K (CBD-9K) are EF-hand proteins that are regarded as calcium buffer proteins together with parvalbumins (PV). CBD is a type III EF-hand protein possessing six EF-hands (see Table 4), all of which seem to be packed in one globular domain, but the folding and packing of individual domains within the globular domain appears to be specific (Linse et al. 1997). The rat CBD9K gene consists of three exons and two introns. Exon 2 contains sequences encoding the first EF-hand and exon 3 for the second EF-hand domain.

CALBINDIN IN NEURONAL POPULATIONS CBD is a calcium-binding protein that is expressed abundantly in a number of cell types, such as neuronal cells, mammalian and avian kidney, brain, and pancreas, and in avian intestines. Wasserman and Taylor (1966) isolated this calcium-binding EFhand protein from the intestine of chicken later found to be a component of the nervous system of a wide variety of species (Celio, 1990; Reifergerste et al. 1993). The occurrence of CBD is predominent in long-axon as well as in short-axon neurones (see Baimbridge et al. 1992; Celio 1990; Frantz and Tobin, 1994; Sequier et al. 1990). By virtue of its calcium buffering ability as well as its ability to modulate calcium channel activity, CBD could be regulating calcium homeostasis in neurones (Albritton et al. 1992). CBD also occurs in the neuroendocrine cells of adult and foetal lung of hamsters. This has been demonstrated by immunochemical staining together with neuroendocrine markers. In contrast, other CBPs, such as PV and calretinin, are not detectable (T. Ito et al. 1998). Calretinin is a protein that bears a high degree of homology with CBD and shows similar calcium-binding properties (Cheung et al. 1993). The occurrence of CBD and calretinin are often mutually exclusive, although many cells may express both proteins at the same time (Rogers and Resibois, 1992). In light of the sequence similarities, it may be suggested by inference that these proteins subserve similar functions and could define similar neuronal properties.

NEURAL CELL LINEAGE AND THE REGULATION OF CALBINDIN EXPRESSION It has been argued that clonally related cortical neurones do not express the same complement of CBPs, and therefore their expression may be controlled and influenced by extracellular factors rather than be genetically endowed. Pappas and Parnavelas (1997) exposed cultures of developing brain cortex from rat embryos to bFGF and the neurotropins NGF, brain-derived neurotropic factor (BDNF), and 125

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neurotropin-3 (NT3). They found an enhanced morphological differentiation of large GABA-containing neurones together with the expression of calbindin, the effects being dependent on age and maturation of the subpopulations. As stated before, calbindin is highly expressed particularly in Purkinje cells of the cerebellum, but it also occurs in other neuronal cell types in other regions of the CNS. The specific expression in Purkinje cells seems to be regulated by a 40-bp element, occurring in the promoter region of the calbindin gene. This regulatory element called PCE1 is present also in the promoter of calmodulin II and, obviously, can regulate the expression of other CBPs as well (Arnold and Heintz, 1997). Cell type-specific transcriptional regulation mediated by CBPs may underlie this phenomenon, and it follows from this that different extraneous factors might differentially affect the expression of these proteins depending on the neuronal subpopulation. These CBPs in turn may regulate the expression of transcription factors that induce the expression of other responsive genes. This could rationalise the presence of calbindin in a wide variety of tissues and their responses to a variety of extracellular stimuli. It should be recognised, nevertheless, that other mechanisms such as enhanced stability of transcripts of target genes might also be involved.

CALBINDIN EXPRESSION IN EMBRYONIC DEVELOPMENT AND AGEING Calbindin expression changes during embryonic development to develop into an adult expression pattern. In the cerebellum, calbindin gene transcripts as well as the protein increase to reach a peak at 2 weeks postnatal stage (Iacopino et al. 1990). CBD is transiently expressed during embryonic development (Andressen et al. 1993). In mouse embryos, cerebellar Purkinje cells appear from 11 to 13 days of gestation (Miale and Sidman, 1961), and soon after, they begin to express CBD (Enderlin et al. 1987). CBD is the major component cellular protein of mature Purkinje cells, and the high protein levels maintained in adult life decrease in ageing animals (Baimbridge et al. 1982; Lledo et al. 1992). In developing human cerebellum, CBD immunoreactivity is first encountered at 14 weeks of gestation, with CBD expression being detectable in a variety of cell types. However, the number of CBD staining cells and the intensity of staining decreases with development. At 21 to 31 weeks of gestation, CBD reactivity is restricted to Purkinje and basket cells of the cerebellar cortex (Yew et al. 1997). A developmental pattern is also discernible in the appearance of CBD in neuroendocrine cells of foetal hamster lung from days 15 and 16 of gestation, but endocrine cells appear on day 13 (Ito et al. 1998). In the hamster brain, CBD transcripts have been reported to decrease markedly (50 to 68%) in 19 to 24 month-old hamster as compared with 4- and 9-month-old animals, but, in contrast, calretinin and PV do not show any changes with ageing (Kishimoto et al. 1998). Calbindin immunoreactivity changes in the cerebellum of frog tadpoles induced to metamorphose by treatment with thyroxin (Uray et al. 1998). The potential role of calbindin in cell differentiation has not received much attention to date. Together with PV, calbindin has been shown to be associated with phenotypic conversion of supporting cells into hair cells (Steyger et al. 1997). However, the extent of their individual contribution to the nonmitotic regeneration

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of hair cells is unclear at present. At any rate, the information available is scanty, when compared with the massive evidence available about the effects of S100 proteins on cell growth and differentiation.

PHYSIOLOGICAL FUNCTION OF CALBINDIN Calbindin expression is modulated in a variety of tissues by several extracellular signals and in an apparently tissue-specific manner. There are strong pointers that calbindin may have several functions in normal cellular physiology and that its expression in these tissues may be regulated by specific extracellular ligands by different mechanisms resulting in specific outcomes. Therefore, calbindins could perform tissue-specific functions (Table 8). Among the functions attributed to these CBPs are facilitation of calcium flux, protection of neurones from degeneration, and as a calcium buffer in restricting calcium signals in nerve synapses and hair cells. Neuronal firing patterns may be determined by calbindin (Chard et al. 1995), and in hippocampal slices it can modify synaptic interactions (Heizmann and Braun, 1992).

TABLE 8 Extracellular Biological Signals Influencing Expression of Calbindins Extracellular Signal

Target Tissue

Comments

1,25-dihydroxy vitamin D3 Oestrogen

Intestine, kidney

Not brain tissue

Kidney

Independently of vitamin D3

Adrenal steroids (corticosterone) Growth hormone

Hypothalamic tissue Intestine

During prenatal development

Butyrate

Rat insulinoma cell line

Calbindin D-9K mRNA, also induced in parallel, mRNAs for vitamin D3 receptor and insulin-like growth factor (IGF)-1 Parallel production of insulin and its secretion

Ref. Christakos et al. (1989) Criddle et al. (1997) Lephart et al. (1997a) Salih et al. (1997)

S. Lee et al. (1994)

The regulation of calbindin expression by VD3 in the kidney and the intestine has been well documented. In contrast, VD3 does not affect calbindin expression in brain tissue. Absorption of calcium and lead in the intestines is greatly affected by serum VD3. Conversely, serum levels of VD3 increase markedly with lead ingestion and calcium deficiency. Also apparent, under these conditions, are changes in the expression of calbindin, which suggests a possible mediation of calcium and lead absorption by calbindin (Fullmer, 1997). In Madin–Darby bovine kidney (MDBK) cells, TPA has been found to enhance CBD expression, and this is preceded by the activation and translocation of PKC-α. Furthermore, PKC phosphorylates CBD in

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a calcium- and phospholipid-dependent manner. VD3 also increases CBD expression as well as up-regulates the expression of PKC. PKC-mediated phosphorylation of two threonine residues of CBD could lead to the functional activation of CBD, which is consistent with the transduction of calcium signal by PKC mediation (Gagnon and Welsh, 1997). Calbindin seems to be involved with insulin secretion by pancreatic β cells. Lee et al. (1994) found that butyrate is able to induce CBD mRNA as well as protein levels in the rat insulinoma cell line RIN 1046-38. This treatment also enhanced insulin production and its secretion. The involvement of CBD in insulin secretion has been confirmed in further experiments by Reddy et al. (1997), who transfected the insulinoma cell line with the CBD gene. In the transfected cells a >20-fold increase in CBD mRNA was found, and at the same time insulin mRNA showed a >20-fold enhancement. Insulin production and release also increased approximately six-fold. It seems possible that this effect of CBD is also mediated by PKC, because activation of PKC can influence insulin secretion. Both CBD and CBD-9K appear to be capable of regulating genetic transcription (Reddy et al. 1997; Fukushima et al. 1998). Possibly, calbindin influences the process of calcium-mediated regulation of transcription factors (see Reddy et al. 1997). It is to be expected that the expression of calbindins themselves would be subject to regulation at the transcriptional level. That the specificity of their expression might be linked with definable transcription pathways is an interesting thought that has been explored experimentally. Arnold and Heintz (1997) have identified a 40-bp element, the PCE1, that is necessary for the expression of the CBD in Purkinje cells. This element seems to regulate transcription of CBD gene. PCE1 also occurs in calmodulin II. Both CBD and calmodulin II are expressed abundantly in Purkinje cells, and PCE1 might be a common component in the regulation of expression of these CBPs in Purkinje cells. CBD and calmodulin II are expressed in other cell types too. Transcription elements other than PCE1 may be involved in the regulation of the genes expression at these sites (Arnold and Heintz, 1997). Oestrogen-mediated induction of CBD expression is attributed to two regions (–1075/–702 and –175/–78) of the mouse CBD promoter (Gill and Christakos, 1995). Similarly, oestrogen can regulate specific regulatory sequences of CBD-9K (Romagnolo et al. 1996). The levels of expression of CBD may be regulated by other means, as indicated by Wang and Christakos (1995), who found that retinoic acid (RA) increased, by 10- to 15fold, both CBD protein and CBD mRNA in the medulloblastoma cell line D283. The RA effect was mediated by RA-specific receptors, which ordinarily would have indicated the regulation of transcription of the target gene by RA (see Redfern, 1997). However, the regulation in this case did not appear to occur at the transcriptional level, but the increased expression of CBD and its mRNA seemed to be due to an increase in the stability of the mRNA. CBD seems to play a role in renal reabsorption of calcium. Ovariectomy reduces the expression of CBD gene transcripts, and this is reversed by the administration of 17β-oestradiol in the ovariectomised animals (Criddle et al. 1997). These alterations in CBD expression show no relation to serum levels of VD3. Thus the role played by CBD in renal absorption may be distinguished from that in intestinal tissue.

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NEUROPROTECTIVE FUNCTION OF CALBINDIN CBD is generally regarded, in common with PV and calretinin, as an intracellular calcium buffer because it has not been found to influence enzyme activity or calcium ion channels (see Baimbridge et al. 1992). Nevertheless, buffer function may modulate intracellular calcium levels and in this way regulate neuronal function. Thus, distinct physiological functions may be identified that are affected by alterations in the intracellular levels as well as the conformational state of CBD. For instance, calretinin deficiency seems to lead to an impairment of motor coordination and marked abnormalities in the firing behaviour of Purkinje cells in vivo. In calretinin null (cr –/–) Purkinje cells, CBD is detected by anticalretinin antisera. This has been attributed to the fact that these antisera detect the presence of CBD that has undergone conformational changes upon being saturated with Ca2+ (Schiffmann et al. 1999). These events clearly implicate an impairment of calcium homeostasis in the cr (–/–) cells in vivo. The pattern of CBD occurrence in ageing neurones has led to the postulate that it may function as a neuroprotective agent. Lally et al. (1997) found that the size and number of CBD-immunoreactive neurones were reduced in Alzheimer’s disease, and this has been suggested to be a consequence of cellular degeneration related to the reduced CBD. Alzheimer’s nerve cells containing CBD are believed to be less susceptible to degeneration than those that have greatly reduced amounts of CBD or no CBD at all. This is supported by experiments with PC12 cells into which CBD cDNA was transfected. These cells were far less susceptible to degeneration caused by serum withdrawal, glutamate, and the neurotoxin 1-methyl-4-phenylpyridinium. However, it would appear that CBD cannot protect these cells from degeneration caused by calcium ionophores (McMahon et al. 1998). Calbindin-null mutant mice show severe impairment of motor coordination (Airaksinen et al. 1997). However, these null mutants do develop normally without any disturbances in calcium metabolism and, therefore, Airaksinen et al. (1997) believe that CBD expression in peripheral organs may not be crucial for normal development.

CALBINDIN EXPRESSION AND THE METASTATIC PHENOTYPE The ability of steroids to influence the expression of calbindins has led to investigations aimed at examining their potential involvement in the development and growth of tumours, their progression, and prognosis. Watanabe et al. (1994) investigated CBD levels of lung cancers. The levels of CBD were low in normal lung tissue, but its expression was higher in lung cancers, with SCLC tissue expressing greater amounts than non-SCLC (NSCLC). Furthermore, CBD levels correlated with progression of NSCLC to advanced stages of the disease involving metastatic involvement of the lymph nodes. Upon investigating cell lines derived from these tumours, Watanabe et al. (1994) noticed that CBD expression might also relate to the expression of neuroendocrine-related paraneoplastic properties of these tumours. They found that the CBD could be used to differentiate classical SCLC from variant

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SCLC. The specificity and sensitivity with which this could be achieved was comparable to the neuroendocrine marker neurone-specific enolase. Significant differences also have been noted in the expression of CBD in human colonic cell lines derived from primary tumours and those derived from metastatic tumours. Sampson-Johannes et al. (1996) found that SW480 cells obtained from primary colorectal tumours did not express CBD, but in sharp contrast, SW620 cells derived from metastatic tumours did. They also demonstrated that SW480 cells were not capable of colonising human foetal lung tissue transplanted into SCID-hu mice, but SW620 cells colonised the grafts upon intravenous introduction into the animals. Furthermore, SW620 cells expressed sialyl Lewis-x and Lewis-a antigens which are conducive to metastatic spread at far higher levels than SW480, which were unable to colonise the foetal lung grafts. On the basis of these results, the authors suggest that the appearance of the metastatic ability could be associated with the acquisition of CBD expression. It ought to be recognised, however, that this experimental model is too remote from the spontaneous metastasis. The differences in the abilities of the two cell types to localise in foetal lung grafts may simply reflect their adhesive capabilities, rather than CBD expression. Some preliminary work has been reported (Castro et al. 1998) that relates the value of CBD expression to prognosis of lung cancer.

9

Calretinin: Its Role in Cell Differentiation and as a Potential Tumour Marker

CALRETININ AND ITS ALTERNATIVELY SPLICED ISOFORMS Calretinin is a neuronal calcium-binding protein. It shows marked homology with CBD, and presumably on account of this, calretinin and CBD show mutually exclusive expression in neuronal populations, although in some cell types both may be expressed simultaneously (Rogers and Resibois, 1992). The pattern of their expression and marked similarities between them with respect to calcium-binding properties (Cheung et al. 1993) could indicate that they perform similar functions in neurones. Thus, calretinin seems to share with CBD certain features such as regulation of expression by growth factors and involvement in cell proliferation, differentiation, and neoplastic transformation. Also, calretinin may possess, in common with CBD, a neuroprotective property. On the other hand, the apparent mutually exclusive nature of their expression might be a reflection of the cooperative effects between calretinin and CBD. The calcium-buffering function of calretinin might result in CBD not being detected by specific antisera. As stated in the previous section, in cr (–/–) Purkinje cells, there is an impairment of calcium homeostasis. As a result, CBD is oversaturated with calcium. This brings about changes in the conformational state of CBD. As a consequence, CBD becomes detectable by specific antisera (Schiffmann et al. 1999). These conformational changes seem to translate into alterations in cell function. Therefore, the experiments of Schiffmann et al. (1999) might suggest a cooperation between calretinin and CBD in function. Calretinin is expressed as two splice variant isoforms, encoding two proteins of 20 and 22 kDa molecular size, truncated at the C-terminal region. The 20-kDa isoform is a result of splicing of exon 7 to exon 9, and the 22-kDa isoform results from splicing of exon 7 to exon 10. Both alternatively spliced messages show frame shift and contain stop codons in exon 9 (in calretinin-20K) and exon 10 (in calretinin22K) (Schwaller et al. 1995). Calretinin-22K contains the first 178 amino acid residues of unspliced calretinin, and this sequence is followed by a C-terminal 14 amino acid sequence that is not found in the unspliced calretinin (Gander et al. 1996). Antibodies raised against this 14-amino acid sequence do not recognise fulllength calretinin, which indicates the novel nature of the C-terminus of calretinin22K. However, in spite of their variant secondary structure, the calcium-binding 131

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properties of calretinin-20K and -22K appear to be unaffected (Schwaller et al. 1995). Calretinin-22K occurs in many colon carcinoma cell lines. However, the significance of the expression of splice variants remains unclear, especially in view of the small numbers of cell lines studied and the absence of information on the stability of the variant isoforms and their incidence in vivo.

REGULATION OF CALRETININ EXPRESSION The expression of calretinin is regulated by a number of neurotropic agents and growth factors and correlates with the appearance of phenotypic features of differentiation. Farkas et al. (1997) found that glial cell-derived neurotropic factor (GDNF) increased the number of neurones expressing calretinin in cultures of embryonic striatal neurones. The numbers of calretinin-expressing cells also is increased by bFGF, alone or in combination with retinoic acid (Pappas and Parnavelas, 1998). The neurotropic factor BDNF induces the extension of neuronal dendrites of hippocampal sections maintained in culture. The subpopulations that respond to BDNF appear to be those that express calbindin and calretinin (Marty et al. 1996). There are also indications that there could be some specificity in the morphological response to the neurotropic factor. No response to BDNF is observed in neurones that express PV. Furthermore, the calretinin content of Cajal–Retzius neuronal cells is regulated by BDNF together with induction of differentiation (Marty et al. 1996), but the latter is not essential for their survival. Calretinin expression in medial basal hypothalamic regions of male rats is reported to be greater than that of female rats at 19 and 20 days of gestation, suggesting a developmental and hormonal regulation of calretinin (Lephart et al. 1997b). There is no question that expression is developmentally regulated as shown by Jiang and Swann (1997), in the evolution of the neuronal populations and the emergence of cells expressing calretinin in the mature hippocampus of the rat. The development and maturation of neurones of the chick dorsal root ganglion are closely related to the expression of calretinin. The ganglia become calretinin positive after 9 days of incubation, and the number of calretininexpressing cells increases with the length of incubation (Kiraly and Celio, 1992). Calretinin-staining clusters of cells are also detected in the medullary epithelium of the thymus at day 11 of incubation, coinciding with the functional maturation of the thymus (Kiraly and Celio, 1993). It is of interest that PV is detected in the epithelial cells at day 9, but CBD is not detectable. This would suggest that at least in some developing organ systems, calretinin and CBD may be individually associated with functional maturation, in spite of their molecular homology to each other. The expression of calretinin and CBD in the oxytocin (OT)- and vasopressin (VP)-containing neurones of the supraoptic nucleus has been the subject of a detailed investigation by Miyata et al. (1998). In both types of neurones, there is a marked differential distribution of calretinin and CBD positivity. In OT neurones, 72 to 84% of cells were CBD immunoreactive as compared with ca. 50% staining for calretinin. In VP neurones, no calretinin staining cells were detectable and only 25% of neurones stained for CBD. Whether this can form a functional differentiation between the two CBPs is uncertain. However, because calretinin and CBD do not differ

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133

substantially with respect to their calcium-buffering ability, the possibility that they may yet be functionally different in other respects needs to be explored further.

CALRETININ EXPRESSION IN CELL PROLIFERATION AND DIFFERENTIATION The relationship between calretinin expression and cell proliferation and the state of differentiation also has been reported in nonneuronal cell types. Indeed, studies of several tumour cell lines have provided evidence supporting such a relationship. Gotzos et al. (1996a) found that 8 of 10 colonic carcinoma cell lines expressed calretinin, and the protein was found in rapidly proliferating cells. Upon being induced to differentiate by treatment with sodium butyrate and hexamethylene bisacetamide, WiDR colon adenocarcinoma cells showed marked reductions in cell proliferation as well as expression of full-length calretinin and its splice variant isoform calretinin-22K (Schwaller and Herrmann, 1997). HT29-18 colon adenocarcinoma cells have been induced to differentiate into enterocytes by using a different strategy — glucose starvation. Again, a marked reduction in calretinin mRNA was found in the differentiated cells (Cargnello et al. 1996). These authors read between the lines and suggest that calretinin may maintain an undifferentiated proliferative state characteristic of neoplastic transformation.

CALRETININ AND ITS POSSIBLE NEUROPROTECTIVE PROPERTY In common with CBD, calretinin may be neuroprotective. In Alzheimer’s disease, large pyramidal neuronal cells show a differential susceptibility to degeneration, and specific subpopulations, which express calretinin, might be resistant to degeneration (Hof et al. 1993). The presence of calretinin also seems to provide some protection against serum deprivation of rat cerebral cortex organ cultures (Weisenhorn et al. 1996). A study of the expression of calretinin, together with CBD and PV mRNA, in hamster brain in relation to ageing, has also helped to focus attention on differences between calretinin and other CBPs. Kishimoto et al. (1998) found that whereas CBD transcript levels fell by 50 to 68%, calretinin and PV transcripts remained unchanged. Implicit in these findings is the suggestion that the down-regulation of CBD expression associated with the ageing process might reflect the neuroprotective action of CBD, and further, by inference, that calretinin may not function in this way. However, it ought to be recognised that most of the available evidence is too preliminary in nature to warrant conclusive interpretation.

CALRETININ AS A POTENTIAL TUMOUR MARKER The expression of calretinin in a number of colonic carcinoma cell lines and the loss of its expression upon differentiation, which has been discussed in the previous sections, has inevitably led to the suggestion that it may serve as a potential maker

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of human malignancies. Gotzos et al. (1996b) examined a series of mesotheliomas for the expression of calretinin, PV, and CBD. Calretinin was found in mesotheliomas of the epithelial type and in the epithelial component of mixed tumours. Sarcomatoid mesotheliomas and the sarcomatoid component of mixed tumours did not express calretinin. Neither were PV and CBD expressed in these tumours or in normal lung tissue. Furthermore, none of the three CBPs was detected in adenocarcinoma of the lung. Therefore, Gotzos et al. (1996b) suggest that calretinin expression could provide a useful means of differentiating epithelial-type mesotheliomas from metastatic adenocarcinoma of the lung. Doglioni et al. (1996) have claimed a diagnostic sensitivity of 100% in a series of 44 mesotheliomas investigated. Leers et al. (1998) have advocated a combination of cadherin and calretinin to distinguish between metastatic carcinomas and mesotheliomas and regard this combination of markers as a powerful tool. Calretinin has been proposed as a marker for serous carcinoma of the ovary (Folpe and Gown, 1997). Also, high levels of calretinin-22K have been found in the serum of many cancer patients. Serum levels of calretinin in breast and colon cancer have been reported to be very high. The protein is detectable in epithelial cells, nerve fibres, connective tissue, and mesothelial cells. Calretinin also has been found in ischemic necrosis of the gut (Schwaller et al. 1998). Needless to say, there is much scope for investigating the clinical value of serum calretinin determination in tumour typing. There is virtually no information available about its relevance in cancer prognosis.

10

Calcineurin in Cell Proliferation, Cell Adhesion, and Cell Spreading

MOLECULAR FEATURES OF CALCINEURIN Among more than 30 target proteins activated by CaM is calcineurin. Calcineurin is a serine/threonine phosphatase. It is also known as protein phosphatase (PP)-2B. Together with PP-1, PP-2A, and PP-2C, calcineurin forms an important group of phosphatases that play a critical role in the phosphorylation/dephosphorylation cycles associated with regulation of the activity of a variety of enzymes. Calcineurin is highly conserved as well as widely distributed. Nevertheless, it has restricted substrate specificity. Its major substrates are the NF-AT and other members of the NF-AT family of transcription factors, N-methyl-D-aspartate (NMDA) receptors (Barford, 1996; Tong et al. 1995; also see below), and IP3 receptors (Cameron et al. 1995; Cunningham, 1995). Microtubule-associated proteins MAP-2 and the tau protein (Ferreira et al. 1993; see below for further references) are also among its substrates. The biochemical and pharmacological significance of these substrates have been discussed below in the context of the various physiological functions performed by calcineurin. Calcineurin is a major protein of brain tissue, accounting for approximately 1% of its total protein. In brain tissue it is associated with the cytoskeletal structures or is bound to the plasma membrane. It occurs as a major component of neurones, especially of the neurostriatum and cerebellum. Lymphocytes also contain large amounts of calcineurin (Kincaid et al. 1987). Calcineurin is composed of two subunits: a catalytic subunit known as calcineurin A and a regulatory subunit called calcineurin B. Calcineurin is usually isolated with bound iron and zinc ions, but it still requires the binding of exogenous metal ions to achieve maximum activity. Calcineurin possesses low catalytic activity in the absence of exogenous metal ions. It can be activated by Mn2+, Mg2+, and Ni2+. The activation by Mn2+ seems to involve the catalytic subunit calcineurin A. The calcineurin B sequence is highly conserved, as compared with calcineurin A. Nevertheless, in the latter the catalytic domain is highly conserved in evolution. Calcineurin A is 59 kDa in size and binds CaM. The regulatory 19-kDa calcineurin B contains four EF-hand calcium-binding domains. It is encoded by a single gene and

135

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Calcium Signalling in Cancer

contains 163 amino acid residues (reviewed by Klee et al. 1988). However, alternative splicing might result in a number of variant forms of calcineurin B (C.D. Chang et al. 1994). Three isoforms (α, β, and γ) of calcineurin A and two of calcineurin B have been described (Tumlin, 1997). Calcineurin A-β has been mapped to human chromosome 10q21–22, and calcineurin B to chromosome 2p15–16. The calcineurin A-α occurs on chromosome 4 (M.G. Wang et al. 1996).

CALCINEURIN IN CELL PROLIFERATION AND ADHESION-RELATED PHENOMENA Calcineurin has been implicated in a number of physiological events, such as cell proliferation, cell death, and signal transduction, and in immunosuppression. It also has been implicated in certain functions of the nervous system, e.g., in neurotransmission. The discussion here will be restricted mainly to areas pertinent to the biological behaviour of cancers, although some reference will be made to Alzheimer’s disease, in which cytoskeletal abnormalities occur prominently. The regulation of physiological activity of a large number of biological macromolecules is dependent on phosphorylation. It is to be expected that, as a phosphatase, calcineurin would be a key component in the phosphorylation of some of these molecules. From this it should follow, therefore, that calcineurin would impinge significantly on the biological behaviour of cancers and in other disease states.

PUTATIVE ROLE

OF

CALCINEURIN

IN

CELL CYCLE PROGRESSION

There is a large body of evidence that points to the importance of calcineurin in the progression of the cell cycle. Calcineurin A gene seems to be essential for the G1–S transition of Aspergillus nidulans cells. A disruption of the gene by homologous recombination results in the arrest of the cell cycle in its early phase. Calcineurin A mRNA accumulates in the G1 phase well before the G1–S transition point (Rasmussen et al. 1994). The induction of DNA synthesis in response to growth factor stimulation of cells has been shown to depend on Ca2+ uptake and on the activation of calcineurin (Tomono et al. 1996). A suggested modus operandi is by the regulation of specific cyclins. The initiation of DNA synthesis and mitosis is controlled by two key factors: (1) cyclin-dependent protein kinases (cdk) composed of a cyclin regulatory subunit and a cell division control (cdc2) family kinase and (2) CDK inhibitors (see Sherbet and Lakshmi, 1997b for further information). The involvement of calcineurin in the progression of the cell cycle is demonstrated by the fact that the inhibition of calcineurin leads to an inhibition of fibroblast growth factor FGF-induced expression cyclins A and E in Swiss 3T3 cells (Tomono et al. 1998). Calcineurin seems to be associated also with determining the length of the G2 phase of the cell cycle and entry of the cell into the mitotic phase. The G2–M transition, in Saccharomyces pombe for instance, is dependent on the activation by dephosphorylation of a complex of G2specific cyclin with cdc2 kinase. This process of activation is inhibited by Swe-1 (Wee1 homologue) tyrosine kinase. The transcription of Swe-1 is regulated by Zds-1. Calcineurin and Mpk-1 seem to regulate the transcription and posttranslational modification of Swe-1, respectively (Mizunuma et al. 1998).

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It would be of considerable interest, therefore, to examine whether some of the effects of calcineurin could be explained by its putative participation in the phosphorylation events associated with cell cycle progression. A number of cyclins and cdk are expressed in a cell cycle-related fashion. The cyclin D/cdk-4 complexes are activated by cdk-activating kinases. This activated complex now phosphorylates rb protein and enables the cell to transit into the S phase. The cyclin B/p34cdc2 complex is associated with the G2–M transition of cells. The phosphorylation of p34cddc2 at tyrosine 161 is believed to activate it, which enables the cell to make the G2–M transition. Phosphorylation of threonine 14/tyrosine 15 is believed to produce a negative regulatory effect on cell G2–M transition (see Sherbet and Lakshmi, 1997b for references). Calcineurin, being a threonine phosphatase, could conceivably affect this negative regulation of the G2–M transition and allow cells to enter the mitotic phase. It is also possible that calcineurin functions by means other than altering the cyclins associated with the G1–S transition. Tomono et al. (1998) found that cyclosporin A, at calcineurin-inhibiting concentration, inhibited cyclins A and E, but not DNA synthesis. An interesting aside to this story is that carbachol, a Ca2+-mediated stimulator of tyrosine phosphorylation, induces the formation of actin stress fibres and markedly increases the F-actin/G-actin ratio in smooth muscle cells in vitro. Tyrosine kinase inhibitors have been found to inhibit the formation of stress fibres in the cell system (Togashi et al. 1998). The opposite effects exerted by the phosphorylation of serine/threonine and tyrosine residues could conceivably suggest a regulatory mechanism of stress fibre formation by a process of differential phosphorylation. Such a mechanism is encountered in the control cell cycle progression, where cyclin function is regulated by differential phosphorylation. As alluded to above, the phosphorylation of the threonine residues and inactivation of p34 has a negative regulatory effect on cell cycle progression. In the light of the crucial role played by the cytoskeleton in cell division, further studies of the potential involvement of calcineurin in the cell division cycle and allied areas are clearly warranted and may turn out to be highly fruitful. The progression of the cell cycle is controlled at both G1–S and G2–M transition checkpoints by the phosphoprotein p53. The biochemical basis of p53 function is its ability to regulate transcription by binding to DNA. Calcineurin appears to be able to modulate p53 binding to the long terminal repeats (LTR) of human immunodeficiency virus (HIV-1). The compound PD 144795 inhibits HIV-1 transcription, which correlates with the inhibition of the phosphatase activity of calcineurin (Gualberto et al. 1998). This demonstration of p53-mediated transactivation provides another putative mechanism by which calcineurin could influence cell cycle progression. Calcineurin is known to modulate the function of other transcription factors, e.g., NF-AT3 and GATA-4, by dephosphorylation (Molkentin et al. 1998). We know from the work of Cai et al. (1996) that calcineurin might be involved also in the transcription of the IL-2 gene. They showed that inactivation of calcineurin leads to an inhibition of the transcription of the IL-2 gene. The expression of fibre-type specific genes, of slow- and fast-twitch myofibrils of adult skeletal muscles, seems to be regulated by calcineurin. The transcription of these genes occurs by the

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mediation of NF-AT and MEF2 transcription factors (Chin et al. 1998). The calcineurin inhibitor FK506 reversibly inhibits insulin secretion by HIT-T15 cells in culture, and in parallel, intracellular levels of insulin mRNA and insulin also decrease. The inhibition of insulin gene transcription has also been confirmed using human insulin/CAT reporter gene constructs (Redmon et al. 1996). Recent reports show that IGF, which is involved in muscle growth, regeneration, and hypertrophy, also appears to follow the calcineurin NF-AT, GATA pathway in activating the expression of the appropriate genes. The transfection of IGF-1 gene into skeletal myocytes and its expression at the postmitotic stage induce the expression of calcineurin mRNA transcripts and the protein. The latter becomes localised in the nucleus. IGF-1 as well as activated calcineurin also induce GATA-2 expression. This transcription factor associates with calcineurin and NF-Atc1 (Musaro et al. 1999). In parallel studies, Semsarian et al. (1999) have shown that treatment of skeletal muscle cells with IGF-1 or dexamethasone activates calcineurin and induces the translocation of NF-Atc1 to the nucleus. These observations clearly implicate the calcineurin/NF-AT signalling pathway in the cellular responses generated by these growth factors. Finally, the ubiquitous HSPs come into this picture as well. HSPs have gained much coverage as stress-induced proteins. Besides, they are of exceptional importance to the life of the cell, for they take part in processing of nascent protein, transport, and targeting of proteins to various cellular subcompartments. Furthermore, not only are HSPs expressed in a cell cycle-related manner, but they also interact with many cellular proteins that are involved in cell cycle regulation. It is of much interest, therefore, to cite here some recent work that seems to strengthen the association of both HSPs and calcineurin in the progression of the cell cycle. Someren et al. (1999) have shown that HSP70 and HSP90 both activate calcineurin in the presence of CaM. The requirement of CaM seems to be absolute for the activation by HSP70. Activation of calcineurin does not occur in the absence of CaM. Someren et al. (1999) have also demonstrated that calcineurin co-precipitates with HSP. However, in light of much evidence to be discussed in a later section, it is difficult to establish the precise pathway by which either HSPs or calcineurin might influence the progression of the cell cycle.

THE EFFECTS

OF

CALCINEURIN

ON

CELL ADHESION

AND

MOTILITY

The expression of calcineurin A as well as B is up-regulated in HL-60 cells induced to differentiate by treatment with all-trans RA. A progressive increase in calcineurin phosphatase activity occurs in parallel with granulocytic differentiation and inhibition of cell proliferation (Kihara et al. 1998). Therefore, there may be another pathway by which calcineurin influences cell proliferation. RA is a well-known modulator of cell adhesion and inhibitor of cell motility and the invasive behaviour of cancer cells (Fazely and Ledinko, 1990; Edward and Mackie, 1990; Edward et al. 1989, 1992; Wood et al. 1990). Hendey and Maxfield (1993) found that the motility of neutrophils on vitronectin-coated substratum could be inhibited by using inhibitors of calcineurin. The formation of filopodia and the motility of neurones of chick dorsal root ganglia are

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said to depend on calcineurin. The calcineurin inhibitors cyclosporin A and FK506 delay the formation of neurites and inhibit neurite extension. A targeted and focal inactivation of calcineurin in regions of growth cones affects filopodial retraction and direction of its subsequent outgrowth (Chang et al. 1995). FK506 has also been found to inhibit the transendothelial migration of human lymphoma cells, Nalm-6, in both in vitro and in vivo situations (Tsuzuki et al. 1998). These authors have suggested that the inhibition of migration might be related to an inhibition of vascular cell adhesion molecule (VCAM)-1 and its α4β1 integrin receptor VLA-4. The VCAM-1/VLA-4 system is known to provide the recognition mechanism and adhesive facility for certain tumour cell types to the endothelium. VCAM expression seems to relate to the progression of melanomas (Denton et al. 1992), but not of breast cancer (Fox et al 1995). Overall, the evidence for any general relationship between the expression of VCAM and its receptor and metastatic potential is not compelling. However, calcineurin can stimulate the transcription factor NF-κB and enhance its DNA-binding property (Franz et al. 1994). This transcription factor is also involved in the induction of cell adhesion molecules, and anti-NF-κB reagents are able to block cancer cell adhesion to endothelial cells (Tozawa et al. 1995). Therefore, the effects of FK506 on transendothelial migration described by Tsuzuki et al. (1998) could be interpreted as indicating that calcineurin might itself be influencing the diapedesis by cancer cells. Mohri et al. (1998) have recently provided some direct evidence that shows that calcineurin affects cell adhesion to substratum and, by implication, also modulates the invasive behaviour of cells. They used the colon cancer cell line Colo201 and demonstrated that the protein kinase inhibitors K252a and KT5720 enhanced cell adhesion to and their spreading on substratum. This process was accompanied by the formation of actin stress fibres. The adhesion appeared to be mediated by integrin α2 and β1, as indicated by their accumulation at the sites of focal adhesion. The higher adhesion mediated by K252a and KT5720 could be blocked by cyclosporin and FK506, which also inhibit calcineurin. An increase in the expression of TGFβ1 in peripheral blood mononuclear cells obtained from patients treated with cyclosporin has been reported (Shin et al. 1998). Other cell types, such as A549 cells, seem to respond to cyclosporin in a similar fashion. The increased expression of TGFβ is associated with enhanced cell motility, invasive behaviour in vitro, and enhanced metastatic behaviour, as indicated by inhibition of the cyclosporin-induced effects of anti-TGFβ antibodies (Hojo et al. 1999). It would be worthwhile to point out here that TGFβ produces a variety of effects. Among these are induction of angiogenesis, inhibition of cyclin-dependent kinases, and induction of inhibitors of metalloproteinases (see Sherbet and Lakshmi, 1997b for references). The invasive behaviour of trophoblast cells is believed to be controlled by the induction of TIMPs by TGFβ (Lala and Graham, 1990). These cellular faculties of adhesion to substratum, cell spreading, and cell motility are closely related to cell proliferation, besides being dependent on cytoskeletal organisation. Therefore, one should consider whether calcineurin could be altering cytoskeletal dynamics by altering the phosphorylation status of cytoskeletal components.

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Calcineurin and the CaM-dependent protein kinase II have been reported to regulate the phosphorylation levels of neurofilament subunits and β-tubulin elements of rat cerebellar cytoskeleton (De Mattos-Dutra et al. 1998). The tau protein has been shown to be an important requirement for neurite outgrowth and growth cone motility. The inactivation of tau seems to lead to an inhibition of neurite outgrowth, and other microtubule-associated proteins are unable to compensate for the inactivated tau (C.W.A. Liu et al. 1999). In common with many other proteins, the biological function of tau is regulated by phosphorylation. tau promotes microtubule assembly and inhibits depolymerisation (Drechsel et al. 1992). Phosphorylated tau has a reduced capacity to bind to microtubules (G.V.W. Johnson, 1992). The tau protein of Alzheimer’s disease paired helical filaments (PHF) has been reported to contain 21 phosphorylation sites, of which some are serine/threonine–proline residues (Morishima-Kawashima et al. 1995). The phosphorylation of threonine 231, serine 235, and serine 262 is essential for maximal inhibition of binding of tau protein to microtubules (Sengupta et al. 1998). Mandelkow et al. (1996) believe that the single serine 262 phosphorylation determines the binding of tau to microtubules. This abolishes tau–microtubule binding and greatly affects the ability of tau to stabilise microtubule dynamics. Because calcineurin is a serine/threonine phosphatase, it may be expected to control tau phosphorylation and in this way also regulate its interaction with microtubules. The dephosphorylation of the tau element of the cytoskeleton of PC12 cells appears to be mediated by calcineurin (H.Q. Xie and Johnson, 1998). Cyclosporin A consistently inhibits the phosphatase activity of calcineurin and inhibits axonal extension (Ferreira et al. 1993). Also, tau protein is found in a hyperphosphorylated form in calcineurin A-α knock-out (–/–) mice (Kayyali et al. 1997). Furthermore, calcineurin strongly influences F-actin stability in cultured hippocampal neurones. The exposure of these cells to NMDA produces a rapid loss of dendritic spines together with a loss of actin filaments. This is prevented by actin-stabilising agents and calcineurin inhibitors (Halpain et al. 1998).

CALCINEURIN IN ALZHEIMER’S DISEASE A characteristic feature of Alzheimer’s disease is the formation of neurofibrillary tangles (NFTs). NFTs are intraneuronal and consist mainly of PHF. It has been postulated that the assembly of PHF involves initially a process of antiparallel dimerisation of tau monomers. The dimers then form PHF (Mandelkow et al. 1996). The tau protein is a major component of PHF. Tau is phosphorylated to an abnormally high degree in Alzheimer’s disease. The phosphorylation is three- to four-fold greater in Alzheimer’s tau protein than in the native protein (Mandelkow et al. 1996; Grundke-Iqbal et al. 1986a, 1986b). This abnormally phosphorylated state is regarded as being responsible for the detachment of tau from microtubules and the consequent microtubule instability. Iqbal and Grundke-Iqbal (1996) state that abnormal phosphorylation might precede the polymerisation of tau into PHF. Furthermore, they claim that dephosphorylation of abnormal tau can restore the ability of tau to interact with and promote microtubule assembly. However, others believe that no such causal relationship exists between tau phosphorylation and the formation of

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PHF (Goedert, 1996). Another factor that might be envisaged is genomic mutations. Mutations in the exons as well as introns of the tau gene have been reported in cases of familial dementia and Parkinson’s disease. Mutations in the exons seem to affect the ability of tau to promote microtubule assembly (Hasegawa et al. 1998). Nonetheless, this abnormal phosphorylation state has been attributed to the generation of an imbalance between protein kinases and phosphatases, engendered by a reduction in the levels of phosphatases (Iqbal and Grundke-Iqbal, 1996). More specifically, the high state of tau phosphorylation has been related to calcineurin dysfunction. This is suggested by the findings of Kayyali et al. (1997) that calcineurin A-α (–/–) knock-out mice show hyperphosphorylation of tau. The abnormal tau accumulates, and these mice also exhibit cytoskeletal changes that might affect neuronal function. The neurodegenerative condition of amyotrophic lateral sclerosis (ALS) is characterised by a loss of motor neurones and neurones of the corticospinal tract. This neuronal loss has been attributed to a deregulation of NMDA receptors (Krieger et al. 1993; Plaitakis, 1990). Glutamate-induced apoptosis and necrosis of cerebellar granule cells is prevented by the calcineurin inhibitors FK506 and cyclosporin (Ankarcrona et al. 1996). Abnormal levels of calcineurin have been reported to occur in ALS. This is believed to result in abnormal phosphorylation of NMDA receptors, which in turn has been suggested to result in the pathogenesis of ALS (Wagey et al. 1997). Alzheimer’s disease and Down’s syndrome also are characterised by extracellular occurrence of amyloid deposits in the form of senile plaques and deposits associated with the microvasculature. The fibrillary amyloid is composed of the amyloid-β protein (ABP) with 39 to 43 amino acids. ABP is derived from the integral membrane glycoprotein, the β-amyloid precursor protein (βAPP) The latter shows an abnormally high accumulation in the brain of people with Alzheimer’s disease. Calcineurin seems to be involved in the formation of this peptide (Desdouits et al. 1996).

CALCINEURIN IN IMMUNOSUPPRESSION Calcineurin plays a key role in immunosuppression. There has been an unequivocal demonstration over the past few years that the drugs cyclosporin A, FK506, and rapamycin achieve immune suppression by inhibiting the function of calcineurin. That calcineurin was the target of some of these immunosuppressants was established some years ago. O’Keefe et al. (1992) transfected the catalytic calcineurin A into cells and showed that this raises the IC50 inhibitory concentration of cyclosporin and FK506. Besides, Jurkat cells that overexpressed calcineurin were resistant to the action of cyclosporin and FK506 and showed enhanced gene transcription that depended on NF-AT and related transcription factors (Clipstone and Crabtree, 1992). These immunosuppressant drugs interact with specific intracellular immunophilin receptors, which results in the formation of a drug–immunophilin complex that is capable of blocking the function of its target, which, in this instance, is calcineurin (J. Liu et al. 1991; Ho et al. 1996). When T cells are activated by mitogenic signals or by antigen binding, they proliferate and switch on the expression of cytokine genes. Antigen binding to the TCR produces a cascade of signalling events (Figure 19), which have been elucidated in some detail.

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FIGURE 19 The involvement of calcineurin in a key position in the TCR-signalling cascade. See text for details.

Calcineurin participates in this signalling cascade as a key enzyme. It is responsible for dephosphorylating the transcription factor called NF-AT and this factor then translocates to the nucleus. NF-AT1 contains a regulatory domain, which lies N-terminal to its DNA-binding region, that binds to calcineurin (C. Luo et al. 1996). In this way calcineurin seems to be targeted to NF-AT. The maintenance of NF-AT in the nucleus requires high and sustained levels, not transients, of calcium (Timmerman et al. 1996), probably reflecting its continual calcineurin-dependent activation and translocation. The NF-AT proteins are expressed in many cell types of the immune system, and these constitute a family of proteins that possess this conserved regulatory domain involved in their binding to calcineurin. NF-AT proteins resemble the Rel family of proteins with respect to their ability to bind to the regulatory elements of certain genes coding for cytokines (Rao et al. 1997). In the nucleus, NF-AT forms a complex with the transcription complex AP1 (jun/fos). The AP1/NFAT complex then initiates the transcription of a number of genes, such as genes coding for the lymphokine, IL-2, IFN-γ, and TNF-α, among others. The inhibition of calcineurin by the immunosuppressants inhibits the dephosphorylation of NF-AT and thereby inhibits its translocation to the nucleus and all the downstream events. Batiuk et al. (1997) have fully correlated the effects of cyclosporin A with the various

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143

FIGURE 20 The multiple functions of calcineurin, on the cell division cycle, in the modulation of cell shape, adhesion and motility, and in the regulation of gene transcription. Also indicated are its participation in the functioning of immunosuppressants and its possible association with abnormal phosphorylation of tau in Alzheimer’s disease.

events of the cascade, namely the dephosphorylation of NF-AT, its translocation, binding of the transcription complex to the DNA, and the eventual activation of gene transcription. They also demonstrated the relationship between the extent of calcineurin inhibition, the inhibition of lymphocyte proliferation, and the degree of IFN-γ production in vitro. It would not be out of place to briefly mention here that calcineurin is involved in the autoimmune condition SLE. SLE affects women of child-bearing age. The menstrual cycle and pregnancy seem to affect disease activity (Lahita, 1993; Jungers et al. 1985; Mund et al. 1963). Rider et al. (1998) have shown that T cells from SLE patients respond to oestradiol by an up-regulation of calcineurin mRNA expression, in a dose-dependent fashion. The effect seems to be specific for oestradiol, because progesterone and dexamethasone fail to elicit a similar response. The increase in mRNA expression corresponds with calcineurin phosphatase activity in oestradiol-treated T-cell extracts. As Rider et al. (1998) pointed out, the mechanism by which oestrogen influences calcineurin expression is unclear, because the latter has no identifiable oestrogen response elements in its promoter region. However, they suggest that oestrogen could be exerting control at the level of calcineurin transcription. They cite the findings of H. Becker et al. (1995) that there is an increased NF-AT binding to IL-2 promoter in SLE patients as compared with control subjects. As discussed earlier in this section and shown in Figure 19, calcineurin

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plays a key role in the translocation of the NF-AT transcription factor to the nucleus. Therefore, it is conceivable that oestrogen influences the T-cell signal transduction cascade in SLE. One is fully justified in concluding at the end of this chapter that calcineurin is a highly versatile phosphatase, and its functional repercussions, shown in Figure 20, are discernible in many facets of biological behaviour.

11

Centrins (Caltractins) and Their Biological Functions

Centrin (also known as caltractin) is an EF-hand calcium-binding protein of approximately 20 kDa molecular size. Errabolu et al. (1994) have cloned human centrin cDNA, which contains an ORF of 516 bp and a predicted 172 amino acid residues in the protein. Sequence analysis shows that human centrin is closely related to centrins from plants, protozoa, algae, and Xenopus, and to CDC31 of Saccharomyces. The sequence also suggests the occurrence of four calcium-binding EF-hands. The centrin gene has been mapped to chromosome Xp28 (Chatterjee et al. 1995). However, the identification of several isoforms of centrin suggests the possibility of the existence of a family of centrin genes. Centrin is a component of spindle pole bodies, basal bodies, and centrosomes. Centrin and γ-tubulin are common components of these organelles. A major physiological function of centrin is the organisation of microtubules. The contractile fibres associated with centromeres, for instance, show calcium mediated contraction, and filament contraction occurs independently of ATP (Chiebel and Bornens, 1995). Using immunofluorescence techniques, researchers have shown that centrin localises in the centrosome of interphase cells. The centrosome duplicates during this phase of the cell cycle and centrin then redistributes between the spindle and the polar bodies. Quite obviously, therefore, it plays an important role in the separation of centrosomes during mitosis (Errabolu et al. 1994). Other functions also have been attributed to centrin. It is associated in the yeast with the duplication of the spindle pole body. Vallen et al. (1994) showed that the CDC31 of Saccharomyces cerevisiae, a protein that is closely related to centrin, interacts with the KAR1 gene product in the duplication of the microtubule organising centre. Centrin interacts with KAR1 protein with great affinity. This interaction is regulated by changes in cellular calcium levels, which, in their turn, seem to be influenced by certain amino acid residues within the KAR protein (Geier et al. 1996). In fact, antibodies to the centrin expressed by the flagellate Naegleria gruberi can recognise CDC31 of yeast (Levy et al. 1996). Centrin and related proteins are involved in flagellar severance and the retraction of ciliary apparatus. In Chlamydomonas reinhardtii the excision of flagella occurs by a process of microtubule severance caused by contraction of centrin-containing fibres (Sanders and Salisbury, 1994). The molecular mechanisms involved in the function of centrins are still poorly understood, but centrosome fractions prepared from haematopoietic cells have been 145

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Calcium Signalling in Cancer

found to contain active Ca2+/CaM-dependent protein kinase II. In vitro, this enzyme has been shown to phosphorylate several centrosome-associated proteins (Pietromonaco et al. 1995). However, there is no evidence at present of centrin phosphorylation in relation to its function, apart from the observation that centrin is phosphorylated during prophase and metaphases of mitosis (Lutz et al. 1995). The H6 gene of the plant Atriplex nummularia encodes a protein with a high degree of sequence homology to algal centrin. Three transcripts of this gene, of 1.3, 2.2, and 2.4 kb size, are expressed, and it would appear that these transcripts are differentially expressed in relation to mitotic activity and developmental and extracellular signals. The 1.3-kb transcript is induced by tactile and hyperthermic signals. Expression of the 2.2-kb transcript is related to the state of mitotic activity. The 2.4kb transcript is expressed in response to heat shock (J.K. Zhu et al. 1996). Centrin has been found in the form of a complex with the high molecular weight HSP70 and HSP90 in Xenopus oocytes that are arrested by exposure to cytostatic factors. When the oocytes are activated by electric shock or by means of ionophores, the centrin–HSP70 complexes appear to undergo dissociation (Uzawa et al. 1995). The functional significance of the formation of these complexes is unclear, although the work of Uzawa et al. (1995) appears to suggest a relationship with activation and division of the oocytes. It ought to be pointed out that high molecular weight HSPs may not be involved in the process of cell proliferation, but they may influence the size of the steady-state cell population by protecting cells from apoptosis. In contrast, the lower molecular weight forms such as HSP28 are indeed so involved. HSP28 inhibits cell proliferation. It may form complexes with EF-hand proteins such as S100A4 (see Albertazzi et al. 1998a). HSP70 has been reported to show a cell cyclerelated pattern of expression. Milarsky and Morimoto (1986) found that it is expressed at the G1–S boundary. It can bind also to the cell cycle regulatory p53 phosphoprotein (Pinhasi-Kimhi et al. 1986; Finlay et al. 1988). So, it is conceivable that it is involved in the regulation of the cell cycle. On the other hand, HSPs participate prominently in protein folding and transport. Whether the formation of a complex between HSP and centrin alters the molecular folding of the latter is not known, but such a modulation of molecular shape has the potential to alter the accessibility and binding of calcium to centrin and thus affect its function. Conversely, complex formation with HSPs might be an aspect of centrin function. The formation of the complex could itself depend upon the conformation adopted by centrin upon calcium-binding. Centrin also may be involved, together with other components of the cytoskeletal system, in maintaining cell polarity and motility (Lingle and Salisbury, 1997). Centrins have been found in nonmotile ciliary structures. Wolfrum (1995) has found centrin in photoreceptor cells. There is some preliminary evidence suggesting that the central helix of centrin may be important for its biological function. A study of the mutations of the Chlamydomonas vfl2 centrin gene has revealed that mutation of the glycine residue at 101 to lysine results in the loss of centrin function. However, this suppressor effect is reversed with additional mutations at amino acid residues 96 or 104, and all three residues occur in the central helix of the protein (Taillon and Jarvik, 1995).

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147

Centrin is highly conserved in evolution. Bhattacharya et al. (1993) studied the molecular organisation of centrins of several lower organisms. From the phylogenetic point of view, the EF-hand domains of several centrins show congruence and may have arisen by gene duplication from an ancestral EF-hand domain. Furthermore, the domains of centrin are congruent with those of CaM. These observations provide strong indicators that centrin and related proteins may have evolved from a common ancestral form by means of gene duplication. A similar conclusion can be reached from the pattern of expression of centrin and related proteins in association with a phylogenetically evolving contractile system (Levy et al. 1996). In mammals, three centrin genes have been described: HsCEN1, HsCEN2, and HsCEN3 (Middendorp et al. 1997). These authors found that HsCEN1 was related to the yeast centrinencoding gene CDC31 and further, that HsCEN2 and HsCEN3 possessed a greater degree of identity to algal centrin than did HsCEN1. This is interpreted as suggesting a divergence in the evolution of centrins.

12

Reticulocalbin Family of EF-Hand Proteins

There are two species of calcium binding proteins that are associated with the endoplasmic reticulum. One of them is a non-EF-hand protein, calreticulin. A family of EF-hand proteins, with variable number of EF-hand domains, has been identified over the past few years (Table 9). Among them are reticulocalbin, calumenin, ERC55 and ERC-fc, and Cab-45, and these may be described as belonging to the reticulocalbin family of CBPs. These proteins are expressed ubiquitously in a variety of tissues. Human calumenin occurs at high levels in the heart, placenta, and skeletal muscle, and at lower levels in the lung, kidney, and pancreas. Its levels are very low in the brain and liver (Vorum et al. 1998). Scherer et al. (1996) have isolated Cab45 from mouse adipocytes.

TABLE 9 The Reticulocalbin Family of EF-Hand Calcium Binding Proteins Protein Nomenclature Reticulocalbin

ERC-55 ERC-pf DNA-SCFa Calumenin (human) Cab-45 (murine) CBP-50 Crocalbin

a

Molecular Features

Ref.

44-kDa protein; C-terminal HDEL sequence; 6 EF-hands; gene 13 kbp and 6 exons 11p13 (WAGR locus) 55-kDa protein; 6 EF-hands 40-kDa; 6 EF-hands 45-kDa cytoplasmic, 30-kDa nuclear 315 amino acid residues; 7 EF-hands

Ozawa and Muramatsu (1993); Ozawa (1995a,b)

45 kDa; 6 EF-hands; Golgi location; no signal for membrane anchoring 50-kDa protein; 1 EF-hand motif (?) 315 amino acid residues; 6 calciumbinding domains; binds to phospholipase A2

Kent et al. (1997) Weis et al. (1994) La Greca et al. (1997) M. Kobayashi et al. (1998) Vorum et al. (1998); Yabe et al. (1997) Scherer et al. (1996) Hseu et al. (1997) Hseu et al. (1999)

DNA supercoiling factor, found to show homology to reticulocalbin.

149

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MOLECULAR FEATURES OF RETICULOCALBIN HOMOLOGUES Reticulocalbin and other members of the family possess a variable number of EFhand domains. Also variable is their affinity for calcium ions. The EF-hand domains of reticulocalbin bind calcium with low affinity (Vorum et al. 1998). According to Tachikui et al. (1997), only EF-hands 1, 4, 5, and 6 bind calcium. The EF-hands 2 and 3 do not bind Ca2+. Calcium binding produces conformational changes in reticulocalbin. The reticulocalbins are approximately 40 to 45 kDa in size and show a high degree of homology among themselves, mostly with respect to sequences outside the EF-hand domains. As the name of the family denotes, these CBPs are associated with the ER. Cab-45 occurs mainly in the Golgi complex. These proteins contain a membrane localisation and anchoring sequence HDEL of 4 amino acid residues at the C-terminal region (Ozawa and Muramatsu, 1993; Vorum et al. 1998; Yabe et al. 1997). The deletion of these residues from ERC-55 has been shown to result in loss of anchorage to the membrane and secretion of the protein (Weis et al. 1994). Reticulocalbin also contains an N-terminal signal for its transfer to the lumen of the ER (Ozawa and Muramatsu, 1993). Cab-45, which occurs in the lumen of the Golgi bodies, does not possess the membrane-anchoring signal (Scherer et al. 1996). The mouse reticulocalbin gene spans more than 13 kbp of the genome. It contains six exons. The human reticulocalbin shows more than 95% amino acid sequence homology with the murine protein, in both EF-hand and non-EF-hand regions of the protein. Both human and murine proteins contain six EF-hand motifs. Whereas human reticulocalbin possesses the HDEL sequence, the mouse homologue contains the KDEL sequence at the C-terminal end (Ozawa, 1995a, 1995b). The human reticulocalbin gene has been mapped to chromosome 11p13 in the WAGR locus, between the Wilms’ tumour gene wt1 and the aniridia PAX gene (Kent et al. 1997).

PUTATIVE FUNCTIONS OF RETICULOCALBIN AND ITS HOMOLOGUES There has not been sustained effort to study the functions of the reticulocalbin family of proteins. Three putative functions can be identified. Their association with ER and the evolutionarily conserved ER anchoring sequence suggests that one function may be protein trafficking. M. Kobayashi et al. (1998) have identified two cDNA clones of 1.6 and 1.8 kb, expressed in Drosophila melanogaster. The 1.8-kb mRNA contained an open reading frame (ORF) that showed marked homology to mouse reticulocalbin. The shorter mRNA encoded a protein in which an N-terminal sequence was deleted. Antibodies against the DNA supercoiling factor identified a cytoplasmic 45-kDa protein and a 30-kDa protein in the nucleus. They also found that the 30-kDa nuclear protein interacted with topoisomerase II, which suggests that this protein is the functional form of the supercoiling factor. In early developmental stages, such as the blastoderm stage of embryonic development, the antibodies stain interphase nuclei. M. Kobayashi et al. (1998) have also described the pattern

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151

of immunostaining of the polytene chromosomes of Drosophila, which are essentially interphase chromosomes. The polytene chromosomes have characteristic condensed bands harbouring specific genes. These form puffs when the genes are transcriptionally active. M. Kobayashi et al. (1998) found that immunostaining for the supercoiling factor was associated with naturally occurring puffs or those induced by ecdysone. These data seem to associate the supercoiling factor with gene expression. There is a further suggestion that reticulocalbin may be associated with the invasive behaviour of tumours. It is reported to be overexpressed in breast cancer cell lines with high invasive ability, but not in cell lines that are weakly invasive (Z.D. Liu et al. 1997). Although these authors have carried out in vitro invasion assays in parallel, it is difficult to tie the invasive behaviour specifically to the expression of reticulocalbin. The MBA and MCF7 breast cancer cell lines that they have used are known to differ markedly in the expression of S100A4, growth factor receptors, and plasminogen activator, among others, which are known to affect the invasive ability of these cell lines. Perhaps some gene transfer experiments are warranted before any evidence can be regarded as conclusive. Even then, conceptually, it is difficult to visualise a mode of function at the present stage in our knowledge of the participation of reticulocalbin homologues in cell physiology.

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Calpains in Normal and Aberrant Cell Physiology

THE CALPAIN FAMILY OF CALCIUM-BINDING PROTEINS Calpains constitute a family of calcium-dependent proteases showing ubiquitous distribution in a wide spectrum of animal species. They are intracellular cysteine proteases. They cleave substrate proteins in a manner that might constitute a mechanism of regulation of the protein substrates (Croall and De Martino, 1991; K. Suzuki et al. 1992). Therefore, they have been regarded here as regulatory CBPs. Mammalian calpains were known, on the basis of activity, as µ- and m-calpains (referred to herein with their gene designation as capn1 and capn2). These are the ubiquitously occurring isoforms (Table 10). Other isoforms showing tissue-specific distribution also are known, and these have been designated as capn3 and capn4. Capn5 and capn6 have been discovered recently and, despite their amino acid sequence homology with other calpains, appear to be distinctive in that they do not possess the CaM-like calcium-binding domain (Dear et al. 1997). The tra-3 gene, which encodes the calpain of nematode worms, shows the highest degree of homology to capn5 and capn6. Thus, in spite of the sequence similarities with other isoforms, capn5, capn6, and tra-3 are not dependent on calcium for their activity and may indeed be devoid of all proteolytic activity (Dear et al. 1997; Barnes and Hodgkin, 1996). The human homologue of tra-3, known as the htra-3 gene, has been cloned. This encodes a protein that is highly homologous to the calpain 80-kDa subunit. Furthermore, like tra-3, the htra-3 peptide lacks the CaM-like calcium binding motifs (Mugita et al. 1997). It would seem, therefore, that calpains might have diverse functions that may be related to the existence of some mechanism by which they might be activated. This is compatible with the discovery of a family of calpain activator proteins, which not only resemble one another but also possess chaperonin-like properties (Melloni et al. 1998a, 1998b). The 30-kDa activator described by Melloni et al. (1998a) has been reported to show a rigorous specificity for capn1 and to be totally ineffective on capn2. This activator protein binds to the 80-kDa subunit of calpain and promotes its dissociation from the smaller subunit. The process of activation then takes the form of autoproteolysis, leading to the formation of two forms of calpain of 78 and 75 kDa molecular size. The activator described by Melloni et al. (1998a, 1998b) bears a high degree of sequence homology with another calpain activator that has 153

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TABLE 10 Calpain Family Isoforms Gene/Protein Designation

Chromosome Location

capn1 (m-calpain) capn2 (m-calpain) capn3 (p94; nCL-1)

Chromosome 11q13a Chromosome 1 Chromosome 15q15.1–q21.1b

Lp82 (splice variant of capn3) capn4 (nCL-2) nCL-4 capn5 capn6 capn8 tra-3 htra-3 (human homologue of tra-3) 11q14d

Characteristic Features Active at micromolar concentration Active at millimolar concentration Skeletal muscle specific Rat lens Stomach specific Stomach, intestine, but not uterus

Chromosome 11q13.5–q14c X chromosome

Expressed only in the placenta Murine calpain, expressed in brain, kidney, and the digestive tract Caenorhabditis elegans sex determination High expression in colon, small intestine, and testis tissues

Note: Chromosomal location data from (a) Pang et al. (1997); (b) Richard et al. (1995); (c) Ollendorf et al. (1992); (d) Mugita et al. (1997). Source: Data collated from Ohno et al. (1990); Richard et al. (1995); Schuler et al. (1996); Matena et al. (1998); H.J. Lee et al. (1998); Ma et al. (1999). Isolation of the new capn8 was recently reported by Braun et al. (1999b).

been isolated from goat liver and called UK114 (Ceciliani et al. 1996a, 1996b). UK114 has been regarded as a cancer marker. However, whether an inappropriate activation of calpains can lead to transformation and acquisition of properties that characterise cancer cells would be a fruitful avenue of approach. The possibility that calpains might, however indirectly, control gene transcription certainly makes an exploration of this avenue highly worthwhile.

MOLECULAR ORGANISATION OF CALPAINS The calpain molecule consists of two subunits: an 80-kDa and a smaller 30-kDa subunit. The gene encoding the large subunit of rat capn2 contains 22 exons, of which exons 3 to 21 encompass 33 kb of the rat genome. The corresponding cDNA of 3.2-kb size codes for a protein consisting of 700 amino acids. This protein shows a high degree (93%) of sequence homology with human capn2 and 61% homology with human capn1. The cDNA, when tagged to an expression vector, generated an 80-kDa protein that was found to be identical to human capn2 (De Luca et al. 1993). The 80-kDa subunit has four domains. Domain II is the cysteine protease domain and domain IV is the Ca2+-binding domain. The smaller subunit has two domains:

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domain V, the N-terminal glycine-clustering hydrophobic domain, and the Ca2+binding domain VI (Nishimura and Goll, 1991; Crawford et al. 1993; Minami et al. 1988). The calcium-binding domains (IV and VI of the two subunits) possess sequence similarities to CaM (Ohno et al. 1984). These domains contain four EFhands each (EF-hands 2–5). Two additional EF-hands occur in the 80-kDa subunit, one at the junction between domains II and III and the second one at a position Nterminal to EF-hand 2 (Theopold et al. 1995; Andresen et al. 1991). The association of the subunits occurs through domains IV and VI, and EF-hand 5 may play an important part in maintaining the dimeric organisation (Y. Minami et al. 1988; Kretsinger, 1997).

REGULATION OF PHYSIOLOGICAL EVENTS BY PROTEOLYTIC FUNCTION Calpains are not wide-spectrum proteases. Importantly, the limited number of substrates that they cleave are involved with the activation of specific cellular systems. Notable among their substrates are PKC-γ, the actin-binding proteins, α-fodrin and the homologous α- and β-spectrin, and growth factor receptor proteins (Lofvenberg and Backman, 1999; Saido et al. 1994; Croall and De Martino, 1991; Martin et al. 1995). It is to be expected that calpains would be involved in physiological events merely by virtue of their proteolytic activity. Several such events can be cited. One of these is keratinocyte differentiation. Calpains are actively involved in the proteolytic cleavage of profilaggrin into filaggrin in the process of keratin filament aggregation (Figure 25). They also may regulate the levels of MAPs during neuronal differentiation. During early brain development MAPs occur as MAP-1B and MAP2. MAP-1B occurs as two isoforms differing in the level of phosphorylation, and MAP-2 occurs as two isoforms with different molecular weights. The isoforms of MAP-1B as well as MAP-2 show equal sensitivity to calpain, but overall, MAP-2 is more susceptible to the proteolytic action of calpain than MAP-1B (I. Fischer et al. 1991). Calpain has been shown to be able to induce apoptotic cell death, and this is associated with the cleavage of substrate proteins such as α-fodrin (spectrin), Ca/CaM-dependent protein kinase IV, etc. Neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited condition characterised by a disposition to develop certain intracranial tumours such as vestibular schwannoma and meningioma. The NF2 gene codes for a protein called merlin, also known as schwannomin, and it is believed to function as a tumour suppressor. Mutations of merlin have been found in a significant proportion of NF2-associated tumours and to a lesser extent in sporadic schwannomas and meningiomas. However, NF2 tumours that show no mutations of the NF2 gene also have been found. Kimura et al. (1998), therefore, examined the possibility that the suppressor protein may be inactivated by mechanisms other than mutation. These authors found that a marked activation of calpain occurs in sporadic meningiomas. Merlin is cleaved by calpain, and inactivation of calpain restores merlin in meningioma cells in culture. It would appear, therefore, that proteolytic control by calpain might be a mechanism by which the tumour suppressor function of merlin is regulated. Much of this evidence is

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correlative, but admittedly proteolytic pathways are involved in the regulation of expression of several cellular regulatory proteins such as p53 (see below) and the mdm2 protein (L.H. Chen et al. 1997), which negatively regulates p53 function. Indeed, the interaction of mdm2 with p53 reduces p53 protein levels by targeting it to ubiquitin-dependent degradation (Kabbutat et al. 1997). The phosphoprotein p53 is intricately involved in the checkpoint control at the G1-M as well as at the G2-M transition of cells. p53 has a short half-life. Its degradation has been attributed to ubiquitination as well as to proteolysis by calpain. The PEST (Pro, Asp/Glu, Ser, and Thr) motif is generally regarded as a signal in the substrate that is required for its recognition by an intracellular protease. p53 contains the PEST sequence, which is required for proteolysis by calpain. Ubiquitination of certain substrates, e.g., STE3 receptor of yeast, has been attributed to a PEST-like sequence (Roth et al. 1998), although we do not know if this is the case with p53 ubiquitination. Mutated p53, which has an increased half-life, does not contain these sequences. However, in some instances, p53 may be degraded by calpain in the absence of the PEST sequences. Wild-type p53 is also stabilised when cells expressing it are treated with inhibitors of calpain. However, the presence of the PEST motif may not be an absolute requirement for calpain function (Carillo et al. 1996; Van Antwerp and Verma, 1996). The human papilloma virus (HPV) E6 protein promotes p53 degradation through the ubiquitin-dependent pathway, and this is unaffected by inhibitors of calpain. Overall this suggests that the stability of p53 might be controlled by the proteolytic action of calpain (Kabbutat and Vousden, 1997). Similarly, cyclin D1, another regulator of cell cycle progression, has been reported to be a target of calpain proteolysis. Choi et al. (1997) showed that cyclin D1 is rapidly proteolysed in NIH3T3 cells when deprived of serum, but calpain inhibitors reversed this. These inhibitors also raise the half-life of cyclin D1. Proteolytic cleavage of the cytoplasmic domain of the cytokine receptor γ-chain (γ-c) by calpain may represent an important regulatory or control event in γ-cmediated signalling. Noguchi et al. (1997) demonstrated that the small subunit of calpain cleaves γ-c, which contains the hydrophilic amino acid PEST sequence, but not when the PEST sequence is mutated. Calpain inhibitors inhibited γ-c cleavage in TCR-stimulated murine thymocytes. Furthermore, in these cells, anti-CD3 cleaved γ-c, but calpain inhibitors inhibited this. Calpain inhibitors enhance the proliferative effect of anti-CD3 antibodies, and this is effectively prevented by γ-c antibodies. Cytokines and growth factors transduce their signals by using the Janus tyrosine kinase (Jak) and STAT (signal transducer and activator of transcription factors) pathway (O’Shea, 1997; Darnell, 1997). The binding of these ligands to the appropriate receptors results in the activation of the Jak tyrosine kinase and the activation of latent transcription factors called STAT proteins that occur in the cytoplasm. In fact, STAT-3 appears to regulate G1–S transition of cells in response to cytokine stimulation (Fukada et al. 1998). Calpain inhibitors have been shown to influence cytokine-mediated cell proliferation and STAT protein synthesis (Noguchi et al. 1997). It would appear, therefore, that the proteolytic function of calpain could constitute an intricate part of signal transduction pathways. These are prime examples of specific modes of regulatory function performed by calpain, in which a specific target is hydrolysed by calpain to bring about the

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regulation of a particular physiological event. In addition, one should bear in mind the finding that calpain can degrade transcription factors such as AP1. Hirai et al. (1991) reported that the c-jun and c-fos gene products, which form the AP1 transcription factor, contain the PEST sequence that is the target sequence of calpain action. Calpain inhibitors increase the expression of AP1 (Zhu et al. 1995). Therefore, one could expect calpain would have a generalised effect on the transcription of a variety of genes, merely by virtue of its ability to regulate the function of the transcription factor itself.

INVOLVEMENT OF CALPAINS IN DEVELOPMENT AND DIFFERENTIATION There have been no significant investigations into the involvement of calpains in developmental processes. However, there is some evidence that could be construed as providing a partial burden of proof. For instance, the tra-3 gene has been implicated in the process of sex determination during early development of the nematode C. elegans. tra-3 lacks the CaM-like calcium-binding sites (of domain IV), but EF-hand 6 is conserved. Its involvement in sex determination has been suggested to be a consequence of potentiating the function of another gene, e.g., tra-2 (Barnes and Hodgkin, 1996). The conservation of EF-hand 6 in tra-3 might suggest that the latter is required in calcium signalling in the sex determination cascade. Calpain homologues of Drosophila may be associated with developmental abnormalities. Mutations of the sol gene of Drosophila result in neuronal defects that cause behavioural abnormalities. Delaney et al. (1991) cloned two alternatively spliced transcripts of the sol locus. The predicted sequences of the proteins encoded by them showed similarities to different regions of calpain. The carboxylic region of the larger protein showed similarities to the catalytic domain of calpain, and the N-terminal region contained several zinc finger-like repeats. The smaller predicted protein did not contain the calpain catalytic domain and had only two zinc finger-like repeats. Calpains also have been found in the human brain. Their possible role in differentiation is illustrated by changes in calpain expression in the SH-SY5y neuroblastoma cells induced to differentiate by retinoic acid (RA) treatment. RA induces differentiation and neurite outgrowth in these cells. After 3 days of RA treatment, the levels of activated and precursor forms of calpain have been found to increase by >50% and 26%, respectively, in the particulate and cytosolic fractions of the cells. However, the levels of calpastatin, an endogenous inhibitor of calpain, were unchanged. Thus the process of neurite extension seems to be associated with a net activation of calpain (Grynspan et al. 1997). The calpain–calpastatin system seems to be regulated during processes of proliferation and differentiation of osteoprogenitor cells in response to the bone morphogenetic protein (Murray et al. 1997). Further evidence has been derived from the pattern in the expression of capn3 gene that is discernible during early foetal development. This could be interpreted as an indicator of its association with, if not participation in, morphogenesis. capn3 expression is detected only in skeletal muscle at this time. At an earlier stage, capn3

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expression is found in the heart and it disappears subsequently. Variants of capn3 generated by alternative splicing are also detected in smooth muscle (Fougerousse et al. 1998). The requirement of both calpain and the proteasome proteolytic systems in the differentiation of myoblasts is exemplified by the work of Ueda et al. (1998). L8 myoblast cells grown in the absence of mitogen show enhanced levels of creatine kinase together with the differentiation of myotubes. However, when calpain inhibitor and the proteasome inhibitor lactacystin are added to these cultures, creatine kinase levels are markedly reduced together with inhibition of myotube differentiation. Although calpains have been implicated in neurodegenerative diseases, a diametrically opposite view is held by some that calpains may contribute to processes such as the remodelling of neuronal dendritic structures after neuronal injury. In murine cortical cultures subjected to dendritic injury, calpain did not seem to augment the injury. Quite the contrary, calpain appeared to assist dendritic recovery (Faddis et al. 1997). Similarly, the calpain inhibitor MDL28170 promotes neuronal recovery from injury by moderate hypoxia (Z.F. Chen et al. 1997). There are now clear indications that capn1 may be expressed in the early stages of the development of the skin (Michel et al. 1998, 1999). Michel et al. (1999) have provided definitive evidence for this. capn1 appears at day 54 of gestation of the human foetus, in the basal layer and in the periderm of the developing dermis. By day 125, capn1 is found in the granular layer. A possible link-up with differentiation is indicated by a significant reduction of capn1 expression in biopsies obtained from harlequin ichthyosis, a condition known to result from abnormal terminal differentiation of the skin. The changes in capn1 expression were apparently specific for harlequin ichthyosis and were not encountered in other skin disorders. These findings are compatible with the known ability and involvement of capn1 to influence the proteolytic cleavage of profilaggrin to monomeric filaggrin during terminal differentiation. It follows from the association between calpain and changes in cellular morphology that the cytoskeleton could be an important target of calpains. Emori and Saigo (1994) studied the expression of calpain in developing Drosophila embryos. In the early stages of cleavage of the fertilised eggs, calpain was found in the presumptive cleavage furrows and was co-localised with actin caps. As stated in an earlier section, calpains might regulate the levels of MAP components of the neuronal cytoskeleton (I. Fischer et al. 1991). Chakrabarti et al. (1993) studied the expression of calpains during brain development in rats, especially in relation to myelin formation. They found low capn2 levels during days 1 to 7 (postparturition), but these reached a peak between days 16 and 30. In rats that were more than 30 days old, roughly half of the calpain activity in the brain was found in the myelin component, whereas in 1 to 10-day old rats the majority of capn2 activity was cytosolic. Furthermore, this contrasted starkly with the pattern of expression of capn1, suggesting that capn2 is associated with the formation of the myelin sheath.

CALPAINS IN CELL PROLIFERATION AND APOPTOSIS The involvement of calpain in the cell cycle traverse by proteolytic degradation of p53, which controls the cell cycle transition checkpoints, has been alluded to earlier. It is to be expected, therefore, that calpain will powerfully influence cell proliferation

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and growth. Calpeptin, an inhibitor of calpain has been found to inhibit the growth of the breast cancer cell lines MCF7, T47D, and ZR75 (E. Shiba et al. 1996). These cell lines are oestrogen receptor (ER) positive, but in a subsequent report it has been demonstrated that the calpeptin-mediated inhibition of growth is not due to a suppression of the hydrolysis of ER by calpain. This could be due to one or more of a variety of mechanisms that interfere with cell cycle control. There is a significant inverse relationship between the expression of ER and EGFr in some of these cell lines. It would be interesting to see if the transduction of EGF signal via the cytoskeletal machinery is affected in any way by calpeptin. Aside from that, it is possible that calpeptin influences p53 stability by inhibiting calpain. Apoptosis or programmed cell death is a natural physiological phenomenon. Cells undergoing apoptosis display distinctive morphological changes, such as condensation of nuclear chromatin and shrinkage of the cytoplasm. The cells break up into membrane-bound apoptotic bodies. The nuclear DNA is fragmented by Ca2+/Mg2+-dependent endonucleases. DNAse I and DNAse II have been regarded as putative candidates in DNA degradation, but recently Enari et al. (1998) have identified a caspase-activated DNA nuclease that is involved in the DNA degradation that occurs during apoptosis. The fragmentation of the DNA produces a characteristic ladder pattern of 180 to 200 bp of oligomers. Perturbations of the intracellular calcium levels are an important feature of apoptosis. High intracellular Ca2+ levels produced by calcium influx or its release from intracellular stores can lead to apoptosis (Orrenius et al. 1996). However, there is conflicting evidence about the induction of apoptosis by calcium chelators and calcium ionophores. Nevertheless, the bcl2 family of genes, which is composed of inducers as well as inhibitors of apoptosis is known to regulate intracellular calcium (see Sherbet and Lakshmi, 1997b for references). Calcium signalling is known to activate a number of proteases. Among the notable ones are calpains and caspase. Both proteases have been implicated in apoptosis. Calpains often have been attributed with the ability to induce apoptotic cell death (Figure 21). They are said to be actively associated with T-cell activation and apoptosis (Squier et al. 1994; Sarin et al. 1995; S.J. Martin and Green, 1995). In mature T lymphocytes the induction of apoptosis by TCR is protease dependent (Sarin et al. 1995). The binding of the appropriate ligand of the TNF family to the Fas receptor initiates the activation of caspases and the down-stream protease cascade that leads ultimately to DNA degradation (see below). Dexamethasone is known to induce apoptosis of thymocytes, accompanied by Ca2+-dependent proteolytic activity. This is also accompanied by the autoproteolysis of the capn1 proenzyme, which suggests that calpain activation is taking place. Calpain inhibitors block apoptotic death (Squier et al. 1994). However, inhibitors of calpains have also been found to induce apoptosis. W. Zhu et al. (1995) found that two calpain inhibitors caused apoptosis of human prostate adenocarcinoma cells. However, in L1210 leukaemia cells, inhibition of calpain did not induce apoptosis (Wojcik et al. 1997). Apoptotic death can be induced in rat hippocampal pyramidal neurones in culture by exposing them to ABP and staurosporine. This can be inhibited by the calpain inhibitor MDL28170 (Jordan et al. 1997). There is a preliminary report that prevention of calpain activation produces resistance to necrosis in hepatocellular carcinoma cells

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FIGURE 21 Involvement of the calcium-activated enzymes calpains and caspases in the induction of apoptosis. Raising intracellular calcium levels leads to activation of these enzymes and to apoptosis. In many instances it has been shown that when their activity is inhibited apoptosis is also suppressed. The Bcl-2 family has apoptosis-inducing genes as well as genes that inhibit apoptosis. These regulate intracellular calcium levels and influence apoptosis through the caspase activation mechanism. The ced-9 gene of C. elegans encodes a protein that has apoptosis-inhibiting activity.

(A.S. Arora et al. 1995). However, necrosis and apoptosis are two distinct processes, and it would not be appropriate to discuss, in the same breath, how they are affected by calpain. A possible pathway by which calpain could influence apoptosis has been suggested by Meredith et al. (1998) who showed that during apoptosis of human umbilical vein endothelial cells, the cytoplasmic tail of integrin β3 undergoes limited proteolysis. They showed not only that calpain is activated during apoptosis but also that calpain inhibitors prevent the proteolysis of the cytoplasmic domain of the integrin. Needless to say, more work needs to be done in order to establish the pathways of apoptosis that are influenced by calpain.

CALPAINS IN CELL SPREADING AND MIGRATION As stated often in these pages, the cytoskeletal dynamics are of overriding importance in signal transduction, cell morphology, and cell adhesion and locomotion. Calpain, by virtue of its proteolytic action, is bound to affect the integrity of the cytoskeleton and the adhesive and migratory properties of cells. Cell spreading is inhibited by the calpain inhibitors, calpeptin and MDL28170 (K.A. Potter et al. 1998). K.A. Potter et al. (1998) cloned a variant NIH3T3 cell line that overexpressed (two- to eight-fold) the calpain inhibitor, calpastatin. These cells markedly differed from the

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parent cell line with respect to their morphology, spreading ability, and cytoskeletal characteristics. The variant cells failed to extend lamellipodia and showed abnormal filopodial extension and retraction. The calpain activity in these cells was found to be substantially reduced. Calpain inhibitors have been found to increase cell adhesiveness and reduce cell migration. A Chinese hamster ovary (CHO) cell line that expresses capn1 intrinsically at low levels has a low migratory ability (Huttenlocher et al. 1997). However, cell migration involves transient adhesion to and release from the substratum and these interactions require the linkage of the focal adhesion receptors to the cytoskeleton. The linkage of the cytoplasmic tail of integrin receptors is of crucial importance in the process of cell adhesion to the substratum. Calpain and similar proteins would affect it by cleaving the cytoplasmic domain of the receptor and may be regarded to be negatively regulating adhesion (Meredith et al. 1998). Palacek et al. (1998) have described migration as a function of cyclic membrane protrusion and adhesion at the leading edge and cytoskeletal contraction and detachment at the rear edge. They go on to demonstrate that calpain is involved in dissociation of the integrin receptor linkage with the cytoskeleton. These authors argue that an inhibition of integrin release by calpain decreases the speed of cell motility, and, inter alia, this may be due to the increased density of adhesive bonds or the strength of adhesion. Huttenlocher et al. (1997) have stated categorically that inhibitors of calpain increase cell adhesiveness and decrease the rate of detachment, whereas Meredith et al. (1998) regard calpains as negative regulators of adhesion. Because the adhesion of the leading edge and the retraction of the rear edge will necessitate a differential regulation of calpain, it would be interesting to see if the endogenous calpain inhibitor, calpastatin, might come into the adhesion dynamics. Calpastatin is itself subject to proteolysis by calpain (Nagao et al. 1994). For instance, very little is known about the intracellular localisation of calpastatin in relation to the direction or speed of cell locomotion. Furthermore, as indicated by the effects of calpain on integrin-mediated signal transduction, other components, such as talin, that are involved in the linkage of cytoplasmic domain of the integrin receptors with the cytoskeleton could conceivably form a part of the total picture.

CALPAINS IN INTEGRIN-MEDIATED CELL ADHESION AND SIGNAL TRANSDUCTION The inhibition of integrin function is believed to be responsible for the downregulation of these adhesive and migratory abilities. Integrins are transmembrane proteins constituting focal adhesion plaques. Talin is a protein that links integrins to the cytoskeletal structures. Interestingly, talin has calpain susceptible sites. Therefore, calpain could affect the processes of cell adhesion and signal transduction, which are mediated by integrins. Using antibodies that recognise the calpain-susceptible sites of talin, Inomata et al. (1996) have demonstrated the involvement of calpain in integrin-mediated signal transduction. Calpain is able to cleave the cytoplasmic domain of β3 integrin (Du et al. 1995) and this could interrupt the signalling pathway. The focal adhesion kinase (pp125FAK) is a non-RTK associated with integrin-mediated signal transduction. Cooray et al. (1996) have argued that calpain

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may have a role of terminating signal transduction in human platelets, by modifying pp125FAK by proteolysis and reducing its kinase activity as well as its subcellular location. Calpain indeed affects several events occurring after platelet aggregation, e.g., the attachment of the integrin tail to the cytoskeleton, as well as tyrosine phosphorylation of cytoskeletal protein and fibrin clot retraction (Schoenwaelder et al. 1997). Paxillin is another protein associated with focal adhesion plaques that is degraded by calpain (R. Yamaguchi et al. 1994).

CALPAINS IN CANCER GROWTH AND PROGRESSION Calpain is significantly involved, as discussed above, in a number of physiological processes that are integral features of the development and progression of cancer. We have seen that the suppressor phosphoprotein p53, which is closely linked with the control of cell cycle progression, is degraded by calpain. Mutations of p53 gene are a common event in tumorigenesis, and these lead to the abrogation of normal checkpoint control function exerted by wild-type p53. We have noted also that calpain is able to regulate the levels of cyclin D. Deregulation of the cell cycle using either target will lead to the deregulation of cell proliferation. Besides, one can also envisage a deregulation of the cell cycle under conditions where calpain is overexpressed per se or calpain activators are inappropriately expressed. Besides, there is some evidence that the calpains may influence apoptosis in certain cell lines. This needs to be established beyond reasonable doubt, because there are reports to the effect that both calpain and its inhibitors are capable of inducing apoptosis. Tumour growth could be seen as a net outcome of these opposing factors of proliferation and apoptosis. A dynamic equilibrium between these two can result in growth stasis. Such a state of equilibrium also can apply to the growth of metastases and can lead to a state of dormancy of metastatic deposits. As shown in Figure 22, calpains and caspases may have positive or negative regulatory effects on tumour growth and metastatic spread. Although the expression of calpain in cancers has not been the subject of much investigation, there is some information on the association of calpain inhibitors with cancers. Squamous cell carcinomas of the lung have been reported to express a protein — squamous cell lung carcinoma antigen (SCCA) — that is able to inhibit capn1 (Kato, 1996). However, because SCCA inhibits other proteinases such as cathepsin L, it is difficult to assess the individual merit of calpain inhibition in squamous cell lung carcinoma. It should be borne in mind that the cathepsin family proteinases play an important role in tumour development and progression to the metastatic state, although, admittedly, cathepsin L has not been found to be as crucially important as other members of the family (see Sherbet and Lakshmi, 1997b). Braun et al. (1999a) have reported an enhanced expression of capn1 mRNA in clear cell renal carcinoma. The level of expression related closely with the presence of metastatic tumours in regional lymph nodes. Tumours with metastasis expressed capn1 mRNA at a higher level than tumours that had not spread to regional lymph nodes. Ceciliani et al. (1996a) isolated three low molecular weight proteins — UK101, 114, and 150 — from goat liver. These proteins are membrane associated. Of these,

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Tumour growth Metastases

Apoptosis

Deregulation of cell proliferation

Endothelial cell apoptosis Antigenic response in host

Calpain/Caspase

Endogenous activators

FIGURE 22 Putative pathways of the involvement of calpain and caspase in the growth of the primary tumour and its metastases. The basic position is that these proteases can alter the steady state of population growth in both primary and secondary tumours. Caspase, but not calpain, has been credited with the ability to induce apoptosis of endothelial cells that can assist the entry of tumour cells into the vasculature. Endogenous activators of the enzymes have been suggested as a possible mode of immunising the host that can conceivably lead to loss of enzyme activation and potential loss of growth.

UK150 has been characterised to be a glycoprotein. UK114 has been suggested to be a marker for cancer (Ceciliani et al. 1996b). UK114 is a 14-kDa protein made up of 137 amino acid residues and shows sequence homology with the calpainactivating factor isolated from rat and bovine brain (Melloni et al. 1998a, 1998b). Furthermore, the goat liver UK114 also possesses calpain-activating properties (Melloni et al. 1998a). Bartorelli et al. (1996) demonstrated that administration of UK101 to patients with breast and colon carcinomas elicited significant immune responses from the patients. Antibody titres against UK114 and 150 rose by 63 and 87% in breast and colon cancer patients, respectively. Although there has been very little background data on the levels of UK114 and 150 in patients with cancer, UK101 therapy appears to have been reasonably successful (Mor et al. 1997). It was administered to 217 patients with metastatic colon cancer. Twenty-five patients in this trial showed reduction in metastatic mass, and 40% of the patients showed static disease. Patient survival correlated with both the baseline Karnofsky index score and anti-UK114 titre. Bussolati et al. (1997) have suggested that the antitumour effects are due to the cytolytic action of anti-UK114 antibodies. The latter were found to be cytolytic in vitro and were able to inhibit the growth of human tumours as xenografts in nude mice.

CALPAINS IN MYELODEGENERATIVE DISEASES During early stages of brain development, calpains show a pattern of expression that is compatible with the view that they are mainly associated with the formation of the myelin sheath. Subsequent to this, they are found in the cytosol rather than in the myelin sheath. Myelin basic protein (MBP) and myelin-associated glycoprotein

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are substrates of calpain. Banik et al. (1994) have shown that component I of human MBP is more susceptible to proteolysis by calpain than components II and III. They have identified two major and several minor cleavage sites in MBP. Because MBP is degraded in demyelinating diseases such as multiple sclerosis (MS), the potential role of calpains in the pathogenesis of MS has been studied in several laboratories. The presence of calpains 1 and 2 in myelin sheath was known for some time. High levels of capn1 and capn2 activity in myelin and premyelin were found to correlate with delayed myelination and high myelin turnover in the demyelinating paralytic tremor (PT)-mutant rabbit (Domanskajanik et al. 1992). Calpain activity has been reported to increase in MS tissue and cerebrospinal fluid. Macrophages, lymphocytes, and reactive glial cells are the sources of the enhanced calpain activity. Calpains are found also in myelin-forming oligodendrocytes and Schwann cells (Banik et al. 1994). Both capn1 and capn2 occur in human lymphoid cell lines. Lymphocytic cell lines predominantly expressed capn1, but in monocytic cells the capn2 tended to be the major isoform (Deshpande et al. 1993). Activation of lymphoid cells with PMA and the calcium ionophore A23187 enhances the expression of both calpain protein and the corresponding mRNA (Deshpande et al. 1995a). Deshpande et al. (1995b) demonstrated further that activation of calpains was accompanied by the degradation of MBP and rat CNS myelin in vitro. A large proportion (60 to 80%) of the degradative activity was attributable to calpain, together with contributions from other proteases. It is conceivable that this degradation results in the exposure of antigenic epitopes. On the other hand, calpains occur in normal myelin and are associated with myelin formation during developmental stages. Therefore, calpains could be a normal constituent of myelin and may be involved in turnover of myelin proteins in the normal course of physiological events (Z.H. Li and Banik, 1995). Possibly, activated infiltrating lymphoid cells generate a higher level of myelin breakdown and greater quantities of antigenic products. The status of the calpain–calpastatin system has been studied in experimental allergic encephalomyelitis (EAE), which is an animal model of MS, mainly in the laboratory of Shields and Banik. In Lewis rats with EAE, the optic nerves have been reported to show a marked increase in calpain content, although RT-PCR did not reveal any changes in calpain messenger RNA. Calpain-specific degradation of fodrin increased by 46% and the myelin-associated glycoprotein decreased by 25%. The endogenous inhibitor of calpain, i.e., calpastatin, was unaltered (Shields and Banik, 1998a,b). Human MS plaques reportedly show very similar alterations when compared with white matter from normal subjects (Shields et al. 1999a). Interestingly, alterations in calpain expression occur in peripheral lymphoid organs before the onset of symptomatic EAE (Shields et al. 1999b). MS plaques are characterised by reactive gliosis, infiltration of inflammatory cell, and a focal destruction of myelin and oligodendrocytes. An enhanced expression of calpain is said to occur in glial and inflammatory cells (Shields et al. 1998a, 1998b). These observations putatively implicate calpain in the autoimmune-mediated demyelination process that occurs in EAE and MS. The regulation of the activity of this normal calpain component deserves serious study. Growth factors such as NGF, PDGF, and acidic as well as basic FGF exert different effects on capn1 and capn2 expression in transfected Schwann cells

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(Neuberger et al. 1997). Neuberger et al. (1997) reported that cAMP and NGF inhibited both capn1 and capn2 activity. Both aFGF and bFGF enhanced capn1 activity by 37% and capn2 by 58%. In contrast, PDGF down-regulated both isoforms. Interestingly, the enhanced calpain activity following FGF treatment was associated with markedly reduced levels of calpastatin activity. In comparison, in cells exposed to cAMP and NGF, calpastatin levels were nearly comparable with those of control cells. These observations suggest that growth factors strongly affect the dynamic equilibrium between calpains and their endogenous inhibitor. The experiments of Neuberger et al. (1997) also indicate the possibility of their intracellular translocation. The expression of calpain isoforms as well as calpastatin isoforms has been studied in the EAE model (Shields and Banik, 1998). The transcription of neither the calpain isoforms nor calpastatin isoforms showed an up-regulation in EAE, but the expression of calpain protein increased more than fourfold. Calpastatin translation was also increased in EAE. This suggests another putative mechanism by which the calpain–calpastatin dynamics could be altered in myelodegenerative conditions.

CALPAINS IN MUSCULAR DYSTROPHY Several forms of muscle myopathy are known. The major conditions are Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and limb girdle muscular dystrophy (LGMD). LGMD is recognised to be of two types: the autosomal-dominant type 1 (subtypes 1A–1D) and the recessive type 2, with several subtypes. Calpain has been implicated in the pathogenesis of LGMD-2A.

ASSOCIATION OF CALPAINS WITH DUCHENNE MUSCULAR DYSTROPHY Calpains are activated in many pathological and aberrant physiological conditions that result in muscle wastage. There is much evidence that calpains degrade a number of myofibrillar proteins, and there is consensus that calpains participate actively in the early stages of myofibril breakdown (F.C. Tan et al. 1988). Calpains as well as cathepsins have been associated with muscle fibre degradation occurring in inflammatory muscle myopathy (Kumamoto et al. 1997). The characteristic muscle weakness and the progressive degradation of striated muscle encountered in DMD and BMD appear to be attributable to calpain activity, besides the abnormalities in the DMD and BMD gene loci that alter the function of dystrophin and the dystrophin–glycoprotein complex. DMD is generally associated with a loss of dystrophin, which links the sarcolemma with the actin cytoskeleton. Although the precise function of dystrophin is unclear, its loss is associated with increased calcium influx and high intracellular levels of calcium. Being calciumactivated enzymes, calpains were implicated in the pathogenesis of this chronic degenerative disease many years ago (Arahata and Sugita, 1989). Kumamoto et al. (1995) studied the expression of calpains and calpastatin in DMD and BMD. Calpains occurred at markedly elevated levels in muscle fibres that had atrophied. In necrotic fibres, both calpain and calpastatin were expressed at moderate levels, but hypertrophic and opaque fibres manifested no activity at all. Ueyama et al. (1998) have recently confirmed the increase of capn1 and capn2 proteins and their mRNAs

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in progressive muscular dystrophy as well as in ALS. However, capn3 appeared to be unaffected. These findings suggest a possible deregulation of the calpain–calpastatin system as the cause of progressive muscle degeneration. Fasting has serious consequences in terms of calpain activation. A two-fold increase in the degradation of myofibrillar protein of rabbit skeletal muscle occurred in response to fasting, together with an increase in mRNA levels encoding capn1 and capn2 as well as calpastatin mRNA levels (Ilian and Forsberg, 1992, 1994). Fasting raised the mRNA levels of cathepsin D and proteasome. These changes appear to be specific for skeletal muscle. Ilian and Forsberg (1994) did not find any changes in the protease mRNA levels in other tissues such as liver, lung, or kidney. The transcription of the genes seems to be up-regulated to meet the need to mobilise muscle protein in response to the physiological insult. Therefore, Ilian and Forsberg (1992) suggest this explains why they did not encounter any increase in capn1 and capn2 protein levels. However, it is essential to know the stability of the messages, as well as the half-life of the enzymes, before relating the findings to the continued requirement for protein mobilisation. It should be borne in mind also that other proteases would have contributed to the process of protein mobilisation as well. At any rate, Kumamoto et al. (1992) confirmed the earlier findings of Ilian and Forsberg (1992) relating to the increase of calpains in response to fasting. They also demonstrated that capn1 and capn2 occurred in the Z-band, where the degradation of myofibrils is initiated. Immunostaining studies showed that the Z-band contained twice as much calpain and calpastatin as the I-band or the A-band.

CALPAINS

AND

LIMB GIRDLE MUSCULAR DYSTROPHY

Three autosomal dominant and several autosomal-recessive loci have been identified. With the view of placing the following discussion in the proper perspective, but at the risk of a minor digression, details about the LGMD subtypes and the molecular features associated with them have been provided in Table 11. LGMD-2A is an autosomal-recessive form of muscular dystrophy. The skeletal muscle-specific homologue capn3 has been implicated in LGMD-2A. The capn3 gene is located in the region of chromosome 15q15.1–q21.1 (Fougerousse et al. 1994). Five distinct genes have been identified with LGMDs, and capn3 as a putative LGMD-2A candidate gene (Chiannilkulchai et al. 1995; Beckmann et al. 1996). Recently, capn3 mutations have been found to co-segregate with the disease in families with LGMD-2A, and this has led to the suggestion that the disease is due to a defect in the enzyme rather than to abnormalities of any structural proteins (Richard et al. 1995). This has been confirmed by recent findings that mutations of the gene result in the loss of proteolytic activity of capn3 (Y. Ono et al. 1998). Therefore, there seems to be a link between this loss of proteolytic activity of capn3 and the pathogenesis of LGMD2A. Spencer et al. (1997) did not find capn3 in muscle biopsies from LGMD-2A patients, but did detect it in control subjects as well as in non-LGMD-2A patients. Capn3 is said to undergo rapid autolysis and it has a half-life of less than 1 hr, which could be one of the reasons for the failure of detection. Sorimachi et al. (1995) found that capn3 also interacts with connectin (titin), which spans the M- to Z-lines of muscle sarcomeres. They have, therefore, suggested that connectin may regulate

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TABLE 11 Limb Girdle Muscular Dystrophy Types and Associated Molecular Features LGMD Type/Subtype

Associated Molecular Features

Ref.

Autosomal Dominant LGMD-1A LGMD-1B LGMD-1C

Caveolin-3 mutations, 3p25 Also in DMD and mdx mice

Minetti et al. (1998); McNally et al. (1998a) Vaghy et al. (1998)

LGMD-1D

LGMD-2A LGMD-2B (Myoshi myopathy) LGMD-2C

Autosomal Recessive Calpain, 15q15.1 Dysferlin mutations, 2p13 γ-Sarcoglycan missense, α-sarcoglycan mutation and loss, 13q12

LGMD-2D

ε-Sarcoglycan mutation, 17q12-q21

LGMD-2E

β-Sarcoglycan missense mutations, 4q12

LGMD-2F

δ-Sarcoglycan mutations, 17q11-q12

Chiannilkulchai et al. (1995); Moreira et al. (1997) J. Liu et al. (1998); Bashir et al. (1996, 1998) Van der Kooi et al. (1998); Jung et al. (1996); Moreira et al. (1997) Ettinger et al. (1997); McNally et al. (1998b); Moreira et al. (1997) Bonnemann et al. (1996); Moreira et al. (1997) Nigro et al. (1996); Duggan et al. (1997); Moreira et al. (1997)

Note: Jung et al. (1996) state that LGMD-2A–2E all show abnormal expression of α, β, γ, and δ-sarcoglycans.

capn3. Obviously, there is a need for much further work to elucidate the role of capn3 in LGMD-2A, especially with a view to relating it to the significance of the association of sarcoglycans with LGMD. As Spencer et al. (1997) have pointed out, capn3 was regarded as the agent responsible for the posttranslational generation of α- and β-sarcoglycans, but it may not be so involved.

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Caspases in Apoptosis, Cell Migration, Proliferation, and Neoplasia

CASPASES IN APOPTOTIC CELL DEATH Calcium-mediated activation of caspases is also associated with apoptosis. The apoptotic pathway involves a cascade of proteolytic events mediated by caspases, their activators, and their repressors. Caspases are cysteine proteases of a family of interleukin-1β-converting enzymes (ICE). A number of proteases — ced-3, ced-4, and ced-9 — have been identified in the nematode C. elegans. Of these, ced-3 is a caspase. A human homologue of the ced-4 protein, called apaf-1, has been described (Zou et al. 1997). Three new forms of apaf-1 have since been identified in mammalian cells (Hahn et al. 1999). The apaf-1 protein appears to promote cytochrome c-dependent activation of caspases (Zou et al. 1997; Kluck et al. 1997; P. Li et al. 1997). Cytochrome c has been found to be involved in caspase activation as an early event in many forms of apoptosis. However, it may not be involved in at least Fasinduced activation of caspase and apoptosis. (Vier et al. 1999). The chromatin condensation and nuclear fragmentation that occur in apoptosis involve, besides the caspases, caspase-activated DNAase (Enari et al. 1998; X.S. Liu et al. 1998). When apaf-1 is stably transfected into HL-60 cells, not only is apaf-1 overexpressed but there is also a marked induction of apoptosis (Perkins et al. 1998). According to two recent reports, apaf-1 (–/–) null mice died at day 15 of embryonic growth and showed reduced apoptotic cell loss and overgrowth of the brain. The null mutants also manifested marked craniofacial abnormalities. Markedly enhanced proliferation of neuronal cells was also encountered in these embryos. Apaf-1 (–/–) cells seemed to resist apoptosis, with an attendant impairment of caspase activation (Yoshida et al. 1998; Cecconi et al. 1998). In oncogene E1A-dependent induction of apoptosis, in which the caspase is activated by the oncogene product, the activated caspase-9 forms a complex with apaf-1 and cytochrome c (Fearnhead et al. 1998). As shown in Figure 21, some members of the bcl-2 family of genes can inhibit apoptosis. A general model has been proposed that the bcl-2 proteins inhibit apoptosis by binding to apaf-1, thereby preventing the activation of caspases. However, this model has been disputed by Morishi et al. (1999), who believe that the inhibition might not be a direct effect of the sequestration of apaf-1 by bcl-2 proteins.

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Caspase-9 might initiate the programme of apoptosis or activate other caspases downstream and ultimately cleave downstream target proteins, which brings about apoptotic disintegration of the cell (Thornberry, 1997; Thornberry and Lazebnik, 1998). Cytochrome c-mediated activation of capase-9 seems to be followed by the activation of a string of other caspases. When cell extracts are depleted of caspase9, the activation of the downstream caspases does not occur. This indicates that activation of capsase-9 is obligatory for the activation of other caspases by cytochrome c (Slee et al. 1999). In Jurkat cells induced into apoptosis, caspase-3 occurs with caspase-6 in the activation complex. HSP60 is also found in this complex (Xanthoudakis et al. 1999); these authors have suggested that this HSP may participate in protein folding and aid in the proteolytic activation of caspase-3. In vitro, HSP60 aided the activation of the caspase by several caspase promoters. As many as ten caspases (caspases-1 to -10) have been identified. These form three natural groups based on their mode of activation from the corresponding procaspase form. Caspases-1, -2, -4, and -5 show autocatalytic activation from their pro-caspase form. The pro-caspases are believed to bind to activator molecules and lead to their oligomerisation and autoactivation. The second group composed of caspases-3, -6, -7, and -9 do not show autocatalytic activation. Lysosomal cysteine proteases may be involved in the activation of some caspases (Ishisaka et al. 1998). The overexpression of caspases-3 and -7 leads to apoptosis of the prostate cancer cell lines called LNCaP. In this system, caspase-7 seems to be activated by proteolysis (Marcelli et al. 1999). Caspases-8 and -9 undergo TNF-R1/Fas (CD95) mediated activation. The Fas ligand is a member of the TNF family. It is expressed in activated T cells. The apoptosis of target cells is induced by the binding of this ligand to its receptor (Nagata and Golstein, 1995; Nagata, 1997). The occupancy of Fas has been found to lead to the activation of caspases (Enari et al. 1995; Longthorne and Williams, 1997; Armstrong et al. 1996; Boldin et al. 1996) and of downstream proteases (Nagata, 1997; Fraser and Evan, 1996). The outcome of the function of this proteolytic cascade initiated by Fas binding is DNA fragmentation. The activation is believed to be due to the binding of caspase to the activated membrane receptor complex. Another line of evidence that strongly supports the role of caspases concerns the resistance of some cell systems to the induction of apoptosis by Fas receptor binding or other factors. Perara and Waldmann (1998) have used peripheral blood monocytes, which undergo spontaneous apoptosis unless the culture medium is supplemented with serum, growth factors, bacterial LPS, or cytokines. This induction of resistance to apoptosis is accompanied by a marked down-regulation of caspase8. Deficiency of caspase-8 also makes the Jurkat cell line JB-6 resistant to Fasmediated apoptosis (Kawahara et al. 1998). T.S. Zheng et al. (1998) deny any role for caspases in the induction of Fas-mediated apoptosis of liver cells, but recognise that caspase-3 may be instrumental in bringing about morphological changes by cleaving substrate proteins. Equally, as suggested by Kawahara et al. (1998), cell killing triggered by Fas binding could take two pathways. One of these is via caspase8, and the other is more akin to necrosis that does not involve caspases. It has been suggested also that HSPs may interfere with apoptotic cell death. HSP70, for instance, seems to ensure cell survival by inhibiting caspase-3-dependent events of

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apoptosis (Jaattela et al. 1998). However, the involvement of HSPs must be interpreted with some caution, especially in light of HSP60 being shown to be possibly associated with the activation complex of caspases. Caspases have several substrates such as poly (ADP-ribose) polymerase (PARP) (see below), lamin A, and several other cytoplasmic and nuclear proteins (see review by Zhivotovsky et al. 1997). The actin-binding proteins, α-fodrin and gelsolin, are cleaved by caspase-3. Actin itself is cleaved by caspase. According to Mashima et al. (1999), this results in the generation of two actin fragments of 15 and 31 kDa. When introduced into cells, the 15-kDa actin fragment brings about morphological changes that are akin to apoptosis. The retinoblastoma susceptibility rb protein is also degraded by caspase. Caspase-3 cleaves CAMPK within its catalytic site (McGinnis et al. 1998). Nedd4, a ubiquitin–protein ligase, is cleaved by several caspases (Harvey et al. 1998). The cleavage of these proteins is associated with apoptosis, but the significance of the process is not understood. The proteolytic cascade involves endonucleases downstream of caspases and calpain in the apoptotic pathway. Because caspase inhibitors also result in the inhibition of DNA fragmentation (X. Liu et al. 1997), it would appear that caspase may expose the DNA to endonuclease function by cleaving the chromatin-associated proteins such as lamin B, histone H1, and topoisomerase II (Neamati et al. 1995; Kaufmann, 1989). In the same vein, nuclear scaffold protease inhibitors block not only the cleavage of lamin but also DNA fragmentation. It seems then, that the cleavage of chromatin scaffold proteins by either pathway results in DNA fragmentation (Lazebnik et al. 1995). Not all caspases are inducers of apoptosis. Caspase-11 is a pro-inflammatory caspase and is activated by the lysosomal protease cathepsin B (Schotte et al. 1998). Apoptotic changes triggered in nuclei by CaCl2 are inhibited by the inhibition of caspase-3 and to a lesser extent by caspase-1 (Juin et al. 1998). Hydrogen peroxide (at 50 µM, but not at higher concentrations) induces apoptosis of Jurkat T lymphocytes and, in parallel, an enhancement of caspase activity is also detected. However, at H2O2 concentrations greater than 50 µM, cells undergo necrosis (Hampton and Orrenius, 1997). Caspase seems to be involved in neuronal apoptosis following ischemic injury, and again caspase inhibitors appear to be able to protect neurones from undergoing apoptosis (Gorman et al. 1998). Overall, it seems reasonable to conclude that caspase plays a significant role in the cell apoptosis occurring in response to a variety of extracellular signals. The evidence is based, as noted, mainly on the protective function of caspase inhibitors and also on the inhibition of the caspase substrate PARP. It ought to be stated, however, that in certain cell types, activation of caspase-3 does not automatically lead to apoptosis (Well et al. 1998). There could be other components in the pathway, e.g., the p36-MBP kinase (MBPK), the activation of which has been demonstrated recently by Kakeya et al. (1998). These authors showed that cytotrienin A, isolated from Streptomyces, induces apoptosis of HL-60 cells by activating MBPK. The broad-spectrum caspase inhibitor, Z-Asp-CH2-DCB, inhibited activation of MBPK as well as apoptosis. However, it did not inhibit the activation of other kinases such as c-jun N-terminal kinase/stressactivated protein kinase and p38 MAPK, which were also activated by cytotrienin A, albeit with a different kinetic pattern from MBPK activation. This suggests that

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activation of MBPK is a component of the apoptosis-signalling pathway. The pathways of caspase and calpain involvement in apoptotic cell death are shown in Figure 21. It is fairly obvious from the above discussion that at present our knowledge of the mechanisms involved is rather limited.

POLY (ADP-RIBOSE) POLYMERASE AS A MARKER OF APOPTOSIS The process of recovery from DNA damage involves the synthesis of poly (ADPribose) in response to strand breaks. The nuclear enzyme poly (ADP-ribose) polymerase (PARP) catalyses the cleavage of NAD+ into nicotinamide and adenosine 5′diphosphoribose (ADP-ribose). PARP further catalyses the covalent linkage of poly (ADP-ribose) to the damaged sites in the DNA. PARP is activated upon binding to DNA strand breaks. It binds to DNA via its zinc finger domains (De Murcia and De Murcia, 1994; De Murcia et al. 1997). It links poly (ADP-ribose) to the nicks in the DNA and maintains its structural integrity until DNA excision repair is carried out by DNA polymerases (Lindahl et al. 1995). Caspases cleave this 113-kDa enzyme into an N-terminal DNA-binding fragment and a C-terminal catalytic fragment. Apoptosis is characterised by the cleavage of PARP and the appearance of the 89-kDa catalytic fragment is regarded as a marker event of apoptosis. There is a large body of evidence that PARP is involved in apoptosis. The ionophore A23187 has been reported to induce apoptosis in PC12 cells, when the ionophore is applied at low concentrations. This is accompanied by the activation of caspase-3. When caspase-3 is inhibited the process of apoptosis is also inhibited (Takadera and Ohyashiki, 1997). Dexamethasone and thapsigargin both induce apoptosis of WEHI mouse lymphoma cells, albeit by different pathways. Nevertheless, the activation of caspase-3 and cleavage of PARP accompanied the induction of apoptosis by both these agents. Both latter events were inhibited by overexpression of the bcl-2 gene (McColl et al. 1998). The protein encoded by the ced-9 gene of C. elegans is an inhibitor of apoptosis belonging to the bcl-2 gene family (Reed, 1997). A similar effect on apoptosis and PARP cleavage by caspase inhibitors had been demonstrated earlier in a different cell system by Bonfoco et al. (1996). The nature of PARP involvement is increasingly being appreciated. Caspase-3 activation and the triggering of PARP cleavage may be described as key events that occur in the induction of apoptosis. The poly (ADP-ribosylation) of nuclear proteins, followed by caspase-3-mediated PARP cleavage, is an early event of the apoptotic process, and it may indeed be an important requirement for apoptosis to proceed. The cleavage of PARP, internucleosomal DNA fragmentation, and morphological changes in nuclei do not occur if cells are depleted of PARP by using antisense strategy. These apoptosis-associated changes take place in cells with wild-type PARP (+/+) genotype, but are conspicuously absent in PARP (–/–) mutants (SimbulanRosenthal et al. 1998). Simbulan-Rosenthal et al. (1999) have since argued that PARP is also a component of the DNA replication complex, which includes several important proteins. The expression of these might be affected by PARP depletion, inhibit DNA replication, and commit the cells to the apoptotic pathway.

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Notwithstanding these studies, the precise role played by PARP is not fully understood. The inhibition of PARP affects apoptosis. The activation of PARP by DNA damage results in NAD+ depletion and a depletion of ATP in the restoration of NAD+ levels. Therefore, cell death can be a result of the loss of ATP. In PARP –/– cells, ATP levels seem to be maintained and the cells are protected from necrotic cell death. However, in both PARP +/+ and PARP –/– mutants, cells can be equally susceptible to apoptosis (Ha and Snyder, 1999). In renal epithelial cells, hydrogen peroxide-induced PARP activation results in necrotic death due to NAD+/ATP depletion (Filipovic et al. 1999). Walisser and Thies (1999) found that endothelial cells exposed to hydrogen peroxide undergo cell death. Presumably, here too cell death may have been caused by NAD+ and consequent ATP depletion. However, when PARP is inhibited these cells switch to apoptosis. Other studies, however, suggest PARP inhibition may indeed protect cells from apoptosis (Richardson et al. 1999; Guo et al. 1998). A putative link-up between the function of caspases and PARP in apoptosis has emerged recently. Aoufouchi et al. (1999) transfected a PARP (–/–) cell line with the DNA-binding domain fragment of PARP (DBD), or mutants: DBDbd (–) which is defective in binding to DNA strand breaks, and DBDcl (–), which resists cleavage by caspases. DBD transfected PARP (–/–) cells showed no changes in staurosporineinduced apoptosis. Cells that had been transfected by both mutants showed marked inhibition of apoptosis. Furthermore, the mutant DBDs inhibited the cleavage of the catalytic subunit of DNA-dependent protein kinase by caspase-3. These results suggest that PARP might be involved in the events leading to caspase activation. It seems, therefore, that it is possible to dissociate the DNA-binding properties of PARP from caspase activation. Aoufouchi et al. (1999) have suggested the possibility that PARP might interact with components involved in caspase activation.

CASPASE-MEDIATED APOPTOSIS AND CELL GROWTH INHIBITION IN TUMOUR EXPANSION The relevance of caspase-mediated apoptosis in tumour development has been emphasised recently by the finding that enhanced expression of caspase-2 leads to a reversion of ras-induced transformation in NIH3T3 cells (Hiwasa and Nakagawara, 1998). Hiwasa and Nakagawara (1998) transfected caspase cDNAs into c-Ha-rastransformed cells. Enhanced expression of caspase-2 alone, but not caspases 1 and 2 together, resulted in a reduction in the ability of the transfected cell to grow in soft agar. This was accompanied by ras protein degradation, suggesting that the apparent reversion of the transformed phenotype was due to the degradation of the transforming gene product by caspase-2. However, Hiwasa and Nakagawara (1998) have not presented any evidence relating to the tumorigenicity of the ras-transformed cells, or any data on possible inhibition of tumorigenicity by the transfection of caspase-2 cDNA into these transformed cells. It is essential to recognise that cell transformation is a process quite distinct from tumorigenicity. Whether caspaseinduced apoptosis occurring in the developing tumour can control the growth of the tumour is still an open question. As stated elsewhere, apoptosis subserves an

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important function in maintaining the kinetics of cell expansion in metastatic growth. By inference, there is no reason why a similar role may not be played by apoptosis in the control of primary tumour growth, or, to put it more precisely, primary tumour expansion. The apoptosis-mediated restriction of a cell population can be differentiated from control by inhibition of growth, although these might not represent totally independent mechanisms. This has been demonstrated in the case of TGF-β, which influences apoptosis as well as cell growth. However, in WEHI 231 cells, the caspase inhibitor BD-fmk can effectively inhibit TGFβ-induced apoptosis but not counteract TGFβ-mediated inhibition of cell growth (T.L. Brown et al. 1998). This apparent dichotomy of mechanisms is obvious also from the pathways of radiation-induced apoptosis. Ionising radiation induces the activation of caspase-3, which is followed by the induction of apoptosis. Ionising radiation induces rapid apoptosis in human lymphoblast cells expressing wild-type p53, but in cells where p53 is mutated or abrogated by viral oncoprotein, apoptosis is delayed as well as reduced (Yu and Little, 1998). In this instance apoptosis seems to occur in conjunction with growth control mediated by wild-type p53. On the other hand, caspases have been shown to cleave p53-induced cyclin-dependent kinase inhibitor p21waf1/cip1 and induce cells to undergo apoptosis (Y.K. Zhang et al. 1999). In the latter case, apoptosis seems to depend on proliferation. In contrast with p53-mediated apoptosis, VD3 has been reported to induce apoptosis of certain breast cancer cells independently of both p53 and caspases. This report is based on the observation that VD3 produces growth arrest and apoptosis of both MCF7 cells, which are p53 positive, and T47D cells that are p53-negative. Furthermore, this apoptotic induction is not inhibited by inhibitors of caspase, whereas TNF- or staurosporine-induced apoptosis is inhibited (Mathiasen et al. 1999). Whatever pathway apoptosis might take, it is inevitable that tumour growth and subsequent processes should be shaped by balancing forces of apoptosis and cell cycle control factors. Nonetheless, it is worthwhile to note here that ICE-like protease expression has been reported to correlate with progression and prognosis of neuroblastoma. The frequency of expression of ICE mRNA was markedly reduced in advanced-stage neuroblastomas as compared with early-stage tumours (Ikeda et al. 1997). The expression of caspase-3 in normal gastric mucosa, gastric adenomas and adenocarcinomas has been studied in some detail by Hoshi et al. (1998). They found that caspase-3 expression decreased from a high level (42% cells staining for caspase) in nonneoplastic gastric mucosa to a lower level (33%) in adenomas, and to a still lower level (17%) in adenocarcinomas. These differences were statistically significant in spite of the large standard deviations of the mean. The caspase-3 positivity, of the three groups, correlated directly with apoptotic indices determined by the TUNEL method and inversely with proliferative indices provided by Ki67 labelling. These data are consistent with the view that a loss of apoptosis-mediated control over cell turnover is an important feature of tumour growth. Conversely, there is an implicit suggestion that ICE-like proteases may be involved in apoptosismediated regression of tumours, in the demonstration by Ikeda et al. (1997) that ICE protease staining could be co-localised in the nucleus with DNA fragmentation. In this context, one should also take note of the recent report that caspase-3 activity was found to increase in colonic carcinomas and adenomas as compared with normal

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mucosa (Leonardos et al. 1999). Obviously, these observations may serve to confirm that the expression of the caspase is related more to the degree of apoptosis taking place in the tumour than to the degree of tumour progression. Donoghue et al. (1999) reported a difference in the pattern of caspase-3 distribution in B-cell diffuse large cell lymphoma. In immunohistochemistry, caspase-3 showed a diffuse distribution in the cytoplasm or a punctate or spotty pattern. The diffused pattern appeared to relate to poor prognosis, and the punctate pattern was associated with complete response to therapy. The authors have stated further that where the percentage of caspase-3-expressing cells was low, prognosis was poor. Not only are some of these findings not compatible per se, but the study seems to raise more questions than it successfully answers. It should be conceded, however, that it would be unhelpful to attribute all apoptotic activity to caspases. Donoghue et al. (1999) found no correlation between apoptosis and caspase expression in the cells, but the degree of apoptosis was associated with poor prognosis. A finding of potential significance is that the caspase distribution pattern might be significant in terms of enzyme activity. Whether the punctate distribution might reflects sequestration of the enzyme is worth further investigation. It should be recognised, nevertheless, that caspase expression may reflect cancer progression and that caspases may actively promote metastatic deposition by a more direct route. Thus, a high proportion of in situ and invasive (58 and 90%, respectively) carcinomas of the breast stain strongly for caspase-3. Caspases-6 and -8 also are expressed at high levels more frequently in carcinomas than in hyperplasia. The enhanced expression correlated well with apoptotic indices in the samples. Furthermore, enhanced apoptosis was associated with poor prognosis (Vakkala et al. 1999a, 1999b). The formation of metastatic lesions depends on a cascade of events; prominent among them is the invasion of the vascular and endothelial systems by cells of the primary tumour. The entry into the circulatory system has been attributed to an active process of transmigration or diapedesis across the endothelial layer, as well as to the inherent structural defects often found in the endothelium. Recently, Kebers et al. (1998) found that several breast cancer cell lines, among them MCF7, MDA-MB231, T47D, and HT1080, induced a four-fold increase in apoptosis of human umbilical vein endothelial cells (HUVEC), with an attendant enhancement of caspase-3 activity. The interaction of MCF7 cells with HUVEC caused a transient increase in intracellular calcium levels (Lewalle et al. 1998), which, presumably, may have led to caspase activation. The induction of apoptosis required cell–cell contact, because media conditioned by the growth of these cells were ineffective. Kebers et al. (1998) have further observed that lymphocytes do not induce apoptosis, suggesting that the apoptosis of endothelial cells might constitute a specific mechanism in the diapedesis of tumour cells. Whether caspase-mediated apoptosis of endothelial cells occurs in vivo is yet to be demonstrated. The outcome of tumour progression has often been assessed in relation to a single given variable as a prognostic factor. As noted above, in the context of caspases we have a paradoxical situation that in both caspase-positive and caspase-negative circumstances some relationship is noticed with tumour progression. As observed earlier, tumour growth and progression are a net outcome of the balancing forces of apoptosis and cell cycle control factors and cell proliferation. Perhaps it would

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be more rewarding to reexamine the question of caspases in tumour progression in this light. For this viewpoint, the recent findings of Volm and Koomagi (2000) are most encouraging. They have examined the relevance not only of caspase-3 but also of c-myc expression in the prognosis of non-SCLC. Volm and Koomagi (2000) have reported that caspase-3-negative patients had a median survival time of 41 weeks as compared with 79 weeks for caspase-3-positive patients. They then looked at cmyc expression and its influence on prognosis. Patients who were c-myc negative had a median survival time of 89 weeks, whereas the median for c-myc-positive patients was 43 weeks. Not only did these two factors correlate inversely with survival, but, myc–/caspase+ patients showed a median survival time of 102 weeks as compared with only 22 weeks for myc+/caspase– patients. This clearly makes the point that more than one factor might influence a given cellular feature and thereby determine the outcome of the disease. The study also serves to further emphasise that it would be unhelpful to try to evaluate a single prognostic factor, while other factors might be present that would impinge on the direction of cellular changes.

CASPASE-MEDIATED PROTEOLYSIS OF FODRIN: IMPLICATIONS FOR APOPTOSIS, CELL ADHESION, CELL MIGRATION, AND NEOPLASTIC TRANSFORMATION Among the several substrates of caspases is the membrane protein called fodrin. The erythroid homologue of fodrin is known as spectrin, with which fodrin shares substantial amino acid sequence homology. Fodrin is attributed with the function of maintaining the structural integrity of the plasma membrane. Fodrin and spectrin form a major component of the skeletal network that underlies the plasma membrane (Levine and Willard, 1981; W.J. Nelson et al. 1990; Bennett and Lambert, 1991; Bennett and Gilligan, 1993). Fodrin (alias spectrin) isoforms are also found in the membranes of the Golgi apparatus (Devarajan et al. 1996, 1997; Beck et al. 1994, 1997; Godi et al. 1998; Fath et al. 1997; Stankewich et al. 1998), lysosomes (Hoock et al. 1997), and intracellular vesicles (Malchiodi-Albedi et al. 1993; Stankewich et al. 1998). Fodrin is an actin-binding protein; therefore, two further putative functions should also be considered. One of these is a presumptive involvement in the process of signal transduction, because fodrin has been found to be able to inhibit phospholipases A2 (PLA2), C, and D (Lukowski et al. 1996, 1998). In comparison, other cytoskeletal proteins such as actin and vimentin are far less efficient than fodrin (Lukowski et al. 1996). Phospholipases are closely associated with the generation of DAG and IP3 from PIP2. These are involved in the activation of downstream pathways of signal transduction that, in turn, involve the activation of appropriate protein kinases, such as PKC, and Ca2+ release from intracellular stores. Another potentially important function that can be attributed to fodrin is in influencing cell adhesion and migration. Spectrin is required for neurite extension in neuroblastoma cells. Sihag et al. (1996) were able to inhibit neurite extension in NE2a/dl neuroblastoma cells with an antibody directed against the N-terminal domain of spectrin. The latter is known to interact with actin. The protein called

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ankyrin mediates the link-up between membrane adhesion proteins and the spectrin cytoskeleton. The expression of some of these adhesion molecules has been shown to cause cell aggregation in which process both ankyrin and spectrin are recruited to the foci of adhesion (Dubreuil et al. 1996). In wound healing of corneal epithelium, fodrin becomes redistributed from its subplasma membrane location to a cytoplasmic location. This redistribution occurs soon after wounding of the epithelium. A similar redistribution occurs in response to PMA treatment and is inhibited by PKC inhibitors (Amino et al. 1995). Presumably these events are related to the cell migration that follows, although there is little direct evidence linking these. As stated above, fodrin (spectrin) is a substrate for caspases. Apoptosis induced by several different pathways has been shown to be accompanied by proteolysis of fodrin. Inhibition of apoptosis also results in the inhibition of fodrin proteolysis (Martin et al. 1995). This proteolysis seems to be produced by ICE/ced-3 proteases (Cryns et al. 1996; Vanags et al. 1996). Kouchi et al. (1997) believe that calpains are not associated with the cleavage of the 240-kDa α subunit of fodrin in the apoptosis of rat thymocytes both in vivo and in vitro. However, Porn-Ares et al. (1998) do implicate calpains. Fodrin occurs in a variety of cell types including keratinocytes, chromaffin cells, and renal epithelium, and in a variety of epithelial and fibroblast cell lines. Fodrin shows a homogeneous cytoplasmic and a discontinuous membrane distribution in benign melanocytic tumours, whereas normal melanocytes at the basal layer of the epidermis only faintly stain for fodrin at the plasma membrane. Overall, neoplastic cells show greater amounts of fodrin than their nonneoplastic counterparts (Tuominen et al. 1996). This observation has been confirmed in an immunohistochemical study of a variety of adenocarcinomas and squamous cell carcinomas by Sormunen et al. (1997). However, malignant melanomas contain subpopulations that do not express fodrin (Tuominen et al. 1996). This does not detract from any putative relationship between fodrin expression and malignancy, because malignant tumours are notoriously heterogeneous with respect to a large spectrum of cellular characteristics. Although much work needs to be done in this area, already there are clear indications that caspase-mediated alterations in fodrin expression and function might be involved in biological processes (e.g., apoptosis, cell adhesion, motility, and modulation of cell shape) that are inherent features of tumour development, dissemination, and metastasis.

CASPASES AND NEURONAL LOSS IN ALZHEIMER’S DISEASE Alzheimer’s disease is characterised by massive neuronal loss, which has been attributed, in recent years, to apoptotic cell death (Barinaga, 1998). The participation of caspases in inducing apoptosis has naturally led to the investigation of these proteinases, together with the bcl-2 family genes, in the pathogenesis of Alzheimer’s disease (Figure 23). Kitamura et al. (1998) showed that the expression of several bcl-2 family genes was up-regulated in Alzheimer’s disease. Caspases are involved in the induction of neuronal apoptosis (Bambrick and Krueger, 1999). Two genes known as PS (presenilin)-1 and PS2 have been associated in a mutated form with early onset of Alzheimer’s disease. The presenilins are integral proteins of the ER.

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FIGURE 23 Possible pathways by which caspases may regulate neuronal apoptosis associated with Alzheimer’s disease. PS, presenilin proteins; PS-P, phosphorylated form of PS protein.

Their mutation could lead to a perturbation of calcium homeostasis in the cell with attendant aberrations of calcium-mediated processes of apoptosis as well as the processing of the amyloid precursor protein. Wild-type PS has been shown to promote neurite outgrowth in neuroblastoma cells grown in vitro (Dowjat et al. 1999). It is conceivable that mutations would affect this process as well. The involvement of PS proteins in apoptosis has been demonstrated by several experiments. For example, overexpression of the mutated PS protein can enhance apoptosis induced by several agents. PS2 antisense mRNA inhibits apoptosis. A new facet might be added to this story of PS2 involvement in neuronal apoptosis. A CBP has been suggested to play a part in this process. Stabler et al. (1999) have reported that a CBP, which they call calmyrin, interacts preferentially with PS2. Calmyrin consists of 191 amino acid residues and possesses two C-terminal EF-hands. It has been shown to interact with the cytoplasmic domain of the platelet integrin αII bβ3, and is regarded as a putative regulator of the function of the platelet integrin (Naik et al. 1997). Stabler et al. (1999) found that calmyrin is myristoylated, and, in the modified form, it interacts with PS2. In HeLa cells, calmyrin is said to co-localise with PS2. The posttranslational modification of the protein could be required for targeting it to the membrane. This is reminiscent of myristoylation of recoverin and its function. Although, in the context of calmyrin, we can only draw inferences as to the significance of complex formation between calmyrin and PS2, some preliminary observations made by Stabler et al. (1999) make interesting reading. They observed an enhancement apoptosis upon co-transfection of HeLa with PS2 and calmyrin. The PS proteins are substrates for caspases. Caspases cleave these at specific sites, e.g., aspartic acid 345/serine 346 and aspartic acid 329/serine 330. Caspase inhibitors as well as mutations at these sites inhibit cleavage of the proteins (Loetscher et al. 1997). Phosphorylation has been found to regulate the caspase sensitivity

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of PS2. Two phosphorylation sites, at serine residues 326 and 329 have been identified in PS2. Both occur close to the site of cleavage by caspase-3. Cleavage of the protein is inhibited by phosphorylation of these residues, and the loss of this ability seems to inhibit apoptosis (Walter et al. 1999). However, there have been no attempts to date to determine if the phosphorylation of PS is related to disease activity. We do know that the sensitivity of the mutated PS1 and PS2 proteins to cleavage by caspases is not affected, which suggests that caspase-mediated modification of PS proteins might not be associated with the disease (Van de Craen et al. 1999). Also, PS proteins carrying mutations at the caspase cleavage site have been found to be functional (Brockhaus et al. 1998). Furthermore, the participation of PS genes themselves might have been overemphasised. Nonetheless, it has emerged from recent studies that caspases-2 and -3 show a marked increase in the brains of Alzheimer’s patients (Shimohama et al. 1999). In that study, Shimohama et al. (1999) also examined the expression of these caspases in developing rat brain. Their data have suggested some differential regulation of expression of these proteases. For instance, caspase-3 is highly expressed from 19-day-old embryos to 96-week-old embryos. However, high expression of caspase-2 begins only in approximately 4week old embryos. Although it does seem that there is a developmental regulation in rat embryos, the relation between the expression of these caspases and the process of ageing is not quite obvious. In any event, these data are at present insufficient to base any conclusions about whether the pathogenesis of Alzheimer’s disease is related to changes in the regulation of caspase expression. Another observation worthy of note is that caspase-3 can cleave PS2 at arginine 329 and generate a Cterminal peptide that might protect cells against apoptosis (Vito et al. 1997). Thus, caspase-mediated promotion of apoptosis by the modification of PS proteins could contain a self-regulatory mechanism. Vito et al. (1997) have suggested that the apoptosis-promoting function and the negative feedback signal provided by the Cterminal fragment generated by caspase activity could eventually determine the outcome. As discussed elsewhere Alzheimer’s disease is also characterised by abnormalities of the tau protein. This protein have been identified as a substrate for both caspases and calpain. The cleavage and dephosphorylation of tau has been shown to occur in neuronal apoptosis (Canu et al. 1998). Similarly, the amyloid precursor protein (APP), again a prominent feature of the disease, seems to be cleaved by caspase-3 (N.Y. Barnes et al. 1998). Not only does caspase-3 occur at high levels in Alzheimer’s brains, but the product of APP degradation co-localises with the caspase in degenerating neurones (Gervais et al. 1999). These observations represent another pathway involving caspase function in the pathogenesis of Alzheimer’s disease.

15

Parvalbumins in Neuronal Development, Differentiation, and Proliferation

The neuroepithelial ventricular zone of telencephalic ventricles gives rise to the majority of neurones and the glial component, which form the mammalian cerebral cortex. It has become increasingly obvious over the past few years that the various neuronal CBPs, such as calretinin, calbindin, neurocalcin, and parvalbumins, may each uniquely identify neuronal subpopulations across the cortical regions. Subpopulations with different CBPs may indeed represent metabolically distinctive cellular subtypes, and the presence of specific proteins may hold functional implications for the particular subtypes. These proteins may be playing different calcium-signalling roles in different cell types under different biological parameters. Parvalbumins are CBPs with three EF-hand domains. Three isoforms of PV may be distinguished: α, β, and CPV3. α-PV, with its high affinity for Ca2+ is regarded as a calcium buffer. Furthermore, its distribution is much wider than that of the other isoforms. PVs occur in a neurone-specific fashion and are used as markers in the development and differentiation of neurones. A study of the localisation of PV in extraocular neurones has revealed moderate anti-PV immunoreactivity in motor neurones, but their axons show heavy staining, suggesting an intracellular segregation of PV (De La Cruz et al. 1998). De La Cruz et al. (1998) also state that PV is the only marker for extraocular motor neurones. The expression of PV seems to be related to differentiation. PV, among other calcium binding proteins such as calbindin D-28K and calretinin, shows age-related changes in the developing brain (Kishimoto et al. 1998; Majak et al. 1998). It has been reported that rhabdomyosarcoma cells that have been induced to differentiate, not only exhibit characteristic dendritic processes but also show a parallel increase in PV together with small increases in vimentin, desmin and neurone-specific enolase (Pappas et al. 1996). The regeneration of hair follicle cells, not involving mitosis, is another system in which the differentiation of supporting cells into hair cells has been shown to be accompanied by changes in the expression of CBPs, e.g., calbindin and parvalbumin (Steyger et al. 1997). Whether there are any tissue specific mechanisms regulating PV expression is unclear at present. Some preliminary work by 181

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Castillo et al. (1995) does suggest this. These authors produced transgenic mice to express PV under the control of specific promoters. It is of some significance that PV expression driven by a neurone-specific enolase promoter was at the highest level in the brain, but lower levels of expression were detectable in other tissues. It is also notable that ectopic PV expression in these transgenic mice did not produce developmental or behavioural abnormalities. β-PV is referred to often as the onco-developmental parvalbumin or oncomodulin (OM). OM occurs in trophoblast cells, preimplantation embryos and neoplasms. Its distribution is far more restricted than that of α-PV. The β-PV isoform known as CBP-15, which has been reported to occur in the guinea pig organ of Corti (inner ear) (Thalmann et al. 1995), is identical to OM with respect to its 30 N-terminal amino acid residues. The identity between OM and CBP-15 has now been confirmed by the demonstration that isoform-specific anti-OM antibodies do cross-react with CBP-15 (Henzl et al. 1997). The OM gene has been mapped to the long arm of chromosome 5 of the mouse (Staubli et al. 1995). The calcium ion affinity of OM is lower when compared to that of α-PV. OM may therefore have a calciumdependent regulatory function. Some investigators have addressed the question of a putative role for PV in cell proliferation. Blum and Berchtold (1994) have reported the expression of OM mRNA transcripts as well as the protein increases at the G1–S boundary in two neoplastic cell lines (T14 and T10) that they tested. The increase was less marked in the T14 line when mitotically synchronised. However, because CaM shows a similar rise at the G1–S interphase, the significance of the rise in the levels of OM expression cannot be evaluated at present. It may be pointed out, however, that the expression of CaM and OM appeared to be differentially affected by the levels of extracellular calcium. When extracellular calcium levels were reduced, CaM expression increased by up to 60%, whereas OM levels decreased and the effects on both were reversed when the extracellular Ca2+ levels were increased (Klug et al. 1994). It is noteworthy that Klug et al. (1994) also state that the low extracellular calcium and the high CaM levels associated with it appeared to be best suited for cell growth. Andressen et al. (1995) transfected PV cDNA into a human ovarian carcinoma cell line but found a reduced mitotic rate in the cell lines carrying the exogenous PV cDNA. Furthermore, there were distinct alterations in cellular morphology and motility. Needless to say, further investigations into the influences exerted by PV on these aspects of cellular behaviour seem worthwhile. CPV3 is an avian PV whose expression seems to be restricted to the thymus (Hapak et al. 1994a, 1994b). CPV3 has an isoelectric point (pI) of 4.6, which is somewhat lower than the pI of muscle PV. A full-length clone of CPV cDNA has 671 bp, and the protein product bears 68% sequence homology to OM. The CPV3 molecule contains cysteine residues at positions 18 and 72 and hence can form oligomers by means of disulphide bonds (Hapak et al. 1994b; Henzl et al. 1995).

16

Osteonectin in Cell Function and Behaviour

MOLECULAR STRUCTURE OF OSTEONECTIN Osteonectin is a secreted glycoprotein associated with the extracellular matrix. It is also known by other epithets, such as SPARC (secreted protein acidic, rich in cysteine) and BM (basement membrane)-40. The protein is encoded by a single gene (Mason et al. 1986). Human osteonectin has a predicted molecular size of approximately 32 kDa (Swaroop et al. 1988). Human osteonectin has been cloned by PCR from an endothelial cell (EC) cDNA library, and this soluble biologically active monomer expressed in E. coli has 293 amino acid residues (Bassuk et al. 1996). Several homologues of osteonectin have been isolated, which share a high degree of amino acid sequence homology, with 61 to 65% of the 200-odd residues at the C-terminal region being identical. But they do show great deviation in the secondary structure of the Nterminal region (McKinnon et al. 1996; Soderling et al. 1997). According to McKinnon et al. (1996) the mouse homologues possess an exon structure similar to that of the osteonectin gene. Hafner et al. (1995) have reported the occurrence of a purine-rich stretch in the 5′ end of the osteonectin gene. This region of bovine and murine genes shows marked sequence similarities, and it contains two GGA boxes with an intervening pyrimidine-rich spacer sequence. Hafner et al. (1995) also have shown that this region contains several regulatory domains. The GGA box 1 of the bovine promoter appears to be sufficient for maximal transcription of the gene, and the pyrimidine-rich spacer element seems, in contrast, to down-regulate gene expression. However, the GGA box does not seem to regulate gene expression, as in bovine cells. The transcriptional machinery obviously needs much further characterisation, especially to elucidate whether the regulatory domains described by Hafner et al. (1995) contribute to cell type-specific expression of osteonectin. Osteonectin has a C-terminal EF-hand domain (Yost and Sage, 1993; Maurer et al. 1995). Maurer et al. (1995) have identified two distinct domains (i.e., an acidic domain and a follistatin like [FC] domain), besides the EF-hand domain. Follistatin is a regulatory protein with multiple functions, notably inhibition of follicle-stimulating hormone. Follistatin has a characteristic and highly conserved amino acid sequence motif known as the follistatin domain. This domain has been shown to occur in a number of proteins, and these have been deemed as forming the follistatin family of proteins. Because osteonectin contains a follistatin domain, it has been regarded as a member of the follistatin family (Maurer et al. 1995; Phillips and

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De Kretser, 1998). The EF-hand domain binds calcium with high affinity. Osteonectin from C. elegans, however, binds calcium with far less affinity as compared with the mammalian counterpart (Maurer et al. 1997). Calcium binding results in conformational changes in the molecule (Bassuk et al. 1996; Maurer et al. 1995; Takita and Kuboki, 1995). The EF-hand domain also mediates the Ca2+-dependent interaction of osteonectin with cells and the ECM. Other calcium-binding sites, which bind the cation with far less affinity, are also known to occur (see Table 13).

FUNCTIONS AND FUNCTIONAL DOMAINS OF OSTEONECTIN Osteonectin is a highly conserved protein and is known to be expressed in a variety of organisms, from invertebrates to mammals (Damjanovski et al. 1997; Schwarzbauer and Spencer, 1993; Lane and Sage, 1994). It is not surprising therefore that, despite its restrictive nomenclature, it participates in a variety of cellular functions and cell behaviours (Table 12).

TABLE 12 Osteonectin in Cell Function and Behaviour Embryonic differentiation and development Remodelling and rebuilding of the ECM Angiogenic and anti-angiogenic function Cell spreading Modulation of cell shape Intercellular and cell–substratum adhesion Cell proliferation Tumour development and progression Source: Based on references discussed in the text.

Several individual molecular domains that participate in this physiological panoply have been identified with specific functions, by using peptide segments derived from different regions of the osteonectin molecule and antibodies raised against these peptides (Jendraschak and Sage, 1996). These domains and their putative functions are shown in Table 13.

REGULATION OF OSTEONECTIN EXPRESSION The expression of osteonectin is regulated by several growth factors that can be closely identified with specific cellular properties such as cell proliferation, angiogenesis, etc. For instance, TGFβ induces the expression of osteonectin mRNA and protein in fibroblasts and other cell types (Wrana et al. 1991; M.J. Reed et al. 1994;

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TABLE 13 Functional Domains of the Osteonectin Molecule

Domain

Amino Acid Stretch

Subdomain

I

3–51

1.1

II

52–132

2.1

2.3

III IV

132–227

3.2 4.2

Sequence

Putative Function

Low-affinity Ca2+ binding; inhibition of cell adhesion, spreading; increase of PAI-1 expression QNHHCKHGKVCELDESNTP Ca binding; inhibition of EC proliferation; loss of focal adhesion; effect on cell cycle progression TLEGTKKGHKLHLDYIG Ca binding; follistatin homology; stimulation of EC proliferation and angiogenesis; plasmin sensitivity KNVLVTLYERDEGNNLLTEK Induction of MMP expression DLDNDKYIALEEWAGCFG EF-hand domain; inhibition of cell spreading, proliferation, and focal adhesion; binding to endothelial cells QTEVAEEIVEEETVVEETGV

Note: Domains III and IV might be involved in binding collagens (see text for references). PAI-1, plasminogen-activator inhibitor; EC, endothelial cell; MMP, matrix metalloproteinases. Source: Based on Jendraschak and Sage (1996).

Blazejewski et al. 1997). Also, PDGF, cytokines, and IGF-1 stimulate osteonectin synthesis and secretion (Chandrasekar et al. 1994). However, S. Nakamura et al. (1996) found that, in rabbit articular chondrocytes, IL-1β and -1α decreased osteonectin levels. In fact, IL-1β decreased osteonectin mRNA levels as well as glycosylation of the protein. S. Nakamura et al. (1996) also have shown that several other factors, among them TNF-α, PMA, and bFGF, can down-regulate osteonectin expression. The down-regulation of its expression by bFGF might be related to a destabilisation of the osteonectin mRNA (Delany and Canalis, 1998; Delany et al. 1996). H. Shiba et al. (1995) found that bFGF reduced osteonectin synthesis as well as osteonectin mRNA in dental pulp cells. They suggested that the effects of the growth factor on odontoblast differentiation might occur at least in part at some pretranslational stage. In this context, it might be of interest to note that bFGF has been implicated in cell motility, proliferation, and angiogenesis, all features in which osteonectin is also putatively implicated. Furthermore, different growth factors and cytokines seem to affect the expression of ECM components, such as fibronectin, laminin, and collagens, in a differential manner (Reed et al. 1994; H. Shiba et al. 1998). This provides additional support to the view that the effects on osteonectin

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produced by the various biological response modifiers might be identifiable with their individual responses. It is small wonder, therefore, that osteonectin expression in cancers has been investigated with some vigour, albeit not with the degree that could be deemed appropriate in the context of the multifarious biological effects of osteonectin.

OSTEONECTIN IN THE REMODELLING OF THE EXTRACELLULAR MATRIX The significant role that is played by components of the ECM in cell motility and morphogenesis has been keenly appreciated by the scientific community. A secreted glycoprotein, osteonectin has been a prime candidate for participation in these processes. It was reported to be closely associated with the ECM in several normal as well as abnormal tissues (Dahlback et al. 1986). Several enzymes that are prominently associated with the membrane, refashion and remodel the ECM and in this way influence cell behaviour. Among these are plasminogen activator (PA) and its inhibitor PAI, and a host of cathepsins MMPs (see Sherbet and Lakshmi, 1997b for review) (Figure 24). That osteonectin could be responsible for pericellular proteolysis and ECM remodelling is supported by several lines of evidence. Osteonectin is known to bind to a number of ECM components, such as collagen types IV and V and plasminogen (Mayer et al. 1991; Kelm et al. 1994; R.L. Xie and Long, 1996). Mayer et al. (1991) reported that osteonectin bound mainly collagen type IV, with other collagens (I, III, V, and VI) bound far less competitively. These binding interactions may involve specific regions of the molecule, as shown for collagens IV and V binding by osteonectin (Mayer et al. 1991; R.L. Xie and Long, 1996). Binding to ECM components could be a means of anchoring osteonectin to the ECM, so that it can participate in ECM remodelling. It also could serve as a cofactor in the formation of plasmin mediated by tissue plasminogen activator (tPA) (Kelm et al. 1994). On the other hand, osteonectin can induce the expression of PAI-1 as it has been shown to do in the case of bovine aortic endothelial cells (Hasselaar et al. 1991). A possible relationship between the expression of PAI-1 and osteonectin also has been encountered in senescent human diploid cells, where they appear to be up-regulated in parallel (S. Wang et al. 1996), although no causal linkup can be suggested on this basis. This would lead to a conservation of ECM components. The proteolytic feedback loop is completed by the demonstration that plasmin itself is capable of hydrolysing osteonectin in a limited way (Sage et al. 1984). Tremble et al. (1993) found that osteonectin up-regulated the expression of several MMPs, which included MMP1, MMP3, and MMP9, in rabbit synovial fibroblasts. Tremble et al. (1993) also identified domains of osteonectin that were involved with the regulation of MMP1 expression. It is unclear if up-regulation of MMPs alone is sufficient for ECM remodelling. In some systems, MMPs require activation. In human glioma U251.3 and fibrosarcoma HT1080, the MMP2 proenzyme is activated by MMP membrane type 1 (Deryugina et al. 1998). Whatever the downstream events may be, MMPs would be expected to refashion the expression and disposition of a number of ECM proteins. Furthermore, MMPs have been found

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to produce a limited cleavage of osteonectin. Such cleavage is accompanied by a greatly enhanced binding of osteonectin to collagens I, IV, and V (T. Sasaki et al. 1997). The involvement of a specific site of cleavage (in helix C of the C-terminal calcium-binding module) has prompted T. Sasaki et al. (1997) to postulate that this enhanced affinity for collagens might be akin to a process of activation.

FIGURE 24 Feedback loop involving serine proteinases and matrix metalloproteinases (MMPs) in the remodelling of the extracellular matrix (ECM) by osteonectin. PAI, plasminogen-activator inhibitor; tPa, tissue plasminogen activator; uPa, urokinase-type plasminogen activator. (Based on references cited in the text.)

A remodelling of the ECM partly mediated by MMPs and their inhibitor TIMPs has recently been shown to be related to the expression of S100A4 in B16 murine melanoma and human astrocytoma-derived cell lines. The enhancement of invasive behaviour and metastatic spread, which occurs upon up-regulation of S100A4 expression, has been putatively linked with these events associated with the ECM (Merzak et al. 1994b; Lakshmi et al. 1997). It is not surprising therefore that, in light of the ECM changes produced by osteonectin, its role in embryonic development and differentiation, and other cell membrane-associated phenomena, such as cell adhesion, modulation of cellular morphology, cell proliferation, and wound healing, should have been investigated. It is also not surprising that such studies should be extended to neoplastic development, invasion, and dissemination.

OSTEONECTIN IN EMBRYONIC DEVELOPMENT AND DIFFERENTIATION Osteonectin is secreted by a variety of tissues and its expression appears to be developmentally regulated. Osteonectin is highly expressed in the germ layers of the early mouse embryo, and the modulation of its expression follows a definite pattern in the course of embryogenesis (Holland et al. 1987; Nomura et al. 1988;

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Sage et al. 1989a). Interference with this temporally controlled activation of osteonectin results in developmental abnormalities. Thus, when osteonectin antibodies are introduced into the blastocoel of Xenopus embryos, defective neurulation and organogenesis occur (Purcell et al. 1993). Microinjection of specific peptides derived from osteonectin induces abnormalities in the establishment of the embryonic axis in morphogenesis. The injection of osteonectin peptides containing FC domains and copper-binding sequences did not affect this process, but peptides containing the disulphide-bonded Ca2+-binding domain profoundly inhibited axial formation, leading to ventralisation of the embryos. The disulphide bonding seemed to be essential for axial inhibition, because peptides lacking cysteine residues were unable to induce axial abnormalities (Damjanovski et al. 1997). However, the recent work of Gilmour et al. (1998) suggests that osteonectin deficiency may not damage developmental processes. They disrupted the osteonectin locus in murine embryonic stem cells and showed that the osteonectin-deficient mice developed normally and were fertile. However, severe abnormalities of the eye, especially cataract formation and rupture of the lens capsule, developed around the age of 6 months. S.Y. Kim et al. (1997) have described the association of osteonectin in the sequence of events occurring during the development and maturation of chicken retina. Osteonectin is not only widely present in embryonic tissues, but it is also closely associated with organogenesis. In foetal rat lung, osteonectin is associated with epithelial cells of the airways during the pseudoglandular stage of lung morphogenesis. It is also found in the canalicular and saccular stages of branching morphogenesis of the lung (Strandjord et al. 1995). Using an in vitro model, Strandjord et al. (1995) were able to demonstrate that osteonectin antibodies inhibited the process of branching morphogenesis, resulted in the formation of dilated airways. The patterns of expression of bone matrix proteins, including osteonectin, suggest that these matrix proteins play different biological roles in the development and differentiation of mineralised tissues (Sommer et al. 1996). Distraction osteogenesis (bone lengthening) has afforded an excellent model that has indicated the involvement of osteonectin in various stages of differentiation (M. Sato et al. 1998). In cartilage differentiation, tissue transglutaminase catalyses the cross-linking of osteonectin into oligomers in situ, and this has been suggested as a major mechanism in the stabilisation of the cartilage matrix (Aeschlimann et al. 1995). Glycine residues 3 and 4 have been identified as the major amine acceptor sites, of which at least one is conserved in vertebrate osteonectin (Hohenadl et al. 1995).

MODULATION OF CELLULAR ADHESION, CELL SHAPE, AND MOTILITY BY OSTEONECTIN It is to be expected that any modulation of the character of the ECM should be reflected in changes in cellular features such as cell–substratum and intercellular adhesion, as well as in changes in cell shape and motility. It was reported several years ago that osteonectin inhibited cell spreading and consequently altered cell shape, which indicated that cell–substratum interaction was being inhibited (Sage et al. 1989b). Subsequently, Lane and Sage (1990) identified the osteonectin domains

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responsible for this inhibition of spreading to be the N-terminal domain I and a sequence occurring in the C-terminal EF-hand domain. They raised antibodies against these peptides and demonstrated that the antibodies blocked the ability of osteonectin to inhibit cell spreading. Experimental overexpression of osteonectin, produced by transfection, in F9 embryonal carcinoma cells correlated with a rounded cell morphology, whereas transfection of antisense osteonectin cDNA constructs was found to restore cell spreading (Everitt and Sage, 1992). However, transfection of human osteonectin cDNA into human 293 and HT1080 cell lines has given somewhat contradictory results. Nischt et al. (1991) found no changes in cell spreading, proliferative rate, or adhesion behaviour of the transfected cell clones, even though they expressed osteonectin mRNA at high levels and secreted large quantities of the protein. Osteonectin can induce a loss of focal adhesion of bovine aortic endothelial (BAE) cells, and it appears to achieve this by a reorganisation of the submembranous cytoskeleton (Goldblum et al. 1994; Murphy-Ullrich et al. 1995). It would be a natural extension of the efforts described above to inquire into the mechanism by which osteonectin inhibits cell adhesion and spreading. As discussed earlier, osteonectin can alter the structural and functional characteristics of the ECM by inducing proteolytic degradation of its components, and thereby modulate cell–substratum interaction. Some clues are provided for the presence of another mechanism by the observation that osteonectin brings about changes in the submembranous cytoskeleton. Extracellular signals may be transduced to the cytoskeleton via integrin receptors. Integrins are heterodimeric transmembrane glycoproteins. They are composed of two subunits: the α and β subunits. A large number of these subunits has been identified. They combine to form a vast array of integrins that function as receptors for ECM components such as fibronectin, laminin, tenascin, osteopontin, thrombospondin, and vitronectin, as well as cell adhesion molecules such as VCAM and ICAM (Dedhar, 1990). These integrins form a link between the ligand and the cytoskeleton. There are at present no indications that the function of osteonectin is mediated by integrins, although we do know that it can bind to certain ECM components such as collagens and vitronectin, which do function through the agency of integrin receptors. Vitronectin is an adhesion-mediating protein. It is a secreted glycoprotein of predominantly hepatocyte origin. It occurs in the ECM of blood vessels and skin and has also been reported to occur in many tumours (Dahlback et al. 1986). It binds to αvβ3 integrin. The expression of this integrin has been associated with the progression of cutaneous tumours and some neuroectodermal tumours (see Sherbet and Lakshmi, 1997b). In light of these observations, it is significant that osteonectin can interact with vitronectin and modulate the adhesive properties of the latter (Rosenblatt et al. 1997). Interaction seems to occur between the heparin-binding region of vitronectin and the C-terminal calcium-binding domain of osteonectin, which is regarded as the region actively involved in the inhibition of cell adhesion and spreading. Rosenblatt et al. (1997) have demonstrated also that PAI-1 induces the binding of osteonectin with vitronectin. As stated previously, osteonectin is able to induce the expression of PAI. Thus the inhibitor seems to participate in the modulation of cell adhesion by two distinct pathways: by a direct route of inhibiting

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PA-mediated excision and modification of ECM components, and by an indirect route of inducing the interaction between osteonectin and vitronectin. Whether other ECM components are similarly targeted by osteonectin largely remains to be investigated. Some ECM components such as fibronectin and osteopontin can be eliminated. The binding of these to their integrin receptors is dependent on the presence of the RGD (arginine–glycine–aspartic acid) motif, which appears to function as a recognition signal for the binding of these ligands to their respective receptors. Sage (1992) has shown that inhibition of cell spreading by osteonectin is not affected by the RGD peptide. It may be suggested on the basis of this finding that neither fibronectin nor osteopontin enters into any interaction with osteonectin. However, one cannot yet exclude this possibility, because the adhesion of some cell types, such as osteocytes and osteoblasts, to osteonectin and osteopontin is strongly inhibited by the RGD peptide (Arden et al. 1996).

MODULATION OF CELL PROLIFERATION BY OSTEONECTIN Osteonectin can inhibit or stimulate cell proliferation. This faculty appears to depend on the cell type and possibly also on the physiological environment. Osteonectin expression has been found in some instances to closely correlate with the processes of cell proliferation and differentiation, upon exposure to hormonal or growth factor milieus. The modulation of endothelial cell proliferation by osteonectin, pursued rather relentlessly by the research group of Sage, is a prime example of its function. Some of their work is discussed below. Osteonectin can inhibit proliferation of microvascular endothelial cells induced by VEGF (Kupprion et al. 1998). In sharp contrast with effects on ECs, in dental pulp mesenchymal cells, bFGF enhances osteonectin expression and also acts as a potent mitogen (H. Shiba et al. 1995). In osteoblasts, oestrogen-induced differentiation and inhibition of cell proliferation is totally unrelated to the levels of osteonectin (Robinson et al. 1997). However, wound healing, where cell proliferation and migration are important processes, does involve osteonectin. Osteonectin is a component of the alpha granules of platelets, and along with thrombospondin it participates in platelet aggregation (Kelm and Mann, 1990; Clezardin et al. 1991). A high level of osteonectin immunoreactivity has been observed in the healing epithelium of the cornea, during a 6-day period following wounding, and then osteonectin reactivity falls off (Latvala et al. 1996). As shown in Table 13, two domains have been identified that correspond with these putative functions. Osteonectin has been shown transiently to inhibit the progression of vascular endothelial cells at the mid-G1 phase of the cell cycle. A similar inhibition is produced also by the peptide 2.1 (Funk and Sage, 1991). This inhibition was not accompanied by a rounding-up of cells. This is not surprising, because rounding-up of cells is a prelude to cell division, and cessation of division is usually followed by a phase of cell spreading. Peptides from domain IV containing the EF-hand motifs produced strong growth inhibition, e.g., peptide 4.2, which seemed to cooperate with peptide 2.1 (Sage et al. 1995). Funk and Sage (1991) noticed further that the inhibition of cell spreading was produced by peptide 1.1,

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which did not inhibit cell cycle progression. However, peptide 2.1 stimulated the proliferation of fibroblasts derived from human foreskin and foetal bovine ligament. This stimulation was strictly concentration dependent and occurred only in the range of 0.1 to 0.4 mM of the peptide. Higher levels of the peptide resulted in growth inhibition (Funk and Sage, 1993). Another peptide; i.e., peptide 2.3, stimulated the proliferation of fibroblasts. Overall, osteonectin appears to be able to control the process of proliferation in a cell type-specific fashion, in which different domains of the molecules participate. Although the function of different peptides in vitro cannot directly form the basis for inferring the function of osteonectin in vivo, it is possible that differential effects on cell proliferation, adhesion, and spreading could be seen as an attribute of different configurations of the molecule assumed upon calcium binding. There will be significant changes in the α-helical content of the molecule in relation to calcium binding. As pointed out by Maurer et al. (1996) conformational changes may occur within a domain. Furthermore, different distant domains may be linked and thereby alter their orientation relative to one another. One also needs to take into account the posttranslation changes, such as glycosylation and phosphorylation, which profoundly affect biological activity. Glycosylation, for instance, has a regulatory effect on the binding of osteonectin to collagen V (R.L. Xie and Long, 1995, 1996).

EFFECTS OF OSTEONECTIN ON ANGIOGENESIS Neovascularisation is a complex process consisting of several events, such as the endothelial cell proliferation and their migration toward the source of the angiogenic factor. The endothelial cells align themselves end to end to form a sprout, which then develops a lumen (reviewed by Sherbet and Lakshmi, 1997b). The currently available evidence for the induction or inhibition of angiogenesis by osteonectin is still somewhat indirect and rather scanty. This is based on the observation that osteonectin influences the proliferation of endothelial cells. Further, certain growth factors that are known to be angiogenic also enhance osteonectin expression, and factors that have been shown to be able to inhibit angiogenesis also inhibit osteonectin expression. Thus, TGFβ induces both angiogenesis and osteonectin expression, whereas IL-1 inhibits both. Some factors such as PA, PAI, and TIMPs might indirectly affect osteonectin expression and also influence angiogenesis. The uncertainty arises when one considers the effects of factors such as bFGF and TNF. bFGF is an inducer of angiogenesis. However, it down-regulates osteonectin expression in endothelial cells, although its effect is manifestly the opposite in mesenchymal cells. As discussed above, there is evidence that VEGF, which is demonstrably angiogenic (Zhang H.T. et al. 1995), inhibits osteonectin expression. Similarly, TNF induces angiogenesis but down-regulates osteonectin. The presence of osteonectin in the ECM of vascular structures and its proven ability to induce loss of focal adhesion of endothelial cells, and to reorganise the submembranous cytoskeleton, are clear indications of a positive effect on endothelial cell motility (see Table 14). Unfortunately, there is very little direct evidence that osteonectin induces angiogenesis. Some findings could be construed as direct evidence. For instance, the presence of osteonectin in enhanced quantities has been found to accompany the

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TABLE 14 Correlation between Angiogenesis and Modulation of Osteonectin Expression by Growth Factors and ECM Components

Effector Agent TGFβ PA IL-1 TIMPs Thrombospondin PAI VEGF bFGF TNF

Effect on Angiogenesisa

Effect on Osteonectin Status/Effect of Osteonectin on Effector Agent

+ + – –c – – – + +

+ +2b – –b – – –b –d –

Note: The table provides a summary of the evidence that links, directly or indirectly, angiogenesis with osteonectin expression. Although osteonectin contains domains that putatively possess both angiogenic and anti-angiogenic properties, the table, constructed on the basis of work discussed in the text, underlines the need for more definitive experiments in order to establish whether osteonectin is involved in the regulation of angiogenesis. a

+, up-regulation; –, down-regulation of expression. Osteonectin influences the expression of the effector agent. c TIMPs 1–4 are known to inhibit angiogenesis (Sherbet and Lakshmi, 1997b; Blavier et al. 1999). d bFGF is believed to up-regulate osteonectin in mesenchymal cells. b

formation of capillary-like structures by endothelial cells both in vitro and in vivo (Iruela-Arispe et al. 1991a, 1991b; Lane et al. 1994). Mendis et al. (1998) noticed an enhanced expression of osteonectin mRNA in the early stages of the formation of blood vessels following injury to adult rat cerebral cortex. However, one does not know whether changes in osteonectin levels occur as a consequence of induction of angiogenesis by some other factor, and whether osteonectin itself is a causative agent or merely an epi-phenomenon. One could concede that, prima facie, osteonectin seems to be involved in the regulation of angiogenic processes, but there is an acute need for more substantive data, such as, for instance, by transfecting osteonectin into appropriate recipient cancer cells and determining if the expression alters microvascular density. Technically this could be demanding, notably because osteonectin contains domains with both angiogenic and anti-angiogenic properties, but it is certainly an achievable goal.

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OSTEONECTIN EXPRESSION IN CANCER DEVELOPMENT AND PROGRESSION Osteonectin seems to be able to influence most of the biological properties of the cell that are highly relevant in the context of cancer development, growth, and dissemination. The most obvious reasons for this are that osteonectin is able to initiate changes in the ECM, cell adhesion and spreading, and also in the cytoskeleton. It has been found to influence cell proliferation, and apparently, it can regulate vascularisation. The elucidation of these properties has inevitably led to the study of its expression in cancer development and progression. Osteonectin reportedly occurs in a wide spectrum of cancerous tissues from human subjects. Porter et al. (1995) found high osteonectin immunoreactivity in invasive tumours of the GI tract, breast, lung, kidney, ovary, brain, and adrenal cortex. They state that normal tissues show low levels of reactivity. It is somewhat paradoxical that trophoblast cells, which are a highly invasive cell component of the placenta, show low levels of reactivity. Nevertheless, bone extracts and osteonectin itself have been found to enhance the motility in vitro of prostate epithelial cells as well as prostate cancer cells (Jacob et al. 1999). The presence of osteonectin has also been demonstrated in normal as well as adenoma of human thyroid (BurgiSaville et al. 1997). However, there is a reasonable body of evidence that suggests a close association of osteonectin expression with the progression of human melanomas. Osteonectin is not expressed by normal melanocytes and it is weakly expressed in a small proportion (4/25) of nevocellular nevi. The level of osteonectin expression is moderate in a majority (13/14) of dysplastic nevi. However, the expression occurs invariably and at a very high level in both primary (7/7) and metastatic (29/29) melanomas (Ledda et al. 1997). A few studies have also been carried out on the relevance of osteonectin as a marker for the progression of breast cancer. According to Bellahcene and Castronovo (1995), osteonectin is only weakly expressed in benign breast disease, but the expression is very strong in both in situ and invasive carcinomas. The presence of oestrogen receptors in breast cancer is regarded as an indicator of differentiation and good prognosis, and their absence is suggestive of clinically aggressive disease. Apparently, there is an inverse relationship between ER status and osteonectin expression. Tumour samples that were low in ER content tended to contain fourfold higher levels of osteonectin mRNA as compared with tumours with high ER levels (Graham et al. 1997). It would be of much interest, in this context, to examine whether osteonectin expression relates directly to the presence of EGFr, because ER-negative breast cancers often tend to be EGFr positive. There is thus an apparent relationship of osteonectin levels to disease progression. This has been confirmed in another study, in which Podhajcer et al. (1996) found that the osteonectin gene is expressed at a high level in invasive human breast carcinoma and also in metastatic lymph nodes. Osteonectin transcripts were found in the fibroblast stroma. Furthermore, high levels of expression of stromelysin-3 also accompanied high osteonectin expression. Osteonectin seems to be able to activate MMP2 in the invasive breast cancer cell lines MDA-MB231 and BT549, but not in the noninvasive MCF7 cells.

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This ability was associated with the osteonectin peptide 1.1 (Gilles et al. 1998). Although Gilles et al. (1998) also found some attenuation of TIMP-2, they have suggested that the invasive propensity of the breast cancer cell lines is most likely attributable to osteonectin-mediated activation of MMP2. There can be little doubt that metalloproteinase expression and the expression of their inhibitors corresponds closely with invasive potential of cancers (see Sherbet and Lakshmi, 1997b). The apparent relationship between osteonectin and MMP does not necessarily reflect a causal connection. There is the distinct possibility that a common effector, such as bFGF, may regulate both. Nonetheless, the outcome could be an alteration in the invasive nature of the tumour. Bellahcene and Castronovo (1995) have stated also that high expression of osteonectin was associated with calcification of the lesions. However, contrary to the findings of Bellahcene and Castronovo (1995), Hirota et al. (1995) reported no correlation between osteonectin expression and the development of foci of calcification. It ought to be stated, nonetheless, that in other cellular systems a relationship does seem to subsist between osteonectin levels and calcification. Human dental pulp cells maintained in tissue culture express osteonectin, and they also contain calcified nodules. The level of osteonectin closely correlates with the level of calcification. When these cells are treated with bFGF, osteonectin expression is reduced and calcification of the ECM is abolished (H. Shiba et al. 1995). M. Sato et al. (1997) used a human salivary cancer cell line and have reached similar conclusions. These cells produce tumours when implanted into nude mice. When treated with VD3, tumour growth rate was reduced and calcified foci appeared in the tumours. In parallel, M. Sato et al. (1997) also found the expression of osteonectin mRNA in these treated tumours. Whether the expression of osteonectin, together with other bone matrix components such as osteopontin and bone sialoprotein, could have some bearing on the propensity of breast tumours to metastasise to the bone, is currently being debated. Osteopontin has been implicated in tumour cell motility (Xuan et al. 1995; Sung et al. 1998). Oates et al. (1996, 1997) transfected Rama-37, a rat mammary epithelial cell line, with genomic DNA fragments from a human mammary carcinoma cell line. The transfectants were found to produce tumours with metastasising ability. They then isolated a cell line from a metastatic tumour and compared its mRNA profile with a control cell line, by subtractive hybridisation. One of the mRNAs strongly expressed, (nine-fold greater in the metastatic cell line as compared with the nonmetastatic parent line), was that for osteopontin. An increase in the level of expression does not constitute irrefutable evidence of a relationship to metastatic ability. Oates et al. (1996) did show that similar transfection of Rama-37 cells with DNA from benign tumours did not result in elevated expression of osteopontin. Some of these early studies have been confirmed recently. The levels of osteopontin have been found to be low in nontumorigenic cells and tumour cells with low metastatic ability. The osteopontin-transfectant cells as well as cells exposed to exogenous osteopontin have been reported to make marked gains in invasive ability (Tuck et al. 1999). What is even more interesting is the observation by these authors that, under both experimental conditions, the gain in invasive ability was accompanied by increases in the expression of uPA mRNA as well as the uPA protein. This

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has marked similarities with the effects of osteonectin on cell invasion. One should recognise, nonetheless, that VD3 can act independently of osteopontin, because although it can enhance the expression of both osteonectin and osteopontin, VD3 indeed inhibits cell proliferation and invasion. Its ability to inhibit invasion can be directly linked with the down-regulation of PAs and MMPs together with an upregulation of the endogenous MMP inhibitors. These arguments simply emphasise the need to include osteopontin in studies of osteonectin involvement in cancer cell invasion and metastasis. It would be relevant to cite here the observations of Jacob et al. (1999), who found that bone extracts as well as osteonectin enhanced the motility of tumour cells that normally metastasised to the bone, e.g., breast and prostate cancer. However, cell lines derived from tumours that do not normally metastasise to the bone did not respond in this way. Jacob et al. (1999) postulate, in consonance with the discussion above, that this apparent differential effect of osteonectin on cell motility could be due to the ability of osteonectin to induce tumour-associated metalloproteinase activity. An activation of uPA together with enhanced proliferative potential has been reported in MCF7 cells exposed to soluble factors secreted by the osteogenic cell line SaOS-2. MCF7 cells are normally only weakly invasive, but they appeared to become more invasive when cultured on an ECM produced by SaOS-2 cells. Furthermore, this acquisition of invasive potential seemed to be related to the ability of the ECM to induce uPA expression in MCF7 cells (Martinez et al. 1999). Although these findings are of considerable significance in the context of cancer invasion, it is necessary to take into account a number of related facts and factors, lest one be led into an alley of overinterpretation of the data. It should be recognised that both MCF7 and MDA cells do synthesise uPA, although MCF7 cells do so at a far lower level than do MDA-MB231 cells. It should be recognised also that ability to synthesise uPA is not itself directly related to the invasive ability of cancer cells. Urokinase receptors as well as PAIs enter into the equation. Undoubtedly there is a large body of correlative evidence derived from the study of plasminogen activator expression in a host of human tumours. Nonetheless, all potential interacting factors need to be checked before one can be certain that one is, indeed, dealing with uPA as the major instigator of invasive potential. Martinez et al. (1999) state that the enhancement of invasive behaviour was found only in the ER-positive MCF7 cells, but not in the ER-negative MDA-MB231 cells. This is consistent with the experiments described by Hachiya et al. (1995), who found that oestrogen enhanced uPA as well as tPA expression and also enhanced the invasive ability of breast cancer cells. They also demonstrated that PA expression is regulated by oestrogen, because tamoxifen blocked the production of PA and also inhibited the invasive ability. Among other factors that come into the reckoning is the hepatocyte growth factor (HGF). HGF bears sequence homology to PA. PA is known to activate HGF (Mars et al. 1993). Furthermore, HGF is a potent mitogen and can induce angiogenesis as well as vascular invasion (Hildebrand et al. 1995). It would be worth recalling here that the ER status correlates inversely with EGFr in these cell lines. EGF is another modulator of the invasive behaviour of cancer cells. Whether the changes in the invasive behaviour might have been mediated by EGF receptors is a moot point.

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As stated previously in this section, osteonectin expression is said to be inversely related to ER status, with high expression of osteonectin being found in ER-negative cells. Furthermore, it also has been claimed that osteonectin stimulates MMP expression in MDA cells, but not in MCF7 cells. It may be that uPA is more relevant in the context of breast cancer cell invasion than are MMPs. Overall, there is a reasonable body of evidence to suggest the ectopic expression of these bone matrix proteins might have serious implications for the osteotropic metastasis of breast cancer. However, the mechanisms involved remain to be elucidated. Although the above discussion suggests a direct relationship between malignancy and levels of osteonectin expression, in ovarian epithelial cells an inverse relationship has been reported. Mok et al. (1996) found high osteonectin expression in normal ovarian epithelial cells, but this was markedly reduced in ovarian carcinoma cells. They also transfected osteonectin cDNA into SKOV3 cell, which is an ovarian carcinoma cell line. This resulted in reduced growth rate in vitro, and furthermore, these cells were less tumorigenic when implanted into nude mice. These results suggest and impute a tumour suppressor property to osteonectin. This is supported by recent work by Vial and Castellazzi (2000). The expression of osteonectin is down-regulated when cells are transformed by oncogenes. Such a downregulation is noticed in chick embryo fibroblasts transformed by v-src or v-jun oncoproteins. When the protein is reexpressed, these cells retain the transformed phenotype but lose their ability to form fibrosarcomas in vivo (Vial and Castellazzi, 2000). In other words, in the experimental model osteonectin does seem to behave like a tumour suppressor. Nonetheless, it would be reasonable to expect further confirmation of the possible tumour suppressor function. Some of these uncertainties are compounded by the view expressed in some quarters that osteonectin might not be a reliable marker for osteosarcoma (Grundmann et al. 1995; Park et al. 1996). An overall view of the present status of osteonectin in relation to cancer progression ought to be ambivalent. There is a need for far more extensive investigation of human tumour types. Above all, much more experimental work is needed to establish the various postulates that bring together the putative functions of osteonectin with the altered biological properties of the cancer cell. In a sense, therefore, it would be premature to dive into investigations of clinical material without appropriate groundwork. This is especially important with respect to osteonectin function, because the protein contains domains that reputedly possess antagonistic functions. The scientific community has not even begun to unravel the mechanisms by which these antagonistic functions become expressed in the physiological setting. Equally, it would be unreasonable to delay the investigation of the relevance of osteonectin in tumour classification, or its potential in clinical management of patients, if indeed it has even the semblance of predictive value.

OSTEONECTIN INVOLVEMENT IN OTHER DISEASE STATES A number of nonneoplastic diseases have been linked with osteonectin. Among these are rheumatoid arthritis (RA) and osteoarthritis (OA). Immunostaining studies of

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cartilage and synovial specimens from patients with RA and OA have been reported (S. Nakamura et al. 1996; Nanba et al. 1997). Osteonectin immunoreactivity is found in chondrocytes in the superficial and middle zones of the cartilage from RA and OA joints, but these zones of normal cartilage are either devoid of any activity or stain only too weakly. Osteonectin expression is greatly increased in synovial cells from these patients. S. Nakamura et al. (1996) have also reported that, on average, the osteonectin content of synovial fluid from RA patients was ten-fold greater than that of synovial fluid from OA patients. Nanba et al. (1997) have made the interesting and valuable observation that osteonectin is detected in the early stages of OA, and they point out that this could be significant in terms of disease progression, because cartilage calcification is closely related to the stage of the disease. Liver fibrosis appears to be associated with enhanced levels of osteonectin. According Inagaki et al. (1996), osteonectin is virtually undetectable in normal liver, but osteonectin gene transcripts were abundantly expressed, mainly in the lipocytes, which markedly increase in number in fibrosis. Similar increases in osteonectin levels have been described by Blazejewski et al. (1997). These authors reported that cultured myofibroblasts from liver synthesised osteonectin. They further observed that in normal liver myofibroblasts occured in small numbers, but that they proliferated markedly in fibrosis and synthesise ECM proteins. Consistent with this, osteonectin mRNA expression was low in normal liver but was markedly enhanced in fibrotic liver. Osteonectin expression is reported to decrease at the early stages of diabetesrelated renal enlargement in experimental animals (Gilbert et al. 1995). As noted by Gilbert et al. (1995), renal enlargement is a mixture of several events occurring in the ECM along with cell hypertrophy and hyperplasia, and one can see how osteonectin might be implicated in the pathogenesis of the disease.

OSTEONECTIN HOMOLOGUES AND THEIR PUTATIVE TUMOUR SUPPRESSOR PROPERTIES Osteonectin is probably a typical member of a large family of proteins whose members share several important structural features and perform similar functions. In recent years several cDNAs have been isolated that show a high degree of structural homology to osteonectin. Hevin (MAST9, SC1), QR1, testin, and tsc36/FRP are members of the osteonectin family. Hevin was first identified in endothelial venules of lymphoid tissue (Girard and Springer, 1995, 1996). The hevin gene is located on chromosome 1 (Claeskens et al. 2000). The hevin cDNA encodes a protein that shows a substantial sequence homology to osteonectin, SC1 (of murine origin), and QR1 isolated from quail (Girard and Springer, 1995). Monomeric hevin is 75 kDa in size and possibly forms dimers in vitro. Bendik et al. (1998) have characterised the full-length cDNA, which has 2808 nucleotides and an ORF of 1992 nucleotides corresponding to a 75-kDa protein. Overall, all osteonectin homologues identified thus far show a molecular organisation similar to that of osteonectin, and, in particular, possess the characteristic FC domain and EF-hand motifs in the calcium-binding domain.

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Hevin is expressed ubiquitously in most normal tissues and in diseased tissues of nonneoplastic origin. There may be differences in the patterns of their distribution in normal tissues, as demonstrated for SC1 (Soderling et al. 1997). The expression of hevin has been reported to be down-regulated in many neoplasms, e.g., in nonsmall cell lung carcinome (NSCLC) (Bendik et al. 1998) and adenocarcinoma of the prostate (P.S. Nelson et al. 1998). Claeskens et al. (2000) transfected hevin cDNA into HeLa-35 cells, which do not express indigenous hevin. The transfected hevin cDNA negatively regulated proliferation and seemed to block G1–S transition of cells. Therefore, Claeskens et al. (2000) suggest that hevin may be a putative suppressor gene. However, it might be premature to label a gene as a suppressor gene merely on the basis of possible inhibition of cell proliferation. It ought to be stated, in defence of the postulate, that hevin does possess anti-adhesion properties, can inhibit focal adhesion of cells and confers a rounded morphology on cells (Girard and Springer, 1996). Needless to say, that this area is worthy of pursuit and should provide interesting results relating to the relevance of these molecules in cell migration, diapedesis, and cancer dissemination.

17

S100 Proteins: Their Biological Function and Role in Pathogenesis

There has been an unremitting effort by the scientific community in the past two decades aimed at identifying the genetic determinants of the invasive and metastasising properties of cancer cells. This has been based on the premise that the acquisition of these seemingly aberrant abilities flows from the expression of a candidate gene or a group of genes that might be connected with the process of metastasis. However, one can muster very little direct evidence for the “metastasis gene” concept. Indeed, a review of recent research would suggest that alterations of the proliferative status, motility, and cell–cell and cell–substratum adhesion of cells, may be seen as the basic requirement for cancer growth, invasion, and metastasis, as well as other nearly analogous processes such as cell differentiation, growth, and morphogenesis. Quite obviously, therefore, a vast array of genes can be identified whose expression profoundly affects cancer cell behaviour as well as cell differentiation, growth, and morphogenesis (Sherbet and Lakshmi, 1997b). It can be argued that neoplastic transformation of cells, accompanied by the acquisition of enhanced proliferative potential, motility, and heterotypic adhesion properties, can be attributed to an overexpression or inappropriate expression of genes that are associated with extracellular signal transduction, and those required in normal physiological function. The S100 proteins, which form the main focus of this section, have been shown to be capable of modulating enzyme function and altering cytoskeletal dynamics. They can bind to a variety of cellular target proteins, and possibly by this means control cell cycle progression. Furthermore, much evidence has accumulated that shows these proteins are associated with terminal cell differentiation; they can promote remodelling of the ECM, alter cell shape, motility, and enhance the invasive behaviour, and metastatic spread of cancer. Here we attempt to link the physical and physiological alterations occurring in a cell consequent to overexpression of S100 proteins with the salient phenotypic aspects of biological behaviour of cells. The S100 family of proteins comprises a large number of CBPs. As can be seen in Table 15, a majority of them are found in a 2.05-Mbp segment of human genomic DNA of chromosome 1q21 region (Marenholz et al. 1996; Mischke et al. 1996; Schafer et al. 1995). The S100 family of genes, including those coding for profilaggrin (FLG)

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TABLE 15 S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function S100 Nomenclature S100A1 (S100α1)

S100A2/S100L S100A3/S100E

S100A4 (18A2/mts1, CAPL, p9Ka, metastasin, calvasculin, FSP1) S100A5/S100D S100A6 (calcyclin, 2A9, CACY, caltropin) S100A7 (psoriasin)

S100A8 (MRP8)a S100A9 (MRP14)a

S100A10 (p11, calpactin light chain, 42C) S100A11 (S100C)

S100A12 (calgranulin C, P6, CGRP, CAAF1)

Chromosomal Location (Human)

Putative Physiological Function and in Pathogenesis

1q21 (Dorin et al. Associated, together with S100B, with 1990; Engelkamp cardiomyopathy; differentially modulated in tissues et al. 1993; Schafer in experimental diabetes (Zimmer et al. 1997); see et al. 1995) text 1q21 (Engelkamp et Putatively associated with tumour suppression; see al. 1993) text 1q21 Specifically expressed in the skin; participation in the differentiation of hair follicles; found in glial tumours 1q21 See text

1q21 1q21 1q21 (Hardas et al. 1996) 1q21

1q21 (Mooglutz et al. 1995; Wicki et al. 1996a) 1q21 (Wicki et al. 1996a; Yamamura et al. 1996)

Associated with acute myeloid leukaemia (Calabretta et al. 1986a,b) and melanomas; see text Psoriatic skin and other skin diseases; regarded as a potential marker for squamous cell carcinoma of the bladder; see text Together with MRP-14, associated with cystic fibrosis, rheumatoid arthritis (Odink et al. 1987; Fanjul et al. 1995); inflammatory bowel disease, allograft rejection, recruitment of neutrophils and monocytes to delayed-type hypersensitivity inflammatory sites (Dunn et al. 1996); might be involved in the regulation of the inflammatory process, transendothelial migration of monocytes (Kerkhoff et al 1991a, 1999b)

Differentially expressed in uveal melanomas and cell lines derived from them (Van Ginkel et al. 1998). Gene with 3 coding exons; exons 2 and 3 encode peptide of 92 amino acid residues (Wicki et al. 1996a, Yamamura et al. 1996a); 2 EF-hand domains; isolated from neutrophils, bovine amniotic fluid (Guignard et al. 1995; Hitomi et al. 1996, 1998); also found in foetal epidermal keratinocytes, squamous epithelia; oesophageal epithelium; differentiation-related expression, but no known association with cell proliferation; associated with Mooren’s ulcer

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TABLE 15 (CONTINUED) S100 Family of Calcium-Binding Proteins: Their Chromosomal Location and Putative Physiological Function S100 Nomenclature

Chromosomal Location (Human)

CAAF2

Putative Physiological Function and in Pathogenesis 2 EF-hand domains; ca. 30% sequence homology with CAAF1; 63% homology with S100A7 (Hitomi et al. 1996) ca. 50% homology to S100A5 and ca. 60% to S100A12; mouse A13 shows ca. 87% homology to human A13; mRNAs expressed at high levels in skeletal muscle, heart, kidney, ovary, pancreas (Wicki et al. 1996b) Associated with Alzheimer’s disease; Down’s syndrome, neurite extension factor; occurs in glial and Schwann cells, cytoskeletal disruption; possibly influences cell cycle progression via activation of Ndr protein kinase Isolated from human placenta (Becker et al. 1992; Emoto et al. 1992); cutaneous sensory signal transduction; progression of prostate cancer; see text Filaggrin IF monomers, aggregation of filaments in keratinocyte differentiation; reduced expression in and possible aberrant interaction with keratin filaments in ichthyosis; rheumatoid arthritis

S100A13

1q21 (Wicki et al. 1996b)

S100B/S100β

21q22 (Duncan et al. 1989)

S100P

4p16 (Schafer et al. 1995)

FLG (profilaggrin)

1q21 (Marenholz et al. 1996; Mischke et al. 1996)

Trichohyalin (THH)

1q21 (Fietz et al. 1992) Mouse chromosome Overexpression in mouse skin tumours (Krief, P., 3 (Krief et al. personal communication) 1997) Xp22.2

Repetin (rptn) (mouse)

Calbindin D-9K (intestinal calcium-binding protein) a

MRP8 and 14 are regarded as myeloid-specific proteins. Shapiro et al. (1999) have identified a B-cellspecific antigen, which appears to possess N-terminal sequence homology with human MRP8. The antigen also shows homology to other S100 proteins too, albeit at a far lower level. Shapiro et al. (1999) have argued, on rather tenuous grounds, that because MRP8 is not expressed in lymphocytes, this antigen might represent a new member of the S100 family.

Source: Data collated from Heizmann (1996), Sherbet and Lakshmi (1997b), and references cited in the table and the text.

and trichohyalin (THH), which are associated with terminal differentiation of keratinocytes, are arranged in the following order: 1 cen–S100A10–S100A11–THH– FLG–IVL (involucrin)–LOR (loricrin)–S100A9–S100A12–S100A8–S 100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A13–S100A1–1qtel. S100B is an exception in that it occurs on chromosome 21q22 (Duncan et al. 1989).

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A similar clustering and synteny of S100 genes is found in mouse chromosome 3, and the organisation shows a great resemblance to that of the human S100 gene cluster. Eight mouse S100 genes are organised in the same way as the human genes. The linkage between S100A8 and S100A9 and the linkage of the array S100A3–S100A4–S100A5–S100A6 are conserved. However, S100A1 and S100A13 are separated, unlike as in the human gene cluster. Therefore, much of the organisation seems to be conserved in evolution, with a degree of rearrangement of some of the genes (Ridinger et al. 1998). S100 proteins are in general of a small molecular size. Although some of them, e.g., S100B(β) and S100A(α), have been known for a long time and much information has accumulated over the years, the function of the majority is still largely unknown. There have been significant advances in recent years in our understanding of the involvement of some S100 proteins in normal and aberrant physiology. Especially noteworthy are the close association some of these proteins show in the abnormal behaviour of cancer cells, and the effects of their elevated expression in tumours and the consequent modulation of cytoskeletal dynamics, cell mobility, and cell proliferation.

S100 PROTEINS IN CELL DIFFERENTIATION, MOTILITY, AND CANCER INVASION PROFILAGGRIN (FLG)

IN

KERATINOCYTE DIFFERENTIATION

The consortium of genes at the 1q21 region, whose physical mapping has been referred to above, includes a number of genes associated with the terminal differentiation of human epidermis. This collection of genes has been called epidermal differentiation complex (EDC) (Mischke et al. 1996). This complex has been most lucidly described by Marenholz et al. (1996) as being composed of three groups of genes: (1) structural genes such as IVL, LOR, and others; (2) genes coding for the IF-associated proteins FLG and THH; and (3) S100A1–S100A10. FLG and THH are synthesised in the granular layer of the epidermis and participate in the aggregation and aligning of keratin IF during the terminal differentiation of keratinocytes. Both proteins contain EF-hand calcium-binding domains and are functionally associated with Ca2+-dependent processing (see below). Krieg et al. (1997) have cloned a novel gene, the repetin gene, from the mouse. This gene has marked similarities to FLG and THH with respect to genomic organisation and EFhand calcium-binding domain characteristics. Repetin, together with FLG and THH, could represent a subfamily of S100 genes (Table 16). The Molecular Characteristics of Profilaggrin Human FLG is a 400-kDa phosphoprotein that is found in the granular cells of the epidermis. Its N-terminal region contains two calcium-binding motifs similar to the EF-hand calcium-binding domains found in the S100 family of proteins. The two calcium-binding sites differ markedly with respect to their calcium-binding affinity (Presland et al. 1995). The FLG molecule consists of several units of filaggrin linked

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TABLE 16 Genomic and Molecular Features of Profilaggrin, Trichohyalin and Repetin Feature Exons/introns EF-hands Repeat elements

FLG 3 exons, 2 introns Two N-terminal exons I and II Filaggrin units

THH

Two N-terminal Involucrin-like

rptn 3 exons, 2 introns Two N-terminal exons I and II Involucrin-like

Source: Based on Markova et al. (1993); S.C. Lee et al. (1993); Presland et al. (1995); Krieg et al. (1997).

together. The molecule undergoes proteolytic cleavage at the sites of linkage during terminal differentiation, generating monomeric filaggrin units with an average molecular size of 42 kDa. The filaggrin units also acquire specific C- and N-terminal sequences as a result of this proteolytic processing (Resing et al. 1993a; Thulin and Walsh, 1995). The processing of FLG is a two-stage process that generates intermediate products with filaggrin repeats. An endopeptidase, FLG endopeptidase 1 (PEP1), involved at this first stage of the cleavage of the linkers has been identified. The functional specificity of this enzyme has been suggested to be due to phosphorylation of FLG (Resing et al. 1995). PEP1 is a serine protease. Also involved in this proteolytic processing are leupeptin-sensitive cysteine protease and protein phosphatases. The former has features resembling calpain (Resing et al. 1993b, 1995). The calpains are Ca2+-activated cysteine proteases. Yamazaki et al. (1997) have shown that µ-calpain is capable of processing FLG (Figure 25).

FIGURE 25 The generation of filaggrin monomers from profilaggrin, with enzymatic cleavage of linking peptides occurring between filaggrin repeats. One of the enzymes implicated in this process is µ-calpain. This enzyme is made up of a large 80-kDa subunit and a 30-kDa subunit. Calpain is activated by the binding of calcium to CaM-like domains that occur in both subunits. (Based on Resing et al. 1993a, 1993b, 1995; Saido et al. 1993; Yamazaki et al. 1997; Kam et al. 1997).

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The expression of FLG is markedly reduced in ichthyosis. This has been attributed to its regulation at the posttranscriptional level. A decimation of FLG levels in keratinocytes from ichthyosis-affected patients has been reported, but the decrease in FLG mRNA expression amounted to only 30 to 60% of controls (Nirunsuksiri et al. 1995). Another reason for this could be the stability of the gene transcript. The FLG transcripts in keratinocytes of ichthyosis subjects were found to be far more unstable and possessed a shorter half-life than the transcripts in normal keratinocytes (Nirunsuksiri et al. 1998). The normal function of FLG might also depend on the state of its phosphorylation. It has been proposed that phosphorylation may prevent FLG from causing premature aggregation of keratin filaments and their packaging into storage granules. It has been suggested also that phosphorylation might be involved in the proteolytic processing of FLG into filaggrin monomers (see Figure 25). The conversion of the highly phosphorylated FLG into nonphosphorylated filaggrin appears to depend on protein phosphatases PP-2A and PP-1. Keratinocytes show a high level of PP-2A and PP-1 activity, but in harlequin ichthyosis, the activity of these phosphatases is greatly reduced, with a resultant disruption of filaggrin generation (Kam et al. 1997). It has been reported that PKC-δ can mediate FLG phosphorylation. This, together with the solubilisation of FLG, indicates that phosphorylation may be involved in the generation of filaggrin monomers (Old et al. 1997). Filaggrin monomers are formed during terminal differentiation of keratinocytes. A major process occurring at this stage of differentiation is the aggregation of keratin filaments. This occurs in association with FLG monomers. It is believed that profilaggrin itself is unable to accomplish this. Haydock et al. (1993) transfected antisense FLG constructs into rat epidermal cell lines. In transfectants expressing the antisense mRNA, the processing of FLG was suppressed, and the differentiation in vitro of keratinocytes was markedly affected as a consequence. The generation of filaggrin also produces marked changes in cell morphology and affects nuclear integrity. Cell adhesion to substratum also appears to be affected (Dale et al. 1997). FLG occurs in keratohyalin granules, and a recent study by Dale et al. (1997) suggests that the insoluble native form of FLG is required in their formation, which indicates that the linker peptides have a role to play in this process. Profilaggrin and filaggrin might be involved in the pathobiology of rheumatoid arthritis (RA). They show a distribution pattern similar to the antigens recognised by antibodies that are used as markers for RA. These so-called anti-keratin antibodies have been shown to recognise epidermal filaggrin and are demonstrably anti-filaggrin antibodies (Simon et al. 1995). Antibodies against filaggrin have been employed as a diagnostic aid, and their use might complement that of anti-keratin antibodies in diagnosing RA (Vincent et al. 1998). The possibility has been envisaged that the pathogenesis of RA could involve a loss of immunological tolerance to filaggrin (Berthelot et al. 1995). Abnormal interaction between keratin filaments and filaggrin may be involved in some cases of epidermolytic ichthyosis. Here, although profilaggrin/filaggrin are expressed at an enhanced level, no abnormalities of this protein have been encountered (Ishida-Yamamoto et al. 1994). Abnormal expression of FLG as well as K6 and K16 filaments is found in keratinocytes from the congenital condition of

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harlequin ichthyosis. FLG expression is either severely down-regulated or totally undetectable in ichthyosis vulgaris. The possible reasons for this have been discussed above.

TRICHOHYALIN (THH) Trichohyalin is another protein that is associated with IF. THH is a 248-kDa protein that is found in trichohyalin granules and possesses two EF-hand-like calciumbinding domains at its N-terminal end. The protein could occur as a flexible rod approximately 215 nm in length (S.C. Lee et al. 1993). It associates with and forms regular and precise arrays of keratin IF in the medulla and inner root sheath cells of hair follicles (Oguin et al. 1992). THH appears to undergo specific sequential posttranslational modifications to produce highly cross-linked rigid filaments. Tarcsa et al. (1997) have proposed that the insoluble cytoplasmic THH is first modified by peptidyl arginase to convert it into a less organised and soluble molecule. These THH molecules are then cross-linked by the agency of Ca2+-dependent transglutaminases to generate rigid filaments that interact with keratin filaments. THH is also found in nonfollicular epithelia, e.g., filiform papillae of tongue epithelium (E.J. O’Keefe et al. 1993). It occurs, together with FLG, in epithelia that possess IF containing K6/K16 keratins, which has led to the suggestion that THH may specifically mediate aggregation of IF containing these keratins (Manabe and Oguin, 1994). The association of THH with a possible differentiation pathway has prompted studies into THH expression in skin tumours (Manabe et al. 1996). Preliminary findings also are available with regard to the possible involvement of repetin (rptn) in the progression of skin tumours in the mouse (P. Krieg, personal communication, 1998). Apparently, rptn mRNA is expressed at low levels in normal mouse epidermis. Treatment with TPA, which is a tumour promoter, results in a transient overexpression of the mRNA. In benign tumours this overexpression becomes constitutive. Benign papillomas show far higher levels of rptn mRNA than normal epidermis. During progression into carcinomas, the steady-state levels of rptn mRNA are reduced. Compatible with this, in a few squamous cell carcinomas investigated, rptn mRNA has been found to occurs at lower levels as compared with papillomas. There is a hint of the possibility that rptn gene expression may be involved as an early event in the progression of skin tumours. These studies are too preliminary in nature to merit serious discussion at present. However, the recognition of the potential of these genes in the context of cancer development does merit citation. Needless to say, they could lend themselves as possible candidates for markers of differentiation in the development and progression of skin tumours.

EFFECTS OF S100 PROTEINS ON CELL DEFORMABILITY AND CELLULAR MORPHOLOGY The ability to alter the malleability of the membrane and cell shape and regulation of intercellular adhesion are two essential requirements for facilitating cell motility, diapedesis, and invasiveness. Changes in cellular morphology also occur in terminal

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differentiation and when differentiation is experimentally induced. For instance, in neuroblastoma cells exposed to retinoic acid, changes occur in cell shape with attendant changes in S100 family gene calcyclin (Tonini et al. 1991). Calcyclin appears to be associated with the tangential migration of proliferating cells of the subventricular zone of the lateral ventricle into the olfactory lobe and their differentiation into interneurones. A subpopulation of astrocytes in the tangential migration pathway seems to express calcyclin (Yamashita et al. 1997). The migration of subventricular zone (SVZ) cells is believed to be organised by the polysialylated neural cell adhesion molecule (PSA-NCAM). Neural cell adhesion molecules (NCAM) and cadherins have been studied extensively in the context of neurite regeneration and neuronal adhesion. Whereas cadherins are Ca2+ dependent for their function, NCAMs are not. It is of considerable interest, therefore, that the migration of the SVZ cells mediated by an NCAM is associated with calcyclin expression. Intracellular calcium levels regulate cellular morphology. Cells appear to alter their shape in response to subtle changes in intracellular Ca2+. Thus calcium transients produced by the mobilisation of calcium from intracellular stores can have effects that differ profoundly from those induced by influxed calcium. Caffeine induces transient increases in calcium in the dendrites and spines of hippocampal neurones. An increase in the size of existing dendrites and possibly also the formation of new dendrites occurs in association with these calcium transients (Korkotian and Segal, 1999). In contrast, glutamate receptor activation by NMDA and glutamate itself produces a collapse of dendritic spines, together with an infux of calcium. This leads to comparatively large changes in the levels of intracellular calcium. The loss of dendritic spines corresponds with the loss of filamentous actin. Actin-stabilising agents counteract the effects of NMDA (Segal, 1995; Halpain et al. 1998). Influxed calcium and calcium mobilised from intracellular stores might have independent roles (Haverstick et al. 1991). It also has been recognised that calcium released from intracellular stores might activate specific components of the signal transduction pathway (Parker and Sherbet, 1992). One can then hardly overemphasise the potential influence CBPs can exert on cellular morphology. S100 proteins alter cell shape and motility apparently by virtue of their ability to alter cytoskeletal dynamics. Inhibition of S100B, which is predominantly expressed in glial and Schwann cells, has been shown to result in changes in cellular morphology as well as in the organisation of the cytoskeleton of glial cells in culture (Selinfreund et al. 1990). Selinfreund et al. (1990) introduced antisense S100B oligonucleotides, which were placed under the control of a dexamethasone-inducible promoter, into rat C6 glioma cells. When the antisense constructs were induced to express, the levels of S100B decreased with concomitant changes in cell morphology. They assumed a flattened morphology and displayed an organised network of microfilaments. The neurite extension factor, which bears sequence homology to S100B, is a homodimer of S100B-like subunits. This factor actively promotes neurite extension (Kligman and Marshak, 1985). S100 proteins are expressed in the dendritic cells in human transitional cell carcinoma of the bladder, and the invasive potential of these tumours has been found to correlate with the presence of cells expressing S100 protein (Inoue et al. 1993). The occurrence of S100 proteins together with

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smooth muscle actin and vimentin has been reported in a metaplastic carcinoma of the breast invading the chest wall as well as in recurrent tumour (Harb et al. 1995). Reeves et al. (1994) generated transgenic S100B mice in which S100B mRNA expression was two- to seven-fold greater than that in control mice. They found enhanced expression of glial fibrillary acidic protein (GFAP) and also enhanced neurite proliferation in the transgenic mice. Similarly, the expression of S100A4 appears to correlate with the in vitro invasive potential of glioma cells in culture (Merzak et al. 1994b). The ability of S100A4 to interact with nonmuscle myosin supports the view that it takes part in cellular motility (Takenaga et al. 1994a, 1994b). S100A4 is highly expressed during embryonic development in the highly invasive mesenchymal elements (Klingelhofer et al. 1997). These effects appear to be due to an interaction of the S100 proteins with the cytoskeletal machinery of the cell. NGF markedly enhances calcium uptake (see Kozak et al. 1992). Many of the effects of NGF on PC12 cells, for instance, are accompanied by alterations in the expression of S100 proteins. An S100 protein called p11 (S100A10), identified some years ago, which shows a high degree of sequence homology to p42C. The latter is induced in PC12 cells by treatment with NGF (Masiakowski and Shooter, 1988). p11 binds to and inhibits the phosphorylation of a tyrosine residue of annexin (p36). It has been found that a complex composed of p11 dimer and two subunits of annexin p36 is involved with the process of actin bundling and in the linkage of the actin component to the cell membrane (Kligman and Hilt, 1988). Zimmer et al. (1998) transfected S100A1 cDNA in antisense orientation into PC12 cells. These transfectants showed a marked reduction in S100A1 expression. When these transfectant cells were exposed to NGF, there was a marked increase in neurite formation. In parallel, Zimmer et al. (1998) also found increased levels of α-tubulin. The antisense S100A1 clones also showed reduced anchorage-dependent growth. S100B has been reported to form a complex with the cytoskeletal tau protein, and this complex appears to inhibit microtubule polymerisation (Baudier et al. 1982; Baudier and Cole, 1988). S100 proteins show a substantial expression in brain tissues; they are found in astrocytes and oligodendrocytes, and their expression seems to be functionally linked with their morphology and differentiation. These functions may be associated with changes in the cytoskeletal elements, their organisation, and disposition. S100 proteins have several putative target cellular proteins; interactions with these might influence a panoply of cellular features (Table 17). S100B has been shown to inhibit the polymerisation of GFAP (Bianchi et al. 1993). It appears to interfere with the early stages of GFAP polymerisation, reducing the rate of assembly of GFAP subunits, besides actively promoting depolymerisation of GFAP polymers (Bianchi et al. 1994). A specific domain in GFAP interacts with S100 proteins, and a peptide known as TRTK-12, which bears homology to this domain of GFAP, has been shown to be able to counteract the inhibition by S100 of the polymerisation of GFAP monomers (Bianchi et al. 1996). Bianchi et al. (1996) have suggested further that S100B achieves this by sequestering GFAP monomers. This view is in sharp contrast with a recent observation that S100B promotes the extension of MAP-2, and might indeed promote the reassembly and stabilisation of the cytoskeleton (Nishi et al. 1997). S100A4 also has been shown to be associated with cytoskeletal elements

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TABLE 17 S100 Proteins and Their Intracellular Targets S100 Protein

Target Molecule

Functional Association

S100A1

Phosphoglucomutase

Stimulation

S100A4

Myosin II Nonmuscle tropomyosin F-actin

Cell locomotion Cell locomotion

Tubulin monomers

Cell proliferation; locomotion Cell proliferation

p53 S100A8/A9 (MRP8/14) S100A10 (p11) S100A11 (S100C)

S100B

Membrane arachidonic acid Annexin II Binds to actin filaments and inhibits Mg2+ATPase; annexins Phosphoglucomutase GFAP CapZ actin-capping protein Ndr nuclear serine/threonine kinase Calponin

Cell locomotion

Translocation to cell membrane Cytoskeletal reorganisation Reorganisation of the cytoskeleton; putative participation in growth and differentiation Stimulation Inhibition of GFAP polymerisation Actin polymerisation Influence on cell cycle progression by the activation of Ndr kinase Interaction with membrane phospholipids

Ref. Zimmer et al. (1995); Landa et al. (1996) Ford et al. (1997) Takenaga et al. (1994b) Davies et al. (1993); Gibbs et al. (1994) Lakshmi et al. (1993) Parker et al. (1994a, 1994b) Klempt et al. (1997) Kaczanbourgois et al. (1996) Zhao et al. (2000); Naka et al. (1994); Mailliard et al. (1996); see text Landa et al. (1996) Bianchi et al. (1993) Kilby et al. (1997) Millward et al. (1998)

Fujii et al. (1995)

(Davies et al. 1993; Gibbs et al. 1994; Takenaga et al. 1994a). In S100A4 transfected Rama-29 cells, the S100A4 staining pattern markedly resembles that of phalloidin, which binds to F-actin filaments (B.R. Davies et al. 1993; Gibbs et al. 1994). Such an association seems to result in cytoskeletal disorganisation (Watanabe et al. 1992, 1993), possibly related to the ability of the protein to promote microtubule depolymerisation (Lakshmi et al. 1993). Takenaga et al. (1994c) demonstrated that the binding of S100A4 to nonmuscle tropomyosin was calcium dependent and identified amino acid residues 39–107 of S100A4 as the binding site. They showed further by immunoblotting that part of S100A4 associated with Triton-insoluble cytoskeletal components. Indeed, in situ, S100A4 could be localised together with tropomyosin in microfilament bundles. In NIH3T3 and 3Y1 tissue culture cells S100A4 is found diffusely distributed in the cytoplasm, but some of the protein is also found in association with actin stress

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fibres (Takenaga et al. 1994b). S100A4 also has been shown to interact specifically with myosin II at the rate of 3 moles of S100A4 per mole of MHC. However, it does not interact with myosin I or II. Further, this interaction might destabilise myosin filaments (Ford et al. 1997). The S100A4-binding site has been identified. It occurs in the rod region and is mapped to a sequence, between residues 1909 and 1937, at the C-terminal end of MHC (Kriajevska et al. 1998). These authors have shown that S100A4 inhibits PKC-mediated phosphorylation of MHC at serine residue 1917. It seems possible that S100A4 might influence tropomyosin-assisted myosin–actin interactions. The distribution of A and B isoforms of nonmuscle myosin II, as discussed earlier, shows a marked relationship to processes associated with cell locomotion. This might afford a mechanism by which S100A4 could regulate the cytoskeletal machinery and influence locomotion. S100 protein has been reported to show calcium-dependent binding to calponin, a protein that can bind to CaM, actin, and tropomyosin. An N-terminal 22-kDa domain of calponin was involved in the interaction with S100 as well as with actin, tubulin, CaM and tropomyosin (Fujii et al. 1994, 1997). Calponin also interacts with membrane phospholipids, and these interactions are inhibited by S100 (Fujii et al. 1995). Most of these observations come from in vitro studies and thus no inferences can be drawn at present as to their relevance to in vivo situations, especially in light of the finding that calponin does not affect tubulin polymerisation (Fujii et al.1997). Nevertheless, Kilby et al. (1997) showed that certain C-terminal amino acid residues and N-terminal residues valine 8 to aspartic acid 12 of S100B may be the domains that are involved in its interaction with the actin-capping protein CapZ. This lends itself strongly in favour of S100 interactions with their target proteins as being a consistent feature in determining cell behaviour. There is, therefore, a complex series of interactions between S100 proteins and cytoskeletal structures, which in turn may be related to membrane fluidity and flexibility, and this could well influence the shape and motility of cells. These interactions may also be indicative of terminal differentiation, as shown experimentally by Tonini et al. (1991). These authors found an enhancement of S100 expression in neuroblastoma cells that were induced to differentiate using retinoic acid. Zimmer and Landar (1995) studied the expression of S100A and B in the differentiation of PC12 cells and L6S4 skeletal muscle cells. Both proteins are expressed in the undifferentiated as well as the differentiated state. S100A1 protein expression was higher in differentiated cells, but the expression of its mRNA showed no change in differentiated L6 cells, and in fact mRNA levels decreased in PC12 with differentiation. This would suggest that S100 expression is regulated at the posttranscriptional level. The S100 protein MRP14 (S100A9) is phosphorylated during monocytic differentiation. Two phosphorylated isoforms of MRP14 have been detected. MRP14 seems to respond to enhanced Ca2+ levels by undergoing a selective translocation from the cytoplasm to the cell membrane (Van den Bos et al. 1996). In fact, heterodimers of MRP8 and 14 are capable of binding specifically to membrane arachidonic acid (Klempt et al. 1997). The association of S100 proteins with syncytioblast maturation and differentiation in the placenta has been reported recently. S100A10, which was formerly known as p11, forms a heteromeric association with

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annexin II that is linked to cytoskeletal structures, and both annexin II and p11 show a progressive increase in association with placental differentiation (Kaczanbourgois et al. 1996). Apart from the obvious implications for membrane-related activities of the cell, there may also be other functions subserved by the association of S100 proteins with cytoskeletal components. MRP8 and MRP14 are both activated by PKC phosphorylation before they are secreted, and the activated proteins associate themselves with tubulin filaments before release, which seems to suggest that such an association could provide a route for the release of MRPs (Rammes et al. 1997).

CELL ADHESION

AND INVASIVE

POTENTIAL

OF

CANCER CELLS

Adhesive interaction between cells as well as between cell and substratum is an important ingredient of cellular motility. This cellular faculty is dependent on not only the cell membrane-associated adhesive macromolecules but also their temporal and spatial distribution in the cell membrane. It follows, therefore, that alterations in the overall expression of adhesion-mediating components of the cell membrane as well as their topographical distribution should entail alterations in the adhesive property. As a converse of this, one would expect to see changes either in the nature or in the pattern of distribution of cell membrane components in association with the acquisition of invasive potential. In inquiries into the question of whether S100 proteins influence the adhesive abilities of cells, a testable postulate would be to see if they regulated the expression on cell membrane components that participate in cell adhesion. Historically there is much evidence that calcium signalling is an important event in the induction of cell motility. Calcium signalling in processes such as the penetration of the oocyte by the sperm cell involves G-protein-mediated increases in intracellular calcium levels (Florman et al. 1989). The migration of neutrophils appears to depend on the enhancement of intracellular calcium levels (Marks and Maxfield, 1990). Chemoattractants have been known to induce calcium influx (Milne and Coukell, 1991). They produce rapid changes in the intracellular Ca2+ levels together with cell spreading, formation of pseudopodia, motility and its direction, and phagocytosis. However, blocking of the intracellular Ca2+ transients seems to have little effect on these biological features, indicating the possible involvement of other factors (Hendey and Maxfield, 1993). Nonetheless, Hendey and Maxfield (1993) report that cell motility on physiological substrates such as fibronectin and vitronectin are dependent on intracellular calcium levels. Cell surface integrin receptors possess calcium-binding domains similar to EF-hands (Tuckwell et al. 1992). The calcium-binding motifs of the α subunit of αMβ2 integrin of leukocytes show similarities to as well as differences from EF-hand motifs (Oxvig and Springer, 1998). Savarese et al. (1992) found that type IV collagen, which induces cell motility, also brings about increases in intracellular calcium levels, presumably by the release of Ca2+ from intracellular stores. Therefore, it would seem that intracellular calcium transients closely relate to the cellular interaction with substratum mediated by ECM components such as fibronectin and collagen. These interactions could be important events in the initiation of calcium signalling. The S100 proteins MRP8 (S100A8) and MRP14 (S100A9) are expressed abundantly in the cytoplasm of neutrophils.

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These proteins associate with vascular endothelia and presumably aid in the diapedesis of cells across the endothelium. Newton and Hogg (1998) have found that MRP14 regulates the function of β2-integrin, an adhesion-mediating glycoprotein. The function of MRP14 appears to be restricted to just this regulatory process. The function of MRP14 itself may be regulated by MRP8. Apparently, the formation of a heterodimer between MRP8 and MRP14 can inhibit the function of MRP14. However, the nature of β2-integrin regulation by MRP14 remains to be elucidated, although Newton and Hogg (1998) do state that integrin does not function as a receptor for MRP14. Many components of the ECM are associated with and known to participate actively in cell motility and cancer invasion (see Sherbet and Lakshmi, 1997b). More pertinent to the topic under discussion here is the observation by Savarese et al. (1992) that type IV collagen, which stimulates motile behaviour in melanoma cells, also increases intracellular calcium levels. The effect seems to be mediated by the release of Ca2+ from intracellular stores rather than by an influx of extracellular calcium. Another ECM component, the transmembrane glycoprotein CD44, has been the subject of much debate with regard to its putative role in cancer invasion and metastasis (see Sherbet and Lakshmi, 1997b). CD44 is a membrane-associated adhesive glycoprotein that mediates intercellular adhesion by virtue of its function as a hyaluronate receptor. It has been attributed with enhancing the invasive ability of cells (Radotra et al. 1994; Merzak et al. 1994a). In this context may be cited recent studies of CD44 expression in B16 melanoma cells in which the expression of S100A4 was experimentally altered (Lakshmi et al. 1997). CD44 expression did not increase when S100A4 expression was up-regulated. However, an enhanced expression of S100A4 produced a redistribution of CD44 into a patchy focal pattern. Because S100A4 is able to depolymerise cytoskeletal elements, it has been suggested that the redistribution of the glycoprotein could be a result of cytoskeletal depolymerisation and the resultant enhanced lateral mobility of CD44 molecules leading to the aggregation and patching of CD44 receptors (Lakshmi et al. 1997). Fully compatible with this line of argument are the recent findings that CD44 molecules localise in cholesterol-rich domains of the plasma membrane. These molecules enjoy restricted lateral mobility because of the cholesterol-rich nature of the membrane domain in which they are localised and also the integrity of the actin cytoskeleton. A disruption of the integrity of the actin cytoskeleton results in an increase in the partitioning of CD44 to the cholesterol-rich domains (Oliferenko et al. 1999). Hyaluronic acid, which is a ligand for CD44, induces such patching, and these CD44 patches are associated with a preferential accumulation of and binding to plaques of the cytoskeletal protein called ankyrin (Bourguignon et al. 1993; Welsch et al. 1995). The interaction of CD44 with ankyrin occurs when CD44 is phosphorylated by Rho kinase, which, in turn, is activated by the GTPase activity of Rho A bound to CD44v. The phosphorylation of CD44 enhances its interaction with ankyrin. Bourguignon et al. (1999) have shown that microinjection of the catalytic domain of Rho kinase produces membrane ruffling. This can be inhibited by CD44 antibodies and by the microfilament inhibitor cytochalasin D.

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It is obvious from these observations that the induction of cell motility by CD44 is a complex process. The redistribution of CD44 into patches occurring under conditions of high S100A4 could provide the tumour cells with discrete and strongly adhesive foci that may promote their invasive behaviour by increasing and strengthening anchorage and intercellular interaction (Lakshmi et al. 1993). Overall, these data indeed suggest that S100A4 may be linked with the cytoskeleton. Further evidence, albeit circumstantial, that S100A4 might associate with the cytoskeleton, has come from recent work by Keirsebilck et al. (1998a). They investigated the status of E-cadherin and S100A4 in two murine tumour cell lines and found tha E-cadherin expression correlated inversely with S100A4 expression in these cell lines. E-cadherin is a transmembrane CBP, which is linked to the actin cytoskeleton via a number of linking elements (see Figure 13). E-cadherin is a putative suppressor of invasive and metastatic abilities (see Sherbet and Lakshmi, 1997b). Low levels of E-cadherin expression have been reported to correlate with poor prognosis of a variety of human cancers. Therefore, the inverse relationship between it and S100A4 is compatible with the invasion- and metastasis-promoting ability of the latter. The exact mechanics of this relationship have yet to be elucidated. Keirsebilck et al. (1998a) found an alteration in the intracellular distribution of α- and β-catenins, which link the cytoplasmic tails of Ecadherin to the actin cytoskeleton. Some cellular proteins, such as the adherence junction protein, are known to bind to this cytoplasmic domain of E-cadherin. APC protein, which is believed to function as a tumour suppressor protein, is known to compete with E-cadherin for binding to β-catenin (Hulsken et al. 1994). Arguably, the suppressor effect of cadherin does not depend on merely the presence of cadherin per se, but also on its being present in a functional state in its entirety as a complex with the linking proteins. Three forms of cadherin–catenin complexes may occur in cells. The form with one cadherin molecule linked to a β-catenin/αcatenin or plakoglobin/α-catenin is the conventional from depicted in Figure 13. In the second type of complex, the extracellular domains of cadherin dimerise in a parallel fashion. A proposed third type is one in which the extracellular domains of two conventional complexes belonging to two neighbouring cells associate in an antiparallel fashion (Chitaev and Troyanovsky, 1998). Further, Chitaev and Troyanovsky (1998) have suggested that an antiparallel association of dimerised complexes of neighbouring cells is responsible for intercellular adhesion, and that this is dependent on not only extracellular calcium but also the presence of the catenin components. It is apparent from this discussion that suppression of invasion by cadherin is a complex process, and at present it is difficult to envisage how S100A4 and cadherin antagonise each other. If the mechanism proposed by Chitaev and Troyanovsky (1998) were valid, one would not expect an inverse relationship between cadherin and S100A4 expression, for the latter would be expected to promote the dimerisation of cadherin complexes by enhancing the lateral mobility of the complexes in the cell membrane. Nonetheless, it would be of considerable interest to see if S100A4 affects in any way the interaction of E-cadherin with the catenins.

S100 Proteins: Their Biological Function and Role in Pathogenesis

S100 PROTEINS

IN

REMODELLING

OF THE

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EXTRACELLULAR MATRIX

ECM undergoes constant changes in terms of remodelling and renewal in a host of biological processes, such as morphogenesis, cell differentiation, wound healing, angiogenesis, cell motility, and also in the invasive and metastatic behaviour of cancers. A wide variety of proteinases are involved in the remodelling of the ECM. These enzymes and their inhibitors are known to be genetically regulated (see Sherbet and Lakshmi, 1997b). That ECM remodelling might involve S100A4 was demonstrated some time ago by Merzak et al. (1994). In glioma cell cultures, S100A4 expression appears to be inversely related to the expression of TIMP-2 gene, which encodes an inhibitor of tumour-associated metalloproteinases. Interestingly, S100A4 is highly expressed in cell lines derived from invasive gliomas, as compared with cell lines obtained from noninvasive localised brain tumours or foetal brain cells. On the basis of these observations, it has been suggested that the invasive property of glioma cells could be a result of the uninhibited function of tumourassociated proteinases and that this lack of regulation of the proteinases concerned might be due to inhibition of TIMP-2. In a recent report, Bjornland et al. (1999) claimed that TIMP-2 up-regulation can occur independently of the state of S100A4 expression. They isolated several clones from a highly metastatic osteosarcoma cell line, which expressed S100A4 at different levels on account of being transfected with an anti-S100A4 ribozyme. It is somewhat intriguing that in the clones with low or intermediate S100A4 expression, both MMPs and TIMP-1, were down-regulated. With a complex experimental system such as this, it is difficult to visualise how MMP homeostasis could be altered by parallel modulation, in the same direction, also of the MMP inhibitor TIMP-1 (also see below). Several other studies also have suggested that the expression of S100A4 might correlate with that of MMPs. De Vouge and Mukherjee (1992) found that both S100A4 and transin-2 are up-regulated in parallel in rat kidney cells transformed by Ki-ras. K. Andersen et al. (1998) found that IL-1α down-regulated the expression of TIMP-1 in osteosarcoma cells that express S100A4 at high levels. In contrast, in cells with low level of S100A4 expression, IL-1α did not change TIMP expression. K. Andersen et al. (1998) have suggested that the effects of bFGF, which also affects TIMP, might not be related to the levels of S100A4 expression. They suggest that IL-1α functions synergistically with S100A4. It should be borne in mind, however, that both bFGF and IL-1α effects on TIMP could have been mediated by osteonectin, because bFGF is known to regulate the expression of osteonectin. In mesenchymal cells, bFGF is known to up-regulate the expression of osteonectin (Shiba et al. 1995). IL-1α, on the other hand, down-regulates the expression of osteonectin. Osteonectin has been shown to modulate MMP expression, and possibly also that of its inhibitor. There is no evidence at present for the latter. However, in many systems studied to date, the MMP/TIMP system is regulated as a unit, with MMP up-regulation more often than not being a result of down-regulation of TIMP or vice versa. None of the experiments described here provide any indication of how S100A4 achieves TIMP2 inhibition. This remains to be elucidated. It should be recognised that the remodelling of the ECM and the downstream effects of such changes on cell adhesion and motility might be directly linked with

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the reorganisation of the cytoskeleton. We know from the work of Irigoyen et al. (1997) that cytoskeletal reorganisation leads to, probably among other things, an up-regulation of uPA. Irigoyen et al. (1999) have further shown that this up-regulation occurs via the ras/erk-signalling pathway. The ability of S100A4 to alter the organisation of the cytoskeleton has been satisfactorily demonstrated. Besides, De Vouge and Mukherjee (1992) have shown that cell transformation by the ras oncogene results in the up-regulation of S100A4 as well as an MMP. Furthermore, heat shock can down regulate not only S100A4 expression (Albertazzi et al. 1998a) but also the expression of membrane type I MMP together with an inhibition of in vitro invasive ability (Sato et al. 1999). Under these circumstances one would be amply justified in considering this to be a putative link between S100A4 expression and ECM remodelling and the associated changes in membrane properties and cell behaviour. S100A4 is generally believed to be a cytosolic protein. However, its putative participation in ECM remodelling suggests that it might be secreted into the ECM. The findings of Duarte et al. (1999) seem to confirm this view. They reported that S100A4 is secreted under both in vivo and in vitro conditions. They further reported that S100A4 inhibits the process of mineralisation and bone nodule formation in bone marrow cell cultures. It is possible, therefore, that the apparent modulation of TIMP activity and any consequential effects on the character of the ECM described by Merzak et al. (1994) could have resulted from the action of the S100A4 secreted into the ECM. The ECM-modulating effects of S100A4 are also implicit in a set of observations relating to the epithelial–mesenchymal transformation that occurs in vertebrates. An important feature of this transformation is that it is associated with the acquisition of the ability to invade and migrate into the ECM (Hay, 1995). Several genes are switched on during this transformation, among them S100A4, which was called the fibroblast-specific protein 1 (FSP1). Although the fibroblast specificity is debatable, it has been shown that antisense S100A4 suppressed the expression of the gene and at the same time suppressed epithelial–mesenchymal transformation (Okada et al. 1997). Okada et al. (1997) also have gone on to demonstrate that EGF and TGFβ− 1, both capable of inducing cellular motility, also induce S100A4 expression in epithelial cells. A somewhat simplified conclusion that flows from the above discussion is that S100 proteins play a very significant role in cell motility, cell adhesion, and the invasive behaviour of cancer cells. It is also obvious that many questions remain unanswered, especially those regarding the interaction of these proteins with the cytoskeletal elements, i.e., the mode of their function as agents that promote cytoskeletal disassembly or inhibit the polymerisation of cytoskeletal monomeric elements. We do, however, have some significant leads as to how S100 proteins might be involved in the apparent regulation of tumour-associated proteinases.

S100 PROTEINS

IN

CELL PROLIFERATION

There is a general acceptance of the concept that cancers possess a high proliferative potential and that this apparent potential is more a consequence of the loss of

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215

homeostatic growth control than of an increase in the rate of proliferation. Inevitably, S100 genes, whose expression correlates remarkably well with invasive and metastatic potential of cancers, have also been investigated for their relationship to growth deregulation in cancers. It is significant, therefore, that some of the major S100 proteins are expressed in a cell cycle-specific manner. Indeed, some members of the S100 family such as S100A4 were originally cloned from highly proliferative tissues and were described as proliferation-related proteins. Two questions can be posed in order to elucidate the relationship between growth regulation and the expression of S100 proteins. One question is whether the presence of S100 proteins correlates with the growth fraction and proliferative behaviour of tumours, or with tumour cells or normal cells in culture. Recently, S100B has been attributed with a role in the regulation of the cell cycle. The evidence takes the form of the demonstration of the ability of S100B to bind to and activate in vitro the nuclear serine/threonine protein kinase called Ndr, in response to changes in the intracellular calcium levels. An N-terminal regulatory region of Ndr seems to be involved in the Ca2+-dependent binding of S100B, and this region contains sequences that are characteristic of CaM/S100 binding (Millward et al. 1998). S100B also seems to be able to regulate the intracellular activity of Ndr. Millward et al. (1998) have proposed a biological role for this interaction between S100B and Ndr and the regulation of the activity of the latter. In melanomas that overexpress S100B, the CaM inhibitor W7 inhibits the activation of Ndr, but not in those that lack S100B expression. The Ndr kinase was identified by Millward et al. (1995). It seems to belong to a subfamily of protein kinases that shows 40 to 60% sequence similarities among the members of the family. These kinases have been implicated strongly in the progression of the cell cycle as well as in the regulation of cell morphology. Therefore, Millward et al. (1998) have argued that an overactivation of Ndr by S100B might be related in some way to the development and progression of melanomas. However, as Millward et al. (1998) have pointed out, some melanomas do overexpress S100B without also showing an overactivation of Ndr. Therefore, they suggest that a mechanism of negative control of Ndr might exist. This postulate requires that this negative regulation is inactivated by loss-of-function mutations of the Ndr regulator. S100B has been reported to stimulate sciatic nerve regeneration and may function as a growth factor for peripheral nerve axons (Haglid et al. 1997). Growth may be regulated by a homeostasis of cell proliferation and apoptotic loss of cells. S100B has been reported to rescue motor neurones from apoptotic death (Iwasaki et al. 1997). The ability of S100B to influence actin dynamics might be involved in this effect. The status of actin polymerisation has been found to regulate apoptosis. Induction of polymerisation leads to apoptosis, whereas inhibitors of polymerisation block apoptosis (J.Y. Rao et al. 1999). As alluded to above, S100B can inhibit actin polymerisation. Therefore the findings of Iwasaki et al. (1997) are compatible with those of J.Y. Rao et al. (1999). On the other hand, it has been found that S100B may lead to apoptotic death by releasing nitric oxide (Hu et al. 1997). The calcyclin gene is expressed more frequently in epithelial-type or Schwann cells than in neuroblastic cells. This is compatible with the difference in their growth potential. A consequence of the induction of differentiation of neuroblastoma cells

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by retinoic acid is an enhancement of calcyclin expression together with arrest of cell growth (Tonini et al. 1991). It is yet uncertain if calcyclin indeed negatively regulates growth, because, in total contrast, a higher proportion of melanoma cells shows the presence of calcyclin in the vertical growth phase (Weterman et al. 1993). It may be pointed out, however, that S100B expression has been reported to increase in neuroblastoma differentiation (Tsunamoto et al. 1988). Tonini et al. (1991) found an inhibition of growth rate in cells where S100B levels had been experimentally reduced. This is not compatible with the findings of Tsunamoto et al. (1988) that reduction of growth rates invariably accompany the induction of differentiation. Nonetheless, S100A4 expression is up-regulated by positive proliferative responses such as those imparted by growth factors and down-regulated by negative responses elicited by hyperthermia, with parallel changes in the size of the S phase fraction. For instance, human tumour cells that are high expressors of EGFr also tend to overexpress S100A4 (Sherbet et al. 1995). In murine BL6 melanoma cells and human HUT cells, which are a heat-resistant variant derived from HepG2 cells, hyperthermia down-regulates S100A4 gene expression. In parallel, there is a decrease in the size of the S phase fraction and an increase in the doubling time of cells (Sherbet et al. 1996; Albertazzi et al. 1998a). Hyperthermia, which is known to increase intracellular calcium levels (Furukawa et al. 1997), induces the synthesis of several heat shock (cognate) proteins. Among the most prominent in terms of their relevance to cell proliferation are HSP70 and HSP28. The microtubule-interacting protein MIP-90 has been reported to show extensive sequence homology to HSP90 (Cambiazo et al. 1999). HSP70 has been known for some time to show a cell cycle-related expression. This HSP seems to be essential for mitotic division in the early developmental stages of the sea urchin Paracentrotus lividus. HSP70 has been reported to accumulate in the mitotic apparatus, and further, antibodies raised against HSP70 have been shown to inhibit mitosis (Sconzo et al. 1999). It would seem that HSP70 is an important ingredient of cell division and could play a role in the cell division machinery. In sharp contrast, HSP28 seems to exert an inhibitory effect on cell proliferation, and furthermore, HSP28 function might have a genetic basis. This is obvious from the experimental work described by Albertazzi et al.(1998a), where there was a remarkable differential increase in HSP28 as compared with the non-HSP28 component of total cellular HSP. Thus, in the BL6-derived HTG murine melanoma cells exposed to hyperthermia, HSP28 expression increased by 3.5-fold as compared with control BL6 cells. The corresponding increase in the non-HSP component of the HTG cells was only 22%. The enhancement of HSP28 together with a down-regulation of S100A4 (mts1) is compatible with previous observations that HSP28 is a growth-inhibitory protein. HSP28 is induced by hyperthermia in normal lymphocytes and macrophages as well as in leukaemia cells. Its induction has been reported to coincide with peak proliferative activity and the onset of growth arrest (Spector et al. 1992, 1993, 1994). HSP28 is highly expressed in quiescent keratinocytes as compared with their proliferating counterparts (Honore et al. 1994). A low molecular weight heat shock protein, HSP27, has been reported to be expressed abundantly in normal squamous epithelia of the oesophagus, but is markedly down-regulated in Barrett’s metaplasia and adenocarcinoma of the oesophagus

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(Soldes et al. 1999). The HSP27 gene has been transfected into A375 melanoma cells and A431 cells. In transfectant cells obtained from both cell lines, overexpression of the HSP was associated with reduced proliferative rates (Kinder-Mugge et al. 1996). Presumably, HSP27 and 28 are the same molecular species. Antibodies against HSP27 have been detected in sera of breast cancer patients but not of normal subjects. Furthermore, their presence in sera correlated with improved patient survival (Conroy et al. 1998). It may be suggested that this reflects less aggressive disease on account of the growth-inhibitory properties of the HSP. It is interesting to note also that cells over-expressing HSP27 show a delay in tumorigenicity (Kinder-Mugge et al. 1996). This delay is compatible with inhibition of cell proliferation. However, after this initial delay tumours did appear, but these were devoid of HSP27 expression and they showed growth properties similar to control tumours. Again, this supports the close link between HSP27 expression and proliferative potential. It would be interesting to study how HSP28 functions as an intermediary in the inhibition of proliferation accompanying the S100A4 down-regulation. There are two pointers to the possible mode of action of this low molecular weight HSP. HSP27 binds both α- and β-tubulin and is also found in association with microtubules (Hino et al. 2000). We know that S100A4 markedly influences cytoskeletal dynamics. Therefore, Albertazzi et al. (1998a) have postulated HSP28 might be involved in direct interaction with S100A4. There are several reports that HSP can bind to p53. Normal human cell lines exposed to hyperthermia show G1 arrest together with an increase in the expression of p53, but not in rb protein phosphorylation (Miyakoda et al. 1999). In light of these findings, the suggestion by Albertazzi et al. (1998a) that there might exist a complete regulatory loop involving S100A4, HSP, and p53 in the control of the G1–S checkpoint of the cell cycle, seems highly credible (Figure 26).

CELL CYCLE-RELATED EXPRESSION

OF

S100 PROTEINS

The progression of the cell cycle involves a sequence of events that includes calcium signalling. Calcium signalling involves both an influx of extracellular calcium and an elevation of intracellular calcium levels by the release of Ca2+ from intracellular stores. Mitogenic agents generate calcium signals via the IP3-signalling pathway. These calcium signals might control the entry of resting cells into the cell cycle, G1–S and G2–M transitions as well as the exit of the cells from mitosis. Mitogens activate calcium influx into the cells, which does seem to control the progression of the cell cycle (Barbiero et al. 1995). In the mitotic phase, transient increases of Ca2+ occur between metaphase and anaphase. An increase in cytosolic calcium is associated also with the process of cell division, i.e., cytokinesis. Calcium channel blockers can inhibit cell proliferation by arresting cells in the G0G1 phase (Zeitler et al. 1997). Fertilisation of the ovum results in a rapid rise in Ca2+. This is required for the cleavage of the fertilised egg (Whitaker and Patel, 1990), which can be blocked by chelating Ca2+ (Zucker and Steinhardt, 1978). Unexpectedly, mobilisation of cell calcium and calcium channel activation also have been found to inhibit the passage of cells at the G1–S and G2–M checkpoints (Vanolah and Ramsdell, 1996). It may be that calcium sequestration by CBPs controls intracellular levels of calcium

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FIGURE 26 Hypothetical view of interaction between S100A4, HSP28, and p53 in the regulatory loop that might control G1–S transition of cells. (From Albertazzi et al. 1998a.) Reprinted by permission of the publisher Mary Ann Liebert Inc.

and thereby regulates the role of calcium signalling in cell cycle progression. Therefore, much attention has been focused on the potential role of S100 proteins and indeed other calcium-sequestering proteins in cell proliferation. Furthermore, much information is currently available identifying individual CBPs with intervention and function at specified stages of the cell cycle. Ca2+ has been regarded also as playing an important part in the cycle of meiotic cell division. The question of whether S100 proteins regulate growth can be answered in two parts. The first part of the answer is the unequivocal association between the expression of certain S100 proteins and specific stages of the cell cycle. It was noticed some years ago that S100B and calcyclin genes were expressed predominantly in the G1 phase of the cell cycle (Hirschhorn et al. 1984). Selinfreund et al. (1990) showed that intracellular levels of S100B can be reduced by introducing antisense S100B oligonucleotides into cells. This resulted in a decrease in the growth rate of the cells. The induction of differentiation of neuroblastoma cells by retinoic acid enhances the expression of calcyclin mRNA. This is accompanied by arrest of cell proliferation, and cells appear to accumulate in the G1 phase of the cell cycle (Tonini et al. 1991). The mechanism by which calcyclin might be regulating the cell cycle is not fully understood at present. Calcyclin could indirectly affect cell cycle progression. It has been found to interact with great specificity with the annexin CAP50 (Minami et al. 1992; Hidaka and Mizutani, 1993). As discussed elsewhere, the expression of annexins is closely associated with cell cycle progression and,

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furthermore, CAP-50 shows a predominant nuclear location (Hidaka and Mizutani, 1993). Minami et al. (1992) have suggested, in light of the apparent specificity of calcyclin binding to CAP-50, that this annexin could be regulated by calcyclin. Therefore, one cannot exclude the possibility that calcyclin might influence and regulate the progression of the cell cycle and cell growth by an indirect route. S100A4 (mts1) was cloned by Jackson-Grusby et al. (1987), who showed that it could be induced to express by serum stimulation of growth-arrested cells and that its expression increased during the S phase of the cell cycle. The NGF induces the synthesis of two proteins in rat PC12 cells, which were originally referred to as 42C and 42A. 42C and 42A are regarded as the rat homologues S100A10 and S100A4, respectively. Not only does NGF-induced neuronal differentiation induce 42C and 42A, but the cells concomitantly exit from mitosis and enter the G0 phase (Masiakowski and Shooter, 1988). From this it seems not only that some S100 proteins may be specifically expressed in relation to the stage of progression of the cell division cycle, but they could conceivably also regulate cell cycle progression itself. Some definitive evidence is now available that shows that S100A4 may regulate cell cycle progression. As stated previously, in B16 and human HUT cells exposed to hyperthermia, the S100A4 is down-regulated, with a reduction in the size of the S phase fraction (Albertazzi et al. 1998b). Parker et al. (1994b) transfected an S100A4 gene, which was placed under the control of a dexamethasone-inducible MMTV (murine mammary tumour virus) promoter, into B16 murine melanomas. They showed that the transfected cells, in which the gene was switched on by exposure to dexamethasone, contained substantially larger S phase fractions as compared with the control cells. These observations suggest that the S100A4 gene could be driving the cells into the S phase. Another line of evidence is provided by the work of Parker and Sherbet (1992), who found that verapamil, a specific blocker of L-type calcium channels, down regulated the expression of S100A4. Subsequently, several investigators have demonstrated that verapamil can inhibit not only cell proliferation (Brocchieri et al. 1996; Zeitler et al. 1997; Hoffman et al. 1998) but also invasion and metastasis (Farias et al. 1998). Interestingly, verapamil reduces the expression of certain membrane-associated metalloproteinases and urokinase-type PA. An enhanced expression of S100A4, on the other hand, is associated with a remodelling of the ECM in a way that is conducive to enhanced cell motility and invasiveness.

POSTULATED MECHANISM

OF

CELL CYCLE CONTROL

BY

S100A4

The mechanisms by which S100A4 exerts control over the progression of the cell cycle are yet unclear. However, much circumstantial evidence has been adduced to support the concept that this protein might form complexes with certain cellular target proteins, such as the suppressor p53 phosphoprotein that has been regarded as the guardian of the genome. When cellular DNA is damaged p53 protein is expressed, and this appears to block the transition of the damaged cells from G1 into the S phase, until DNA repair takes place. In B16 melanoma cells, up-regulation of S100A4 (18A2/mts1) expression is associated with an enhanced level of p53, as detected by immunohistochemical methods. This has been attributed to the formation

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of a complex of S100A4 with p53 (Parker et al. 1994a, 1994b). Such a complex formation would have the effect of stabilising and enhancing the half-life of p53, and as a consequence p53 becomes detectable by immunohistochemical staining procedures. The formation of a complex between S100A4 and p53 could effectively sequester p53 and abrogate its G1–S checkpoint control, resulting in an increase in the size of the S phase fraction. One might then ask why the cells do not continue to accumulate in the S phase, but successfully transit from G2 into mitosis. It has been shown recently that p53 is also involved in the control of the G2–M transition of cells (Michalowitz et al. 1990; Stewart et al. 1995; Pellegata et al. 1996). One of the proposed mechanisms of this involves the participation of a protein called stathmin. Stathmin is a 19-kDa cytosolic phosphoprotein. It is up-regulated in many neoplasms as well as in highly proliferating normal tissues (Luo et al. 1994; Rowlands et al. 1995; Friedrich et al. 1995; Bieche et al. 1998). Even immortalisation, an event regarded as a prelude to neoplastic transformation, of primary embryonic fibroblasts, results in a four-fold enhancement of stathmin expression and a substantial increase in the rate of proliferation. However, in this experimental system there were no changes in stathmin expression upon oncogenic transformation (Mistry and Atweh, 1999). It would seem, therefore, that modulation of stathmin expression is a feature of cell proliferation rather than of neoplastic transformation. This statement is amply substantiated below. The expression of stathmin is down-regulated when wild-type p53 is expressed. Using inducible p53 constructs, it has been demonstrated that when p53 is switched on the stathmin gene is down-regulated together with the arrest of cells in the G2 phase. Indeed, p53 may be capable of down-regulating the stathmin promoter. Ahn et al. (1999) have shown that a decrease in the expression of stathmin occurs in immortalised human and murine cells in parallel with induction of wild-type p53 by DNA-damaging agents. Of interest in the context of the function of S100A4 is the observation that stathmin destabilises the cytoskeleton (Marklund et al. 1996; DiPaolo et al. 1997). It sequesters free tubulin and inhibits tubulin polymerisation (Andersen et al. 1997; Curmi et al. 1997, 1999). Thus S100A4 and stathmin seem to share several properties, of which the most prominent are the influence they exert on cell proliferation and their postulated interaction with p53. A conundrum, in the observed association between S100A4 and cell cycle progression, is why cells that are induced by S100A4 to enter the S phase do not accumulate in the S phase but successfully negotiate the G2–M checkpoint into mitosis. Some recent work has shown that stathmin expression closely parallels that of S100A4. Indeed, changes in S100A4 expression seem to be tightly coupled with corresponding changes in the expression of stathmin gene. Thus, stathmin expression changes when cell proliferation is inhibited by hyperthermia. This happens also when cells are growing exponentially, i.e., with increasing growth rates, or when full serum supplement is restored to serum starved cells (Sherbet and Cajone, 2000; Cajone and Sherbet, 2000) (Table 18). There are two schools of thought about how stathmin might be involved with cell proliferation. One school believes that the G2–M transition of cells is associated

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TABLE 18 Coupling of S100A4 and Stathmin Gene Expression in Relation to State of Proliferation Experimental Condition

S100A4

Stathmin

Growth Rate

Hyperthermia (HUT cells) (decreased growth rate) HeLa cells, 24-, 48-, and 72-hr cultures

↓ ↑

↓ ↑

↓ ↑

Source: Based on Sherbet and Cajone (2000) and Cajone and Sherbet (2000). Reprinted by permission of the publisher Kluwer Academic Publishers.

with enhanced stathmin expression (Eustace et al. 1995; Jones et al. 1995). The other proposes that phosphorylation of stathmin is the critical event that allows the cells to make the transition (Marklund et al. 1994; Larsson et al. 1995; Duraj et al. 1995; Beretta et al. 1995; Lawler et al. 1998). In the studies cited above, Sherbet and Cajone (2000) and Cajone and Sherbet (2000) have not investigated the state of stathmin phosphorylation, for the correlation between stathmin gene expression and cell proliferation was highly significant. S100 proteins have been shown to be able to modulate PKC-mediated phosphorylation of proteins (Kriajevska et al. 1998). S100A4 might function by inhibiting the phosphorylation of stathmin, which is regarded by some as essential for stathmin function. Therefore, the potential significance of posttranslational modification of stathmin cannot be excluded. As stated earlier, previous work has demonstrated that the interaction of S100A4 and p53 might sequester the latter and abrogate its checkpoint control at the G1–S checkpoint (Parker et al. 1994a, 1994b). However, after this successful transition into the S phase, one would expect them to accumulate in the S phase. This does not seem to happen. Although the size of the S phase fraction does increase upon induction of S100A4 expression, cells do not appear to be held back at the G2–M checkpoint. It is in this context that the parallel modulation of the expression of S100A4 and stathmin genes assumes some significance. Cajone and Sherbet (2000) have postulated that S100A4 might directly induce an up-regulation of stathmin expression, enabling the cell to enter into mitosis. Because wild-type p53 has been known to down-regulate stathmin expression, it has been suggested that S100A4 might sequester p53 and abrogate its control over stathmin function, and thereby enable cells to make the G2–M transition (Figure 27). There is simplicity about this concept, which emphasises the possibility of S100A4 being involved at both G1–S and G2–M transition checkpoints. This postulate needs to be tested rigorously, for this is the first CBP that has been shown not only to have close links with cell cycle progression but also to be intricately involved in the two main control checkpoints of the cell cycle.

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M + p53 Stathmin G2 -

p53

P +

G1

S100A4

+

+ p53

S100A4

S

FIGURE 27 The putative involvement of S100A4 in G1–S and G2–M transition in the cell cycle. As discussed in the text, S100A4 may drive the cells into the S phase by sequestration of p53. Stathmin is up-regulated as well as phosphorylated during the G2–M transition. Wildtype p53 is said to down-regulate stathmin and might block the G2–M transition by this method. Because stathmin expression parallels that of S100A4, the figure represents the postulate that S100A4 might itself up-regulate stathmin and promote G2–M transition, or this effect is routed through p53 sequestration.

S100A ISOFORMS S100A protein occurs as two isoforms made up of homo- or heterodimers of the two subunits called the α and β subunits. These two subunits comprise 93 and 91 amino acid residues respectively, and share 58% amino acid sequence homology (Isobe and Okuyama, 1978, 1981). S100A0 is a homodimer of two α subunits, and S100A is a heterodimer of an α and β subunit. S100B is made up of two β subunits. The S100A isoforms as well as S100B occur predominantly in the brain. S100A0 forms a minor component of S100 proteins in the neurones and peripheral nerves (Isobe et al. 1984). S100A0 is also associated with the sarcolemma and the SR (Haimato and Kato, 1987; Donato et al. 1989). Because the SR holds the intracellular stores of calcium, S100A0 could be involved in the mobilisation of calcium from these intracellular stores. Fano et al. (1989) have shown that S100A0 does induce calcium mobilisation from SR-terminal cisternae, which were isolated from rat skeletal muscle. The levels of S100A0 in the bloodstream of patients with acute myocardial infarction have been investigated on the premise that the protein may be released into the bloodstream from damaged heart muscle. In a group of patients investigated, Usul et al. (1990) found a nearly fourfold increase of S100A0 in the serum that eventually increased to roughly 20-fold higher than the levels of the protein found in control subjects. However, there was considerable variation between patients. Nevertheless, such increases were not encountered in patients with angina pectoris. Usul et al. (1990) have, therefore, suggested that this might provide a method for differentiating between myocardial infarction and angina pectoris.

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223

S100A2 AS A PUTATIVE TUMOUR SUPPRESSOR It is evident from the foregoing discussion that a majority of S100 family proteins appear to possess the ability to promote tumour progression by enhancing the proliferative and invasive potential of tumours. One member of the S100 protein family, namely S100A2, is believed to function as a tumour suppressor. S100A2 was isolated more than a decade ago by Glenney et al. (1989). It is found in certain cell types in the kidney, lung, and breast epithelium. It is moderately expressed in the liver, and cardiac and skeletal muscle, but not encountered in the adrenal gland, the intestine, or the brain. Lee et al. (1991) identified several cDNA clones that were highly expressed in normal tissues but suppressed in corresponding tumour-derived cells. Among these clones was a transcript of an S100 family gene. Subsequently, its expression was reported to be down-regulated in breast tumour cells. Exposure of these cells to 5-aza-2′-deoxycytidine produced a reexpression of this gene, which suggested that its expression was normally suppressed in the tumour cells by hypermethylation (Lee et al. 1992). That hypermethylation of this gene is responsible for the loss of its expression in breast cancer cell lines has been confirmed recently (Wicki et al. 1997). A loss of S100A2 expression has been reported in several breast cancer cell lines (Pedrocchi et al. 1994). A marked loss of expression has been found in human sarcomas (E. Horvig, personal communication, 1998). S100A2 expression has been found in only 7% (of 107) of human sarcomas. In contrast, S100A4 and A6 expression was detected in 38 and 48%, respectively, of the specimens. This suggests a preponderant loss of S100A2 expression. However, there was no obvious correlation with clinical features or patient survival. In human astrocytomas also there is a conspicuous loss of S100A2 expression, whereas, in contrast, several other S100 proteins, notably S100A1, A4, and A6, are markedly expressed (Camby et al. 1999). The suppressor function of S100A2, however, is not so clear-cut either in normal melanocytes or in melanomas. Thus, in normal melanocytes S100A2 is expressed at very low levels or is virtually undetectable. Neither is its expression up-regulated in malignant melanoma (L.B. Andersen et al. 1996). S100A2 staining has been reported in the basal layer of the epidermis and in hair follicles, but none has been found in naevi. Also, only a small proportion (4/39) of primary cutaneous melanomas and none of 14 metastatic lesions stained for S100A2 (Boni et al. 1997). A further report has appeared on S100A2 expression in epidermal cell types and epithelial tumours of the skin. Again the basal cells, epithelial cells of the sebaceous glands, and epithelial cells of hair follicles stained positive for S100A2. Also immunoreactive were basal cell as well as squamous cell carcinomas (Shrestha et al. 1998). Overall, the evidence available to date does not lend itself to a firm interpretation that S100A2 has a suppressor function or that its expression is associated with advanced stages of tumour progression. This view is also supported by the data published by Maelandsmo et al. (1997). The differences in the levels of S100A2 expression between naevi and cell lines derived from primary tumours seem to be more marked than those between the primary and metastatic lesions. This suggests that a down-regulation of the gene could occur in the early stages of development of these tumours. Xia et al. (1997) have cast further doubts about the suppressor

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function of S100A2. They found this gene to be highly expressed in basal and squamous cell carcinomas of the skin and oral cavity, although in situ hybridisation studies have indicated that the amounts of S100A2 occurring in the tumour cells themselves were limited. The majority of the protein was found in the hyperplastic epidermis around the tumour. Xia et al. (1997) found no differences in the expression of the protein in primary tumours and metastatic lesions. Ilg et al. (1996) studied the binding of antibodies against several S100 proteins, including S100A2, and noticed a marked difference in their intracellular localisation. S100A2 was located predominantly in the nucleus, but S100A6 occurred mainly around the nucleus. They also found that the binding pattern of S100A2 antibodies differed from that of antibodies against S100A4, the expression of which has been widely reported to promote tumour-progression (see below). Although there is some evidence supporting the putative tumour suppressor function of S100A2, most of the research deals with its expression in tumour-derived cell lines, and there are no significant studies on the status of its expression in human tumours themselves. The observation made by Xia et al. (1997) relating to the presence of S100A2 in hyperplastic epidermis incidentally serves to emphasise a putative relationship between the expression of this protein and the proliferative state of cells. This relationship now will seem more secure with the finding that EGF up-regulated S100A2 expression in organ cultures of human skin. EGF also markedly up-regulated the expression of S100A2 mRNA in immortalised human keratinocytes in culture. In both cases, the EGF effects could be blocked by using PD153035, which is a specific inhibitor of EGFr tyrosine kinase (Stoll et al. 1998). These experiments demonstrate the requirement of EGFr activation for the up-regulation of S100A2 expression, and therefore, suggest the presence of a direct relationship between S100A2 and mitogenic stimulation. With this background of great ambiguity regarding the suppressor function of S100A2, one should look to some recent evidence that S100A2 function might be mediated by wild-type p53. Tan et al. (1999) have identified putative p53-binding sites in the promoter of S100A2. In vitro, wild-type p53 seems to transactivate S100A2. This transactivation is blocked by dominant negative p53 mutants. This can be deemed as evidence that S100A2 might influence cell proliferation with the mediation of p53. Thus p53 mediation may yet prove to be an underlying mechanism in the regulation of cell proliferation by S100 proteins.

S100A3 EXPRESSION IN CELL DIFFERENTIATION AND NEOPLASIA MOLECULAR FEATURES

OF

S100A3

S100A3 is a cysteine-rich CBP that binds to Ca2+ with low affinity but binds with high affinity to Zn2+ (Engelkamp et al. 1993; Fohr et al. 1995). As many as 10 out of 101 amino acids of the protein are cysteine residues. A subfamily of S100 proteins that binds Zn2+ with high affinity can be identified. These proteins bind four Zn2+ ions per protein monomer (Fohr et al. 1995). S100A3 may bind two Zn2+ ions per monomer. One zinc atom binds to four cysteine residues and the second atom binds

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to one histidine residue. Therefore, a commonality of the zinc-binding domain and its organisation and stabilisation seems to be evident, and this has obvious similarities to the GAL4 zinc finger transcription factor from yeast. However, S100A3 binds to Zn2+ with far less affinity than that displayed by zinc finger transcription factors. The binding of zinc by S100A3 seems to be a well-coordinated process (Fritz et al. 1998), and might have effects on its conformational and functional states. The S100A3 gene occurs in the S100 gene cluster on the human chromosome 1q21 and on mouse chromosome 3.

S100A3 EXPRESSION GLIOMAS

IN

CELL DIFFERENTIATION

AND

HUMAN

S100A3 seems to be expressed specifically in skin and derivative tissues (Kizawa et al. 1996, 1998; Boni et al. 1997) and shows a specific pattern of localisation in the subcompartments in human and murine follicles. High expression of S100A3 has been found in the cuticle sheath and cortical cells undergoing terminal differentiation. This has led to the suggestion that it might participate in the differentiation of the cuticle cell and the formation of the hair shaft (Kizawa et al. 1998; Takizawa et al. 1999). The expression of S100A3 has been studied in a series of human astrocytomas of various grades of malignancy (Camby et al. 1999). These authors have reported that they were able to differentiate between grade I (WHO) pilocytic astrocytomas and grades II–IV tumours on the basis of their expression of S100A3. Further, on this basis they suggest grade II–IV astrocytomas form a group of astrocytomas that are biologically distinct from WHO grade I tumours. On the other hand, the expression of S100A3 could be merely an indicator of the degree of anaplasia.

S100A4 IN CANCER DEVELOPMENT AND PROGRESSION S100A4 EXPRESSION

AND

METASTATIC POTENTIAL

OF

CANCERS

The considerable influence that S100A4 exerts over cell adhesion, motility, and cell proliferation has prompted the investigation of the possible relevance of its expression in tumour development and their progression to the metastatic state. S100A4 is found in a variety of normal tissues of both murine and human origin (Grigorian et al. 1994; Gibbs et al. 1995). It occurs in normal adult rat tissues such as smooth muscle, brown adipose tissue, and the liver. Other tissues, e.g., normal breast tissue, endothelia, absorptive and keratinised epithelia, neuronal, as well as some cells of the haematopoietic system, also contain S100A4. S100A4 is predominantly intracellular in distribution, but Gibbs et al. (1995) believe that in breast tissue, it may occur as an extracellular secreted protein. There could be some speciesspecific differences in tissue distribution of the protein (M. Davies et al. 1995). In spite of this apparently generalised expression of S100A4, it ought to be recalled here that there are reports that S100A4 (FSP1) is expressed in a fibroblastspecific manner. Not only this, S100A4 (FSP1) is described as one of the genes

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whose expression is associated with the epithelial–mesenchymal transformation encountered in chordates (Hay, 1995). The fibroblast-specific expression has been attributed to the presence of a cis-acting element in the 5′-flanking region of the first intron of the gene. This sequence is believed to be active in fibroblasts but not in epithelial cells. Okada et al. (1998) have shown further that the 5-bp sequence TTGAT (–177 to –173) interacts specifically with nuclear extracts obtained from fibroblasts. In addition to its wide distribution in normal tissues, high levels of S100A4 have also been recorded in human as well as in murine tumours with high metastatic potential (Ebralidze et al. 1990). A clear correlation seems to exist between the level of S100A4 mRNA and protein and the metastatic potential of Dunning rat prostate carcinoma cell lines (Ke et al. 1997). Indeed, some of this evidence might be construed as suggesting that S100A4 (FSP1) is a developmentally regulated gene. The aberrant expression of S100A4 in neoplasia could be viewed as reflecting abnormalities in the process of de-differentiation associated with cancer development and progression. In addition to the empirical correlation, substantial experimental work in recent years has allowed a strong link to be established between the levels of S100A4 expression and metastatic potential. In the B16 murine melanoma, modulation of its expression levels alters metastatic behaviour (Parker et al. 1991). Upon transfection with the S100A4 gene, benign rat mammary epithelial cells have been found to become more tumorigenic and acquire a higher potential for metastasis (B.R. Davies et al. 1993). Although these results were unambiguous, the process of transfection of exogenous genes has been subjected often to the criticism that it could lead to genomic perturbation as a consequence of a stable integration of an exogenous gene. Kerbel et al. (1987) noted that calcium phosphate-mediated DNA transfer also changed cellular behaviour. They reported that when CBA/J mouse adenocarcinomas were transfected with a vector carrying only a marker gene but not transforming genes, 17% of transfectant cells showed lung colonisation. Equally, there are reports that transfection of extraneous DNA does not alter biological behaviour in any way (Jamieson et al. 1990a, 1990b). Nonetheless, it is imperative that the expression of S100A4 is linked with changes in the biological behaviour of cells. Therefore, to obviate this potential criticism, B16 melanoma cell lines have been transfected with S100A4 (18A2/mts1) and placed under the control of the dexamethasone-inducible MMTV promoter. In these transfectants, switching on the exogenous gene markedly enhances localisation of the transfectant cells in the lung (Parker et al. 1994b). In the same investigation, Parker et al. (1994b) also transfected the inducible construct into a dexamethasone receptor negative B16 cell line and demonstrated that the exogenous gene is not switched on. These experiments have established conclusively that S100A4 enhances lung colonisation by B16 tumour cells. It is worthy of note, however, that Parker et al. (1994b) did not determine whether S100A4 transfectants were capable of spontaneous metastasis. As is often pointed out, much of this work is still open ended. There are one or two points of criticism that need to be taken into account. One of these is that dexamethasone is known to up regulate the expression of other EF-hand proteins such as calcineurin. Because the S100A4 transfectants were exposed to dexamethasone to switch on

S100 Proteins: Their Biological Function and Role in Pathogenesis

227

S100A4 expression, these cells should have been tested for calcineurin expression. Furthermore, tail vein injection of cells often allows them to bypass the initial hurdles that cells released from a primary tumour have to face, and they have direct access to the lung. Thus, although one can accept that S100A4 enhances the localisation of cells in the lung, there are no data on the effects of S100A4 on the release of cells from the primary tumour, their ability to gain access to the blood vessels, and their ability to withstand or escape the immunological surveillance by the host. The recent studies of Lloyd et al. (1998) have answered some of these questions. They transfected the human S100A4 gene into the rat benign mammary tumour cell line, Rama-37. Transfectant cells that expressed S100A4 mRNA at high levels were capable of forming primary tumours in syngeneic rats and were also able to metastasise spontaneously. In contrast, those transfectant cells that expressed S100A4 at low levels were unable to form secondary tumours. Furthermore, they have shown for the first time that metastatic deposits that expressed S100A4 mRNA were able to form primary tumours that were also capable of metastasising spontaneously. In other words, a constitutive expression of the gene does confer metastasising properties. Another piece of documentation, which supports the metastasis-promoting ability of S100A4, is the generation of S100A4 transgenic lines, e.g., Tg463 and Tg507, of GRS/A mice by Ambartsumian et al. (1996). The GRS/A primary strain rather characteristically shows a high incidence of mammary tumours that do not appear to possess metastatic ability. In contrast, the S100A4 transgenic strains developed metastatic tumours in approximately 40% of the animals. Ambartsumian et al. (1996) also demonstrated the presence of S100A4 in both primary and metastatic tumour. Simultaneously, M.P.A. Davies et al. (1996) carried out similar experiments on transgenic S100A4 mice. In the S100A4-expressing animals, mammary tumours were palpable earlier than in corresponding control animals, and macroscopic deposits of tumours were detectable in the lungs. There is thus adequate proof that high expression of S100A4 is conducive to metastatic dissemination. Conversely, the transfection of antisense constructs into malignant cell lines has resulted in the reduction of metastatic potential (Grigorian et al. 1994). The ability of antisense S100A4 mRNA to suppress metastatic potential has been confirmed in further experiments with highly metastatic lines of the Lewis lung carcinoma. Several clones with transfected antisense S100A4 mRNA have been isolated, and all of them proved to have very low metastatic potential (Takenaga et al. 1997a). In a novel experimental approach, Maelandsmo et al. (1996) transfected human osteosarcoma cells that expressed S100A4 at a high level with a ribozyme directed against the gene transcript. With the destruction of the S100A4 mRNA by the ribozyme, the osteosarcoma cells showed no skeletal metastases when injected intracardially into nude mice. They also found that the degradation of S100A4 mRNA had no effect on the proliferation of the cells either in vitro or in vivo. On the basis of the latter results, Maelandsmo et al. (1996) seem to be suggesting that metastatic ability may be dissociated from proliferative ability. This is an interesting postulate, but, so far as S100A4 is concerned there is little doubt that its high level of expression leads to high growth rates. Compatible with the association of high S100A4 expression with malignancy is the recent demonstration that the S100A4 gene may be down-regulated in benign

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mammary epithelial cells. It would appear that S100A4 transcription is inhibited by a cis-acting sequence, related to the consensus recognising GC factor, occurring 1.3 kbp upstream of the transcription start site. An inverse relationship has been noted between the GC-factor and S100A4 mRNA levels (Chen et al. 1997). In conclusion, one may justifiably regard the currently available body of evidence, relating to the conferment of invasive and metastatic abilities by S100A4, as virtually irrefutable. It ought to be stated, nonetheless, that there has appeared a solitary report to the effect that S100A4 shows no relationship to cell proliferation, invasion, or metastasis. This report described the transfer of S100A4 sense constructs into MCF7 cells, with no apparent effects on in vitro invasion or proliferation. The transfectants produced no metastasis in mice, but there was marked tumour necrosis and abnormalities in tumour vasculature (Onischenko et al. 1996). There are a number of points to be taken into account with regard to this work. Grigorian et al. (1996) also used MCF7 cells for transfection of S100A4 and found that the transfected cells acquired marked metastatic properties. This is in sharp contrast with the findings of Onischenko et al. (1996). MCF7 cells are known to express the putative metastasis suppressor gene nm23 at a high level (Sherbet et al. 1995). S100A4 and nm23 genes might be coordinately regulated (Parker et al. 1991; Hsu et al. 1997). Admittedly, the significance of nm23 expression in breast cancer cells may not be rated high. However, Albertazzi et al. (1998b) have reported recently that the expression status of both S100A4 and nm23 taken together correlated far more powerfully with the clinical aggressiveness of breast cancer than when the status of S100A4 alone was considered. In other words, it would have been advisable for Onischenko et al. (1996) to look also at nm23 expression in their experiments. There have been no other reports so far that S100A4 has any effects on neovascularisation or cell death by necrosis or apoptosis. Without prejudice to their findings, however, it would be worth remarking that these latter effects could be collateral changes associated with the integration of the exogenous gene into MCF7 cells. The chances of this happening have been obviated by Grigorian et al. (1996) by adopting a different experimental strategy of transfecting the gene under the control of an inducible promoter. Some ambivalent views have been expressed also by Chiaramonte et al. (1998). These authors used a murine mammary adenocarcinoma cell line, TS/A, and two murine melanoma cell lines, B16-A and B78H1. All three cell lines responded to IFN-γ treatment with an increase in metastatic ability. However, when they tested these IFN-γ-treated cells for S100A4 expression, no changes were noticed in TS/A cells. Further, of the two melanomas only B16-A showed an enhancement of S100A4 expression. The effects of IFN-γ seem to be somewhat variable. It has been found to enhance transcription in human macrophages (Grigorian et al. 1994). On the other hand, IFN-γ down-regulates S100A4 expression in human colon adenocarcinoma cell lines. This effect is dependent on membrane-associated IFN-γ receptors and is not shown by IFN-γ or -β (Takenaga, 1999). Chiaramonte et al. (1998) have, therefore, quite legitimately argued that different genes may be associated with the metastatic behaviour of different tumour types. It would be useful to pursue these observations, even in the face of the virtually overwhelming evidence for a major role for S100A4 in tumour progression.

S100 Proteins: Their Biological Function and Role in Pathogenesis

229

Whether S100A4 promotes the metastatic spread of tumours by influencing cellular properties at a specific stage of the metastatic cascade is not clear at present. In light of the alleged inhibition of the microvasculature by S100A4, Cajone and Sherbet (2000) have employed heme oxygenase-1 (HO-1) gene as a marker in a series of experiments. Heme oxygenase (HO-1) is a stress–response protein. The induction of HO-1 gene is believed to play a protective role against both heme- and nonheme-mediated oxidant injury. Many agents such as hyperoxia, bacterial LPS, hydrogen peroxide, interleukin, prostaglandins, TPA, and hyperthermia, among others, are known to induce HO-1 (Choi and Alam, 1996). The HO-1 gene expression is regulated by the presence of responsive elements that can bind these agents. Another agent called 4-hydroxynonenal (4-HNE) is also a potent inducer of HO-1 expression. 4-HNE is a product of the peroxidation of membrane lipids and it has been shown to be able to induce the synthesis of HSPs (Cajone et al. 1989; Cajone and Bernelli-Zazzera, 1989). One of the HSPs induced by 4-HNE is a 32-kDa protein (Allevi et al. (1995). This HSP32, apparently associated with oxidative stress, seems to be none other than HO-1. Although total identity between these two proteins has not been fully established, HO-1 has been referred to, not infrequently, as HSP32 (Choi and Alam, 1996). Cajone and Sherbet (2000) modulated the levels of S100A4 expression by using agents that are known to up-regulate HO-1 expression. They found that the HO-1 expression was up-regulated irrespective of the effects of the agent on S1004 expression (Table 19). Grigorian et al. (1994) found that LPS has a variable effect on S100A4 expression in inflammatory macrophages. In these cells it reduced S100A4 expression in the first 3hr but then up-regulated it over the next four hours.

TABLE 19 The Lack of Relationship between Expression of S100A4 and Heme Oxygenase (HO-1) Genes Cells/Treatment

S100A4

HO-1

HUT 37,a – 42°C ↓ ↑ 3T3 normal serum + 4-HNE ↓ ↑ 3T3 serum-starved cells + 10% FCS ↑ ↑ 3T3 + TPA ↑ ↑ 3T3 + 4-HNE ↑ ↑ a HUT is a heat-resistant clone of cells derived from HepG2 cells. Source: Based on data from Cajone and Sherbet (2000). (Reprinted by permission of the publisher Kluwer Academic Publishers.

In other words, there was a total dissociation of the expression of the two genes. This might throw some light on the possible function of this protein in cancer metastasis. HO-1 has been reported to have a marked effect on tumour angiogenesis.

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Transfection of human HO-1 gene has been found to produce a two-fold increase in neovascularisation (Deramaudt et al. 1998). Indeed, some agents that induce HO1 gene expression are also inducers of angiogenesis. For instance, the HO-1 gene contains response elements for LPS, IL-6, and prostaglandins (Camhi et al. 1995, 1996; Kozumi et al. 1995), all of them known inducers of angiogenesis. These are powerful indicators that that S100A4 might not be associated with induction of neovascularisation. However, the effects of S100A4 expression on the cellular properties that are highly relevant in other compartments of the metastatic cascade, such as the phase of expansive growth of the primary tumour, phase of invasion, and possibly also the growth of overt metastases, are virtually indisputable (see also Figure 28 and Table 21).

CLINICAL POTENTIAL PROGNOSIS

OF

S100A4

AS A

MARKER

FOR

CANCER

S100A4 has attracted a great deal of attention in recent years because of the wide variety of physiological events and parameters of biological function that it appears to influence. However, in contrast with several other putative markers of malignancy, investigations into the possible clinical value of this gene are of recent origin. This is because much of the proposed work has been dampened by the somewhat illinformed criticism and reservations expressed about it. Fortunately, a great deal of research into the basic aspects of S100A4 function has been carried out, which has greatly emphasised the importance of S100A4 in the clinical assessment of the disease. Some of the work, such as that of Pedrocchi et al. (1994), Grigorian et al. (1996), and Maelandsmo et al. (1996), used tumour cell lines derived from human tumours, and these authors have shown that the levels of S100A4 expression were related to invasive and metastatic ability. Sustained studies of human tumours aimed at determining the relevance of S100A4 in predicting progression of the disease have followed. These have been carried out mainly in melanomas, breast cancer, and astrocytic tumours.

S100A4

IN

HUMAN BREAST CANCER

Pedrocchi et al. (1994) first described the importance of S100A4 in breast cancer cell lines. Recently, Albertazzi et al. (1998b) have published a detailed study of S100A4 expression, not only in relation to the degree of aggressiveness of the disease, but also as to how it relates to other clinical markers such as the status of oestrogen and progesterone receptors and the degree of tumour differentiation. Albertazzi et al. (1998b) also employed another marker, nm23-H1. nm23 is a putative metastasis suppressor gene identified in murine melanomas (Steeg et al. 1988a, 1988b), of which two human homologues have been cloned (Rosengard et al. 1989; Stahl et al. 1991). Several reports in the literature suggest an inverse relationship between the level of expression of nm23 mRNA or the gene product, nucleoside diphosphate (NDP) kinase, and metastatic potential. Such an inverse relationship has been described in some forms of human cancer, e.g., melanoma (Florenes et al. 1992; Caligo et al. 1994). In colorectal and gastric carcinomas, nm23

S100 Proteins: Their Biological Function and Role in Pathogenesis

231

expression has been reported to be down-regulated substantially in metastatic tumour as compared with the primary carcinoma (Ayhan et al. 1993; H. Nakayama et al. 1993). An inverse relationship between nm23/NDP kinase expression and metastatic potential has been reported to occur in hepatocellular carcinoma (T. Nakayama et al. 1992). Low levels of NDP kinase expression have been correlated with reduced survival of patients with infiltrating ductal carcinoma of the breast (Barnes et al. 1991; Royds et al. 1993). However, several studies repudiate this inverse relationship (Haut et al. 1991; Hailat et al. 1991; Keim et al. 1992; Zhou et al. 1993; Sawan et al. 1994; Saitoh et al. 1996; Srivasta et al. 1996; Aldersio et al. 1998). A study of a large series of breast carcinomas has shown that NDP kinase expression is neither related to disease relapse or patient survival, nor does it correlate with other prognostic factors such as tumour grade, oestrogen and progesterone receptor status and p53 expression (Sawan et al. 1994). Stephenson et al. (1998) examined 412 cases of lung carcinoma for nm23/NDP kinase expression. They found no relationship of its expression to metastatic disease or prognosis. Similarly, overexpression of nm23H1 in epithelial ovarian carcinomas is associated with lower survival and significantly poor prognosis in early stage and well-differentiated carcinomas (Schneider et al. 2000). Some of the problems associated with the observed lack of inverse correlation between nm23/NDP kinase expression and metastatic progression might be due to the tacit assumption that NDP kinase function and the putative metastasis-suppressor properties are interrelated and inseparable. Contrary to this, it has been argued that these properties can indeed be dissociated. H.Y. Lee and Lee (1999) transfected a cDNA coding for a mutant form of nm23-H1 that lacked NDP kinase activity into human prostate carcinoma cells. Nonetheless, the transfectant cells showed reduced invasive ability, in the same way as cells that had been transfected with wild-type cDNA. Although these observations do suggest that the two properties of nm23 are dissociable, conceptually the putative metastasis-suppressor function of nm23 becomes even harder to appreciate. This somewhat equivocal correlation, between nm23/NDP kinase expression and tumour progression, has led to the suggestion and actual demonstration that this gene might be coordinately regulated with the metastasis-promoting S100A4 gene in the B16 murine melanoma (Parker et al. 1991). These authors used MSH and RA to modulate the levels of S100A4 expression and found that the expression of nm23 levels showed corresponding changes. As a result, the ratio of their expression remained virtually constant. Similar results have been described more recently by Hsu et al. (1997) in CH27 human lung cancer cells, which essentially confirm the findings of Parker et al. (1991). Indeed, there is evidence that S100A4 expression promotes the depolymerisation of tubulin, whereas nm23 expression has the opposite effect (Nickerson and Wells, 1988; Lakshmi et al. 1993). Whether there is a direct regulatory link between S100A4 and nm23 remains to be established. It is unclear whether S100A4 is itself involved in some way with the negative regulation of nm23. The 1q21 locus, which harbours the S100A4 gene, also contains the human homologue of the PRUNE gene of Drosophila melanogaster. The prune eye colour phenotype of Drosophila is attributed to null mutations of the PRUNE gene. The occurrence of one mutant copy of awd (abnormal wing disc) (the

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Drosophila homologue of nm23-H1) with the prune genotype is lethal. This suggests that nm23-H1 and the prune protein might interact. The prune protein has indeed been found to interact with nm23-H1 and to negatively regulate the latter. In further confirmation, it has been noticed that the interaction is impaired when nm23-H1 is mutated (Reymond et al. 1999). The emergence of this negative regulation of nm23H1 by the prune protein does not explain the inverse relationship shown to subsist between S100A4 and nm23-H1. Perhaps it would be fruitful in future studies relating to the expression of these genes also to measure PRUNE expression as well. With this background, Albertazzi et al. (1998b) focused on the expression of S100A4 (h-mts1) as well as that of nm23 in human breast cancer. This was with a view to determining their individual abilities to serve as markers, and further to check whether a combination of the status of their expressions might enable one to obtain a more accurate assessment of the metastatic potential of the tumours. The data presented by Albertazzi et al. (1998b) show that high S100A4 expression is associated with metastatic spread to the regional lymph nodes. The expression of nm23 on its own did not show a statistically significant inverse correlation with nodal spread. However, the expression status of the two genes, taken together, strongly correlated with nodal metastasis. Furthermore, the correlation was more significant when S100A4 and nm23 were considered in combination,as compared with S100A4 on its own (Table 20). This suggests that, although nm23 did not seem relevant to nodal metastasis, a more accurate assessment of nodal status could be derived by looking at the expression of both S100A4 and nm23 genes. A further indication that S100A4 expression status might be related to the clinical aggressiveness of the tumour is provided by another line of evidence. Albertazzi et al. (1998b) have reported that breast cancers with no detectable expression of S100A4 were ER and PgR positive. With increased expression of S100A4, the tumours could possibly progress toward a hormone-independent state. Such progression has been demonstrated in vitro. MCF7 breast cancer cell lines that were transfected with and producing S100A4 have been reported to acquire hormone-independent growth (Grigorian et al. 1996). This is compatible with the generally held view that ER/PgRnegative breast cancers are clinically more aggressive and also tend to be EGFr positive. The ER/PgR status of breast cancers generally correlates inversely with EGFr status, which has been regarded by some as an indicator of poor prognosis (Sainsbury et al. 1987a, 1987b; Bolla et al. 1990; Hainsworth et al. 1991). This fits with the findings of Sherbet et al. (1995) that breast cancer cell lines that were high expressors of S100A4 tended also to be high expressors of EGFr. Furthermore, the recent finding that EGF is able to induce the expression of S100A4 in epithelial cells (Okada et al. 1997) may be deemed as supporting the relationship between EGFr expression and cancer prognosis. A correlation between the expression of type 1 growth factor receptor family and prognosis has also emerged from the recent work. Rudland et al. (2000) found that S100A4 positivity of breast cancer correlated significantly with the expression of c-erbB2 and c-erbB3. The prognostic significance of c-erbB2 has been well established and overexpression of this receptor is associated with poor prognosis. However, there are no data relating to the prognostic value of c-erbB3 nor c-erbB4 (Angus et al. 2000). Albertazzi et al. (1998b) also looked at tumour differentiation, but this did not correlate with S100A4 expression.

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TABLE 20 Relationship Between S100A4, S100A4v, and nm23 Expression to Nodal Spread of Breast Cancer Gene Expressed

Relationship to Nodal Spreada

S100A4 nm23 S100A4+nm23 S100A4v+b S100A4–/S100A4v–b

+ – ++ – +

a

+, statistically significant relationship (p < .05); ++, strong relationship (p < .01). b The expression of S100A4v (the splice variant form) alone did not relate to nodal spread; however, tumours that express neither S100A4 nor S100A4v do not tend to spread to the regional lymph nodes. Only 27% of carcinomas expressing neither h-mts1 nor h-mts1v showed metastatic spread to the lymph nodes; 57% (4/7) of carcinomas in which only the variant isoform was detectable showed nodal metastasis. This apparent difference did not reach statistical significance. Source: Based on Albertazzi et al. (1998a, 1998b). Reprinted by permission of the publisher Mary Ann Liebert Inc.

The clinical data, together with the state of expression of steroid receptors and the expression levels of S100A4 and nm23 genes, were analysed using artificial neural networks (ANNs) for accuracy of prediction of nodal spread of the carcinomas. Naguib et al. (1997) previously had demonstrated that ANNs could be used to predict nodal involvement in breast cancer. In that study several established as well as experimental cancer markers had been analysed, and ANN techniques were found to be capable of dissecting and identifying the most powerful predictors of nodal metastasis. The ANN analyses, provided by Albertazzi et al. (1998b) as well as by Naguib et al. (1998), have also supported the conclusion that, overall, S100A4 expression is associated with and indicative of more aggressive forms of the disease. The investigation of Albertazzi et al. (1998b) does emphasise the view that when complemented with nm23, S100A4 could provide a powerful marker for predicting breast cancer prognosis. On the other hand, S100A4 might be a significant and independent marker for prognosis. An investigation of a large series of breast cancer over a period of 16 years has led Rudland et al. (2000) to conclude that S100A4 expression correlates strongly with patient survival. The Rudland group found S100A4 mRNA in both epithelial and stromal components of breast cancers and, further, that the levels of S100A4 are higher in carcinomas as compared with benign breast tumours (Nikitenko et al. 2000). They have also reported that 80% of patients who were S100A4 negative were alive after 19 years of follow-up, whereas only

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11% of S100A4-positive patients were alive at the end of this period. The correlation between S100A4 negativity and survival was highly statistically significant. Interestingly, Rudland et al. (2000) found that S100A4 expression correlated only marginally with axillary lymph nodal involvement. Nevertheless, S100A4positive patients who were also node positive had poorer survival than S100A4 patients with no nodal involvement. The involvement of regional lymph nodes is regarded as the most consistent predictor of prognosis (Angus et al. 2000). The poorer prognosis of the S100A4+/node+ group may be related to whether or not S100A4 is expressed by tumour cells in the regional lymph nodes. Overall, the work reviewed here leaves little room for doubt that S100A4 is associated with tumour malignancy and prognosis. With the large body of evidence of the active participation of S100A4 in a wide spectrum of cellular functions, a demonstration of its association with metastatic cells of human breast cancer would be the coup de grace that is long awaited and that might satisfy the scientific cognocenti. As stated previously, two splice variants of S100A4 have been reported so far (Ambartsumian et al. 1995; Albertazzi et al. 1998c). The larger variant, described by Ambartsumian et al. (1995), occurs in many tissues, although with differences in the levels of expression. Albertazzi et al. (1998c) found only the shorter splice variant (h-mts1v) in the series of breast cancers that they had investigated. They did not detect the expression of either variant in a number of tumour cell lines. The apparently highly specific nature of its expression is somewhat inexplicable. Nonetheless, Albertazzi et al. (1998c) found that h-mtsv expression did tend to correlate with nodal spread of breast cancers, albeit the correlation was not as persuasive as in the case of the unspliced S100A4 transcript. Notwithstanding the positive nature of these findings, it ought to be stated here that assessing the state of expression of the S100A4 protein, not merely that of its mRNA, is equally important for providing a total picture of the relevance of the gene to progression of cancer. It is the occurrence of the functional protein that would determine the nature of the downstream events that define the degree of aggressiveness of the disease. As Ambartsumian et al. (1999) observed recently, S100A4 mRNA is expressed in all organs of S100A4 transgenic mice that they had developed. However, the protein is not expressed in organs that do not normally express the S100A4 gene in the wild-type animals. Therefore, it is imperative that information concerning the expression of the protein is collated at the same time as the mRNA levels are measured. This would take into account the fact that there may exist mechanisms that regulate the translation of the mRNA transcripts and possibly also decay of the protein in the normal course of cellular events.

S100A4

IN

OTHER FORMS

OF

HUMAN CANCER

Among other forms of human cancer that have been studied for S100A4 expression is human melanoma. Maelandsmo et al. (1997) detected high S100A4 levels only in approximately 50% of melanomas, but benign nevi also showed roughly similar levels of S100A4. Quite obviously, S100A4 bears no relationship to the clinical state of disease in this case. A criticism that can be made of this study is the method of assessment of gene expression as undetectable, low, moderate, and high. It would

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have been helpful if the authors had given the actual values of signal densities and provided the range of values of each group. The findings of Shrestha et al. (1998) generally support those of Maelandsmo et al. (1997). Shrestha et al. (1998) found little or no S100A4 in several cell types derived from normal skin. Furthermore, neither did basal cell carcinomas or squamous cell carcinomas show any S100A4 immunoreactivity. The apparent lack of correlation of S100A4 with melanoma progression is in sharp contrast with the high degree of correlation found in B16 melanoma cell lines. One should state, notwithstanding, that a direct comparison of expression of the gene in tissue culture cell lines and tumour material, which is heterogeneous in cell composition, is probably not helpful, nor can it lead to meaningful conclusions. In human gliomas, the expression of S100A4 was found to correlate with the degree of malignancy as indicated by the World Health Organization (WHO) histological grading. However, the changes in S100A4 were far less striking than those occurring in S100A1, which significantly correlated with malignancy (Camby et al. 1999). It would be worthwhile to bear in mind that the histological grading of these tumours is a differentiation-related feature, and often tumour behaviour may not strictly conform to characteristics defined for a particular histological group. Nonetheless, intrinsically the degree of differentiation would be inversely related to malignancy, and therefore the correlation reported between S100A4 and gliomas of different grades may be seen as generally supporting the involvement of S100A4. With the added knowledge that S100A4 expression is higher in cell lines derived from higher grade gliomas as compared with lower grade tumours, it would not be premature to conclude that S100A4 may play an important role in the malignancy of gliomas. The expression of S100A4 in colorectal tumours seems to show a clear correlation with disease progression. The expression of the gene is low in colonic fibroblasts. Normal mucosal tissue and adenomas have been described as showing comparable levels of expression, but expression level is markedly higher in adenocarcinomas. Cells lines derived from adenocarcinomas also show high levels of S100A4 expression. Immunohistochemical studies have generally confirmed these Northern analyses of S100A4 expression. Of much greater interest is the observation that although adenomas are negative, carcinomatous foci within the adenomas have been found to stain for S100A4 (Takenaga et al. 1997b). Quite obviously, the expression of S100A4 correlates well with the progression of colonic tumours.

S100A6 (CALCYCLIN) IN CANCER The pattern of expression of calcyclin, now carrying the new nomenclature of S100A6, has been studied in several forms of human cancer, e.g., melanomas (Weterman et al. 1992, 1993), salivary gland tumours (J.W. Huang et al. 1996), chondro-osseous tumours (Muramatsu et al. 1997), and squamous cell carcinomas of the oral mucosa (Berta et al. 1997). Muramatsu et al. (1997) have reported that calcyclin, together with S100A1 and A4, was particularly strongly associated with the development of chondro-osseous tumours. Enhanced calcyclin expression has been reported in squamous cell carcinoma of the oral mucosa but not in benign

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mucosal lesions. This was accompanied also by an increased expression of the Hras oncogene (Berta et al. 1997). Several genes are differentially expressed in rat mammary tumours, which are induced by the carcinogen N-methyl-N-nitrosourea (NMU). L.H. Young et al. (1996) detected several differentially expressed cDNA clones. Of these, one clone showed strong homology to calcyclin. The expression of this clone was ten-fold greater in tumour tissue than in normal tissue. Pedrocchi et al. (1994) investigated several breast cancer cell lines and noticed marked differences among S100 proteins in their relationship to aggressive behaviour. Calcyclin did not appear to bear any relationship to tumour aggressiveness. However, its expression was higher in the vertical phase of growth of melanomas and correlated with metastatic potential of melanomas (Weterman et al. 1992, 1993). Van Ginkel et al. (1998) found S100A6 to be differentially expressed between normal uveal melanocytes and uveal melanomas and cell lines derived from them. Also differentially expressed were annexins V and VI, CaM, S100A11, and S100B. Although they have suggested that S100A6 expression may relate to the malignancy of uveal melanomas, it would be difficult to dissociate the effects of S100A6 from that of other CBPs. As Sudo and Hidaka (1999) have shown, the CBPs may interact with one another. The correlation of calcyclin expression with metastatic potential has been confirmed by two recent studies (Boni et al. 1997; Maelandsmo et al. 1997). In the first study, all of 39 primary cutaneous melanomas tested showed intense cytoplasmic staining for calcyclin. Also 9 of 14 metastatic tumours were found to be calcyclin positive. In the second study, S100A6 expression was reported to correlate with patient survival times (Maelandsmo et al. 1997). In a series of human astrocytomas, Camby et al. (1999) found the expression of S100A6 to be clearly related to their WHO grading. Thus, grades I and II tumours could be distinguished, on the basis of S100A6 expression, from the more malignant grade III and IV astrocytomas. However, as discussed previously, there is some ambiguity about the relationship between calcyclin expression and tumour growth. Some investigators regard calcyclin as a negative regulator of growth on the basis that induction of differentiation in tumours can enhance calcyclin expression. It may be that the involvement of this S100 protein has other facets in its relationship with tumour aggressiveness than are currently appreciated and investigated. For example, it has been shown to bind to annexin XI-A (Sudo and Hidaka, 1999). This interaction seems to be of a highly specific nature. This may have some functional implication for S100A6, because annexin expression shows a marked relationship to cell cycle progression and, furthermore, the expression of annexins is linked with deregulation of growth of cancers.

THE BIOLOGICAL PROPERTIES OF S100A7 (PSORIASIN) STRUCTURE

AND

MOLECULAR PROPERTIES

OF

S100A7

Psoriasin (S100A7) has been traditionally associated with psoriatic skin lesions. The S100A7 gene coding for this protein has been described as the psoriasis-susceptibility gene. It spans 2.7 kb of the genome and consists of three exons and two introns. The gene has been mapped to the S100 gene family cluster at chromosome

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1q21 (Hardas et al. 1996). The S100A7 protein has been isolated from psoriatic skin. It is a ca. 11-kDa cytoplasmic protein with PI of 6.2. It is also secreted into the extracellular compartment. S100A7 is structurally similar to other S100 proteins. However, there are some important differences, especially with regard to the Nterminally located EF-hand calcium-binding domain. The latter contains a distorted loop, and notably, in S100A7 this EF-hand lacks an important calcium-binding residue. Consequently, it binds no more than one calcium ion per monomer (Brodersen et al. 1998). S100 family proteins are also known to bind other divalent cations besides Ca2+, and their binding to the proteins can often be functionally more significant than their binding of Ca2+. Recombinant S100A7 indeed binds Ca2+, Zn2+, and Mg2+. It can bind seven Ca2+ ions in the presence of KCl and several in the presence of NaCl. Therefore, in the secreted state, S100A7 could bind Ca2+ and, in the cellular compartments, possibly binds higher amounts of calcium. It can bind eight Zn2+ ions in the presence of KCl and four in the presence of NaCl (Vorum et al. 1996). The binding of these cations produces conformational changes in the protein. A subsequent picture of calcium-binding described by Brodersen et al. (1998) suggests a greatly reduced calcium-binding ability, and moreover, their findings are not compatible with the view that calcium-binding brings about conformational changes in S100A7. It may be that insofar as S100A7 is concerned, calciumbinding may be functionally less significant than Zn2+. Recently, Brodersen et al. (1999) have identified a Zn2+-binding site in S100A7 that contains three histidine and one aspartate residues. It is of much interest to note that these residues present the Zn2+ in a way characteristic of certain metalloproteinases, which are important components of the ECM. It also seems from the work of Brodersen et al. (1999), that the absence of Zn2+ may have collateral effects on the organisation of the distorted EF-hand loop. This might result in reduced calcium-binding by S100A7. Although the zinc-binding site occurs in other S100 proteins as well, the reason why calcium-binding is functionally the most significant event in a majority of these proteins could be the absence of collateral Zn2+-induced changes.

S100A7

IN

SKIN PATHOLOGY

S100A7 (psoriasin) was originally isolated from psoriatic skin and seen to be associated clearly with inflammatory disease of the skin. Retinoic acid treatment has been shown to induce S100A7 expression in skin but not in other tissues. This suggests a tissue-specific regulation of its expression. Expression occurs at a low level in untreated epidermal keratinocytes but not in fibroblasts or melanocytes (Tavakkol et al. 1994). S100A7 has been shown to function as a chemotactic factor for CD4+ T lymphocytes and to mediate inflammation. However, it is structurally distinct from conventional lymphokines (J.Q. Tan et al. 1996). S100A7 has been shown to form complexes with the epidermal-type fatty acid-binding protein (EFABP). EFABP, which shows an increased expression in psoriatic skin, is believed to be involved in the transport of cytosolic fatty acids. S100A7–EFABP complexes occur in the cytoplasm of differentiating keratinocytes derived from psoriatic skin (Hagens et al. 1999). The functional significance of the complex is not understood

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Calcium Signalling in Cancer

at present. Two possibilities have been suggested: keratinocyte differentiation and/or the transport and targeting of cytosolic fatty acids. Semprini et al. (1999) have suggested that the S100A7 gene may not be a candidate gene for familial psoriasis susceptibility. Several other genes occurring in the 1q21 cluster are said to be overexpressed in psoriasis (Hardas et al. 1996). This may support the contention that S100A7 is not a candidate gene for psoriasis. Furthermore, the 1q21 gene cluster contains a collection of genes associated with the terminal differentiation of epidermis. As discussed in an earlier section, genes in this cluster are also actively involved in the control of cell proliferation. Primary human keratinocytes exposed in vitro to growth factors show an up-regulation of not only S100A7 but also MRPs 8 and 14, S100A11. Also expressed in these cells are the actin-binding non-EF-hand proteins, gelsolin and annexin (Olsen et al. 1995). Because of the wide-spectrum genetic modulations involved, the relative significance of the overexpression of individual genes of this cluster, is very difficult to assess in relation to pathogenesis of psoriasis. It is inevitable that the overexpression of other CBP genes at the locus will have repercussions on the expression of S100A7, because not only does calcium bind S100A7, but calcium is also able to up-regulate the synthesis of S100A7 (Hoffmann et al. 1994). It follows, therefore, that a significant sequestration of intracellular calcium by other CBPs would affect S100A7 expression.

S100A7

IN

NEOPLASTIC DISEASE

The possibility that S100A7 expression in keratinocytes might reflect the state of their proliferation and differentiation has made it easier to appreciate why A7 is expressed in both normal and malignant keratinocytes in culture (P.H. Watson et al. 1998). Early studies indicated that S100A7 is expressed in breast cancers as well as cell lines derived from breast cancers, but it was not detectable in control tissues (Moog-Lutz et al. 1995). Leygue et al. (1996) compared its expression in ductal carcinoma in situ and invasive ductal carcinoma. They detected S100A7 mRNA only in the in situ carcinomas. It would be inappropriate to extrapolate from these limited studies on the possible relationship between expression of the gene and invasive behaviour of the carcinoma. In this context it might be interesting to note that S100A7 has been found to enhance the adhesion of neutrophils to epidermal cells (Von den Driesch et al. 1998). Unhappily, most of these studies are very preliminary in nature. Nonetheless, the results are significant enough to warrant further investigation and confirmation. There have been tentative investigations of the possibility that S100A7, as a secreted protein, could be a potential marker in certain tumour types. In squamous cell carcinoma of the bladder, urinary S100A7 has been suggested to be a useful noninvasive marker for follow-up of patients (Ostergaard et al. 1999). S100A7 is expressed, together with markers for keratinised stratified squamous epithelia, in squamous cell carcinoma of the bladder. S100A7 expression is localised to the socalled squamous pearls (Celis et al. 1996). As expected, S100A7 is also detectable in the urine of patients. There is a lack of persuasive data on the relationship between S100A7 expression and the state of malignancy of bladder carcinoma. However, the

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239

expression of several other important genes of the 1q21 cluster is often up-regulated with S100A7 as a consortium. This seems to be ample justification for evaluating these genes as a potential noninvasive profile of markers for bladder cancer.

S100A8 AND S100A9 PROTEINS IN INFLAMMATORY DISEASES S100A8 and S100A9 proteins, previously known by the designations MRP8 and MRP14, respectively, are CBPs of the S100 protein family (Kligman and Hilt, 1988; Kerkhoff et al. 1998). They contain two EF-hand domains, one at either end of the molecule, and they are separated by a central hinge region. The EF-hands themselves are flanked by hydrophobic regions. Besides Ca2+, they can also bind Zn2+ ions. The latter might regulate some of the functional properties of these proteins. S100A8 and A9 are regarded as myeloid-specific proteins and show an enhanced presence in activated phagocytes. Increased plasma levels of these proteins have been reported in patients with inflammatory conditions, such as rheumatoid arthritis, cystic fibrosis, and chronic bronchitis. Although localised predominantly in the cytosol, they may be secreted by activated monocytes (Rammes et al. 1997). It would seem that they are translocated from the cytosol to the tubulin cytoskeleton and the plasma membrane. The targeting of these molecules to this cellular compartment could be a result of phosphorylation. Guignard et al. (1996) found that translocation occurred after the proteins were phosphorylated. As an extracellular protein, several functions have been envisaged and attributed to S100A8/A9. Kerkhoff et al. (1999a) have suggested they might be involved in the transendothelial migration of monocytes. Among other suggested roles are growth inhibition and cytostasis. There is some interesting evidence that they may regulate the inflammatory process by virtue of being able to form complexes with arachidonic acid and aid its transport and metabolism (Kerkhoff et al. 1999b). The binding of arachidonic acid by the S100A8/A9 heterodimeric complex has been found to be dependent on Zn2+ binding. Whereas Ca2+ binding seems to be conducive to the binding of arachidonic acid, Zn2+ binding appears to reverse this process. Many of these studies are still in an incipient stage and much further work is required in order to elucidate the mechanisms by which S100A8 and A9 putatively carry out this range of function. Until then, most of the suggested mechanisms will remain in the realm of speculation.

S100A11 (S100C) AND POSSIBLE MODES OF ITS FUNCTION S100A11 was isolated and characterised by Ohta et al. (1991). It is an 11-kDa protein with two EF-hand domains and shows 41 and 37% sequence homology with S100A and S100B proteins. The S100A11 gene has been localised to the S100 gene cluster at chromosome 1q21 (Moog-Lutz et al. 1995; Wicki et al. 1996a,b). The protein has been shown to bind to actin and inhibit actin-activated Mg2+-ATPase

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FIGURE 28 The biological processes influenced by S100 proteins. This is especially true of S100A4, as discussed here. In the context of cancer, the major influences are on cell motility, adhesive properties, and proliferation. The effects exerted by S100 proteins on cytoskeletal dynamics seem to be important in maintaining or modulating cell morphology and also highly pertinent in cell proliferation and signal transduction. Overall, this illustration focuses on the breadth of biological effects brought about by S100 proteins and the need to regard S100 genes, such as S100A4, as normal genes, whose inappropriate expression could lead to aberrant biological behaviour, rather than as “oncogenes” or “metastasis” genes. (From Sherbet and Lakshmi, 1997b, 1998.) Reprinted by permission of the publisher Academic Press.

activity of smooth muscle cells (Zhao et al. 2000). Zhao et al. (2000) have, in fact, shown that the inhibition of the myosin-ATPase enzyme is not due to the depolymerisation of actin filaments but it is a direct effect of actin binding by S100A11. The cytoskeletal binding action is probably not unique to this protein. The association of S100A4 with the cytoskeleton has now been amply demonstrated (Figure 28 and Table 17). Moreover, S100A4 also binds to the rod region of myosin and inhibits actin-activated ATPase (Ford et al. 1997). The function subserved by S100A11 is yet unclear. Apart from the obvious consequences of Mg2+-ATPase inhibition to the functioning of the smooth muscle, it is of interest to note that S100A11 interacts also with annexins (Naka et al. 1994; Mailliard et al. 1996; Seemann et al. 1996), a property it shares with S100A10 (Kaczanbourgois et al. 1996). These interactions of S100A11 and S100A10 could be important indicators of their cellular function. PKC-mediated regulation of annexin function involves certain amino acid residues of the S100 proteins that have been implicated also in the interaction between annexins and S100A11 and S100A10. The interaction with cytoskeletal elements quite obviously implicates them in the regulation of the cytoskeletal machinery. Furthermore, there is a potential, albeit indirect link between these S100 proteins and cell proliferation and differentiation, and, by analogy with S100A4 function, it might be involved in cancer invasion too. This aspect of potential influences of S100A10 and S100A11 has received almost no attention to date.

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S100P IN CANCER PROGRESSION S100P was isolated from human placenta and characterised some years ago (T. Becker et al. 1992; Emoto et al. 1992). It has received much attention in recent years. S100P consists of 95 amino acid residues and possesses two EF-hands that bind calcium with different affinity. The high-affinity EF-hand occurs at the Cterminal and the one with low affinity occurs at the N-terminal region of the protein. The protein also binds other divalent ions such as Zn2+ at the C-terminal region and Mg2+ at the N-terminal end. The binding of Ca2+ and Mg2+ has been shown to alter the conformational state of S100P, and it has been postulated that the biological properties of the protein may be altered by a Ca2+//Mg2+ switch (Gribenko and Makhatadze, 1998; Gribenko et al. 1998).

S100P

AND ITS

PUTATIVE FUNCTIONS

The presence of S100P in skin sensory corpuscles has been described by some investigators (Delvalle et al. 1995; Albuerne et al. 1998). Intense immunostaining for S100P has been encountered in the lamellar cells of Meissner corpuscles and the inner core cells of Pacinian corpuscles (Albuerne et al. 1998). In the avian Herbst corpuscles also S100P is known to be associated with the inner core cells. These studies have found no evidence of S100P in the central axons of the sensory corpuscles. The protein may be putatively involved in sensory signal transduction. Both these studies also refer to the occurrence of other S100 proteins. That S100P might contribute, together with S100A, to the structural integrity of the sensory apparatus is suggested by the finding that the pattern of expression of S100 proteins, including S100P, was not altered in skin samples obtained from patients with spinal cord injury. However, there was a reduction in the number of sensory corpuscles that expressed these proteins (Albuerne et al. 1998). Frank and Wolburg (1996) studied the events associated with wound healing and tissue repair following injury to the optical nerve in the rat. They have reported the appearance of reactive astrocytes staining for S100P about 6 days after the nerve injury, and these astrocytes might be involved in the restructuring of the nerve fibres. Some preliminary observations have been reported about the expression of S100P in relation to the progression of carcinoma of the prostate. Androgens are actively involved in the growth and function of the prostate. Carcinomas of the prostate have notably been described as being androgen dependent at early stages of their development, but becoming independent of this hormone during progression of the disease. Averboukh et al. (1996) found that the androgen-responsive cell line LNCaP-FGC, derived from prostate cancer, expresses S100P gene, but this is downregulated within 30 hr after androgen deprivation. On the basis of these observations, they suggest that S100P might be involved in the aetiology of prostate cancer. One would concede that the expression of the gene might be regulated by androgen, but this is a far cry from demonstrating its implication in the pathogenesis of prostate cancer. One could even tentatively suggest that S100P might conceivably function as a suppressor gene. A number of true metastasis-suppressor genes, as contrasted with tumour-suppressor genes, have been cloned.

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Calcium Signalling in Cancer

In evaluating the potential role of S100P one should also take account of the fact that androgens regulate the expression of PSA. They regulate PSA gene transcription by means of the androgen-responsive elements that occur in the promoter region of the PSA gene (Riegman et al. 1991). Probably cis-acting elements are involved also in the AR-mediated transcription of PSA (J.Y. Zhang et al. 1997). The PSA-related protein, human glandular kallikrein-1, is also regulated by androgens. The gene that encodes it contains an androgen-responsive element (Murtha et al. 1993). A proportion of PSA occurs in the form of a complex with other proteins such as α1-antichymotrypsin. The ratio of bound to free PSA is greater in carcinomas than in BPH. Therefore, the proportion of free PSA found in the serum has been regarded as a reasonable tumour marker (Becker and Lilja, 1997; Stenman et al. 1999). Sartor et al. (1997) studied the rate of increase of PSA and have concluded that a rapid increase in the level of PSA indicated metastatic disease, in contrast with a moderate rise in PSA, which was associated with local recurrence. However, Bangma et al. (1995) did not find any relationship between PSA expression and progression of the disease. Chopra et al. (1996) found PSA expression in normal prostate, BPH, and carcinomas. Some recent experimental work has shown that PSA can inhibit endothelial cell proliferation and also inhibit the angiogenic effects of FGF and VEGF. In the mouse model, PSA has been found to reduce metastatic deposition of tumour cells in the lungs (Fortier et al. 1999). The apparent antiangiogenic and anti-metastatic effects of PSA have raised further doubts about its reliability as a marker of tumour progression. However, because PSA forms complexes with proteases other than antichymotrypsin, there is room for refinements of its utility as a diagnostic agent. PSA is a kallikrein-like serine protease and can conceivably bring about changes in the ECM that might be conducive to tumour cell invasion. Indirect evidence for this comes from the observation that the in vitro invasive behaviour of LNCaP cells is inhibited when the proteolytic activity of PSA is experimentally suppressed (Webber et al. 1995). In vivo, the invasive behaviour of prostate carcinoma correlates with PSA concentration in serum (Bostwick et al. 1996). The PSA-related prostatespecific kallikrein is also regulated by androgen (Murtha et al. 1993), and its expression has been reported to increase from benign epithelium through intraepithelial neoplasia to carcinomas (Darson et al. 1997). Therefore, PSA alone could be viewed as being responsible for the invasive behaviour and metastatic spread. However, one must reconcile with the increasing androgen independence of the process of progression of this cancer. In the midst of contradictory findings, there seems to be a consensus view that PSA expression might in fact be maintained or even increase with progression. However, the uncertainty about the relationship between PSA and tumour progression might yet allow one to dissociate the potential influence on tumour progression of S100P from that of PSA. Therefore, in spite of these various complications arising from androgen-mediated regulation, S100P deserves to be studied further. For example, it would be of much interest to know the state of its expression in BPH, and whether there are any discernible changes in expression in the progression of disease from the state of intraepithelial neoplasia to invasive adenocarcinoma.

S100 Proteins: Their Biological Function and Role in Pathogenesis

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Of considerable practical significance is the recent report by Bertram et al. (1998) of the strong association between S100P, and to a lesser extent S100A4, expression and drug resistance. S100P was highly expressed in doxorubicin-resistant cells as compared with those sensitive to the drug. The reasons for this association can only be speculated. At any rate, the need to exploit the practical value of this finding should far outweigh the desire to understand the underlying mechanisms.

POTENTIAL VALUE OF S100 PROTEINS AS MARKERS OF CANCER PROGRESSION AND PROGNOSIS The establishment of a marker for monitoring disease progression and possibly as an aid in predicting prognosis should contain two basic elements. One of these is that the expression of the putative marker should show clear and unequivocal empirical correlation with disease state. The second, more important element is that the putative marker should manifestly function via a mechanism that involves one or more fundamental processes of the life of the cell. Whether measuring the expression of S100 genes has any role to play in the clinical management of human cancer is an area that now clearly warrants investigation. Early indications are that this could be useful. Serum levels of S100 proteins have been measured in melanomas. Elevated levels of generic S100 proteins may be related to metastatic outcome (Buer et al. 1997; Drummer et al. 1997). S100 proteins were detectable in the serum of 79.3% patients with metastatic disease as compared with 4% of stage I/II patients and 21.4% of stage III patients. Furthermore, there was a sharp decline in S100 levels in two patients in remission (Drummer et al. 1997). The detection of higher serum levels also has been reported in all stages of cutaneous melanomas, as compared with levels found in normal subjects (Abraham et al. 1997). Immunohistochemical staining of cutaneous tumours has revealed intense staining for S100A6 in all primary lesions and in 64% (9/14) of metastatic lesions studied; but staining for S100A4 was found to be weak in these neoplasms (Boni et al. 1997). S100A4 expression has been studied in human colorectal neoplasms. Normal colonic mucosa and adenomas have been found to contain comparable levels of S100A4, but adenocarcinomas express considerably larger amounts of S100A4 mRNA. Furthermore, immunohistochemical analyses have revealed metastatic tumours to be strongly S100A4 positive (Takenaga et al. 1997b). It has been reported recently that, in human infiltrating ductal carcinomas of the breast, S100A4 (h-mts1) expression correlates strongly with the potential to metastasise to axillary lymph nodes. S100A4 expression also inversely correlates with oestrogen and progesterone receptor status. These observations suggest that S100A4 (h-mts1) expression might reflect the aggressiveness of breast cancers (Albertazzi et al. 1998b). Albertazzi et al. (1998b) also reported the occurrence of a variant hmts1v, a shorter transcript of S100A4 (h-msts1) in which exon 1 (1a, 1b) is spliced out (Albertazzi et al. 1998c). Their data have suggested the possibility that the expression of this variant might also be indicative of aggressive disease, albeit not as powerful a marker as S100A4 (h-mts1) itself. Further work needs to be done on

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the expression of both S100A4 (h-mts1) and h-mts1v in breast cancer as well as in other forms of cancer in order to consolidate these findings before pronouncing on the clinical value of S100A4 expression in tumours. Nevertheless, there are firm data concerning the ability of S100A4 to enhance the invasive and metastatic propensities of experimental tumour models as well as in certain human neoplasms. Of the S100 family proteins, S100A4 seems most satisfactorily to fulfill both the criteria stated above. It appears to function via the modulation of the cytoskeletal machinery of the cell and by controlling the progression of the cell cycle. These observations provide considerable support for the thesis that S100A4 expression might be a powerful marker, from the S100 family of proteins, of cancer progression and prognosis.

Epilogue The range of biological parameters influenced by calcium-binding proteins in general and S100 proteins in particular is indeed most impressive (see Figure 28). CBPs are highly versatile proteins, and highly significant is their mediation of several critical events closely aligned to and identifiable with specific compartments of the metastatic cascade (Table 21). Six compartments can be identified in tumour development, invasion, metastatic dissemination, and the development of overt metastatic lesions. In all these, CBPs and S100 proteins, in particular, will influence the cellular properties that are highly associated with the behavioural or phenotypic event relevant to that specific metastatic compartment. This seems to begin at the beginning, in the initiation of neoplastic transformation and the expansive growth of the tumour. The transduction of signals originating from aetiological agents or down-stream signals of TGFs or mitogenic stimuli often takes the calcium-signalling pathway. The flow of information is mediated by CBPs. The CBPs may be regarded as the translators and the harbingers of the calcium messages. They undergo conformational changes upon binding to Ca2+. A consequence of this seems to be the acquisition of the ability to recognise target proteins and translate the calcium signal into a biochemical function reflecting a phenotypic property. Also often involved in the pathway is a coordinated functioning of kinases and phosphatases, as, for instance, in the transduction of the TCR binding by its antigen, in which calcineurin phosphorylation plays an important part in information flow.

TABLE 21 Metastatic Compartments and Relevant Cellular Properties Influenced by Calcium-Binding Proteins Metastatic Compartment A.

Aetiology

B.

Cell proliferation, apoptosis

C.

E.

ECM remodelling, modulation of cell adhesion, cell shape, motility, cytoskeletal dynamics Cytoskeletal dynamics, cell membrane malleability, cell motility, angiogenesis Heterotypic adhesion

F.

Cell proliferation, apoptosis

D.

Relevant Events of the Metastatic Cascade Neoplastic transformation Tumour development, expansive growth Invasion Extravasation from vascular system Arrest of cells at metastatic targets Development of overt metastases

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The expansive growth of tumours is a reflection of a net increase of the cell population size, which is a function of total cell proliferation and apoptotic cell loss. The deregulation of the cell cycle seems to be the prime cause of this expansive phase of tumour development, and it is very appropriate that North (1991) described cancer as a disease of the cell cycle. As we have seen, S100 proteins not only show cell cycle-related expression, but individual proteins such as S100A4 might be closely linked with the regulation of cell cycle progression in conjunction with other regulatory proteins such as p53, rb, and stathmin. The activity of these proteins is exquisitely coordinated to achieve effective regulation. Calcium signalling is an integral part of the process of apoptosis, and the apoptotic pathway contains several target enzymes activated by calcium. This is equally important in the development of metastatic deposits, but the kinetics of cell proliferation and apoptotic loss in primary tumours and metastatic deposits are not strictly comparable. We have discussed the potential effects of Ca2+-mediated activation of caspases and calpains in the deregulation of cell proliferation and induction of apoptosis. The influence of VD3 on cell differentiation with attendant inhibition of proliferation is a good example of the interplay of VDRE-mediated activation of gene transcription and the control over this exerted by osteocalcin. The latter in turn is regulated by TGFβ. The invasive phase of the metastatic cascade is replete with examples of CBP activity. There is much empirical evidence that high levels of S100 family proteins occur in association with enhanced invasive propensity. Besides, some of these proteins appear to take part in the remodelling of the extracellular matrix, which, as we have noted, figures prominently in cell adhesion and invasive behaviour. S100A4 and osteonectin are prime examples of ECM-modulating CBPs. Both seem to participate in its remodelling by having recourse to regulating the proteolytic activity associated with the ECM. The regulation of the contractile machinery of the cell is obviously of considerable importance in cell motility. The regulatory component of the actomyosin assembly, i.e., MLC, binds calcium in its function of myosin-ATPase regulation and generation of contractile forces. MLCs themselves undergo reversible phosphorylation through the agency of Ca2+/CaM-dependent kinases and phosphatases. The phosphorylation of MLCs seems to be involved in the invasion of vascular endothelium by activated PMN. As we have noted already, not only does calcium signalling via the activation of calpains and caspases severely affect tumour development, but we have also established the involvement of these enzymes on the adhesive and invasive behaviour of cells. The transmembrane glycoprotein cadherin forms a complex with β-catenin and α-catenin and links-up with the actin cytoskeleton. This complex is a complete example of calcium signalling mediated by cadherin, which has calcium-binding domains in the extracellular part of the molecule, and the catenin serving an adhesion function as well as providing a signal transduction machinery. Furthermore, there might be a feedback mechanism present, with cadherin negatively regulating the signalling function of β-catenin. The cadherin–catenin complex, at the same time, is also essential for the physical process of formation of adherens junctions. The cellular properties of heterotypic adhesion and deregulation of cell proliferation kinetics would apply equally also to the development of overt metastatic deposits. Nonetheless, one should recognise that the adhesive properties required of a tumour

Epilogue

247

cell trying to gain entry into the vascular system are probably totally different in character from those required for extravasation of the tumour cell in the target organ. On the other hand, one might speculate that the seeding of tumour cells at the target organ might be a reflection of the induction of apoptosis of endothelial cells rather than by a conventional form of diapedesis across the epithelium. Notwithstanding the downstream pathway of calcium signalling, a retardation of the initial event of calcium influx into cells seems to affect profoundly the invasive behaviour of cells as well as prolong survival times of mice bearing human tumour xenografts. The vascularisation of the tumour and the decimation of tumour cells that have gained access to the vascular compartment are the major gaps in our understanding of CBP activity. Osteonectin has been attributed with both angiogenic and antiangiogenic abilities. There is very little direct evidence that one can cite for either attribute. Some indirect evidence is available that suggests that S100A4 may not be involved with tumour vascularisation. However, there is also a report that claims that S100A4 can reduce the size and density of tumour-associated microvasculature. It might be recalled here that activation of calcium influx brings about major changes in the shape of endothelial cells, which can lead to enhanced permeability and promote the diapedesis of leukocytes. Activation of calcium influx also induces angiogenesis. Furthermore, certain agents that inhibit calcium signal transduction also seem to be capable of inhibiting angiogenesis. Whether CBPs intervene in this aspect of signal transduction is uncertain at present, but this is a distinct possibility. We know that calcium-dependent NOS is involved in the remodelling of vascular endothelia and in angiogenesis. Furthermore, the expression of endothelial NOS in carcinomas correlates with tumour grade. Hence, one of the strategies attempted to control metastatic spread was aimed at inhibiting angiogenesis by blocking the calcium-signalling pathway mediated by calcium-dependent NOS. That calcium signalling might be implicated in the regulation of angiogenesis is, therefore, not altogether in the realm of scientific speculation. There is much scope for intensive investigation of this aspect of the function of CBPs in the metastatic spread of cancer. It is now safe to say that the study of CBPs in general and of S100 family proteins, such as S100A4, in particular have much to offer in the understanding of cell behaviour in a normal cellular environment as well as their behaviour upon neoplastic transformation. This conclusion is based on the extensive evidence that links CBPs, beyond reasonable doubt, with cellular properties that are essential features of tumour growth, differentiation, invasion, and metastatic dissemination. There is much to learn and much to be gained.

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Index A ABP, see Amyloid β-protein ACTH, see Adrenocorticotropic hormone α-actinin, 51, 103 Action–myosin interaction, 117 Actomyosin assembly, 112 ACV, see Acyclovir Acyclovir (ACV), 61, 62 Adenomatous polyposis coli (APC) protein, 104 Adenylyl cyclase, 22 Adhesion cell–substratum, 58 intracellular, 58 -mediating proteins, 59, 94 Adrenal cortex, osteonection immunoreactivity in invasive tumours of, 193 Adrenocorticotropic hormone (ACTH), 24 Alzheimer’s disease calcineurin in, 136, 140 calretinin and, 133 caspases and neuronal loss in, 177 CBD-immunoreactive neurones in, 129 Amyloidosis, gelsolin expression in, 42 Amyloid precursor protein (APP), 178, 179 β-Amyloid precursor protein (βAPP), 141 Amyloid β-protein (ABP), 141 Androgen, 241 Androgen receptors (ARs), 101 Angina pectoris, 222 Angiotensin signals, 76 Annexin(s), 35 II, 208 cell cycle-related expression of, 39 function, PKC-mediated regulation of, 36 in human foreskin fibroblasts, 37 interaction between S100A11 and, 240 Antibodies, anti-keratin, 204 Antichymotrypsin, 242 Anti-keratin antibodies, 204

APC protein, see Adenomatous polyposis coli protein Apoptosis calpains in, 158 caspase-mediated, 173 dexamethasone-induced, 18 PARP as marker of, 172 APP, see Amyloid precursor protein βAPP, see β-Amyloid precursor protein Arabidopsis thaliana, 96 Arachidonic acid, binding of, 67 ARs, see Androgen receptors Aspergillus nidulans, 136 Astrocytomas, expression of S100A3 studied in human, 225 Atriplex nummularia, 146 Autoimmune conditions, 53 Autoimmune diseases, 84

B BAE cells, see Bovine aortic endothelial cells Barrett’s adenocarcinoma, villin expressed in, 45 Barrett’s metaplasia, 45, 216 Basement membrane (BM), 183 Basic fibroblast growth factor (bFGF), 76 BDNF, see Brain-derived neurotropic factor Becker muscular dystrophy (BMD), 165 Benign prostatic hyperplasia (BPH), 100 bFGF, see Basic fibroblast growth factor Bifonazole, 78 4,5-Bisphosphate, 10 Bladder, squamous cell carcinoma of, 238 BM, see Basement membrane BMD, see Becker muscular dystrophy Bombesin, 19 Bone disease, metastatic, 60 metabolism, osteocalcin in, 54 scans, 61 Bovine aortic endothelial (BAE) cells, 189 349

350 BPH, see Benign prostatic hyperplasia Bradykinin, 19 Brain -derived neurotropic factor (BDNF), 125 osteonection immunoreactivity in invasive tumours of, 193 Breast cancer(s), 70, 228 aggressiveness of, 243 ER-negative, 193 ER/PgR-negative, 232 ibandronate treatment of metastatic, 61 prognosis, marker for predicting, 233 carcinomas, NDP kinase expression in, 231 ductal carcinoma of, 98 epithelium, neoplastic transformation of, 111 osteonection immunoreactivity in invasive tumours of, 193 Butyrate, 127

C Cadherin, 246 –catenin complexes, forms of, 212 as marker for serous carcinoma of ovary, 134 Caenorhabditis elegans, 89, 102, 154 Caherin, 107 CAI, see Carboxyamido-triazole Calbindin D-9K (CBD-9K), 125 D-28K (CBD), 125 immunoreactivity, 126 neuroprotective function of, 129 Calbindin, structure and biology of, 125–130 calbindin expression in embryonic development and with ageing, 126–127 calbindin expression and metastatic phenotype, 129–130 calbindin in neuronal populations, 125 neural cell lineage and regulation of calbindin expression, 125–126 neuroprotective function of calbindin, 129 physiological function of calbindin, 127–128

Calcium Signalling in Cancer Calcimedin, 31 Calcineurin, in cell proliferation, cell adhesion, and cell spreading, 135–144 calcineurin in Alzheimer’s disease, 140–141 calcineurin in cell proliferation and adhesion-related phenomena, 136–140 effects of calcineurin on cell adhesion and motility, 138–140 putative role of calcineurin in cell cycle progression, 136–138 calcineurin in immunosuppression, 141–144 molecular features of calcineurin, 135–136 Calcium /calmodulin-dependent protein kinases (CAMPKs), 13 capacitative energy of, 10 Calcium-binding proteins (CBPs), 1, 5 cell proliferation, 245 EF-hand, 29 gelsolin, 40 molecular configuration of, 65 natural classification, 29–33 neural, 79 neuronal, 131 non-EF-hand, 30–31 photoreceptor-specific, 32 posttranslational changes of, 66 S100 family of, 200 Calcium signalling pathway, 5–28 architectural aspects of signal transduction machinery, 25–28 cyclic AMP in calcium signaling, 21–25 deregulation of inositol 1,4,5trisphosphate pathway, 18–20 homeostatis of cell calcium, 5–10 deregulation of calcium homeostasis as primary event in carcinogenesis, 9–10 plasma membrane Ca2+-ATPase pump, 5–7 sarcoplasmic–endoplasmic reticulum Ca2+-ATPase pump, 7–8 voltaged gated calcium channels, 8–9 inositol phosphates in calcium signal transduction, 16–18

Index phospholipid signalling, 10–11 protein kinase C and isoforms in signal transduction, 14–16 protein kinase C pathway, 13–14 PTEN phosphatase in regulation of lipid signalling, 11–13 ryanodine and related receptors in calcium mobilisation, 20–21 Calcyclin, 64, 235 Caldesmon, 121 Calelectrin, 31 Calmodulin (CaM) family, of calcium binding proteins, 75–85 calmodulin and physiological function, 75–79 calmodulin and cell proliferation, 77–78 calmodulin-mediated signal transduction, 76–77 calmodulin in neoplasia, 78–79 structure and mode of action of calmodulin, 75–76 guanylate cyclase-activating proteins, 85 recoverin subfamily of neural calcium binding proteins, 79–85 G-protein signalling pathway, 79–80 mode of action of recoverin, 82 post-translational modification of recoverin, 82–83 recoverin and cancer-associated retinopathy, 83–85 recoverin and function, 80–82 Calmyrin, 33 Calnexin, 49 Calpains, in normal and aberrant cell physiology, 153–167 calpain family of calcium-binding proteins, 153–154 calpains in cancer growth and progression, 162–163 calpains in cell proliferation and apoptosis, 158–160 calpains in cell spreading and migration, 160–161 calpains in intergrin-mediated cell adhesion and signal transduction, 161–162 calpains in muscular dystrophy, 165–167

351 association of calpains with Duchenne muscular dystrophy, 165–166 calpains and limb girdle muscular dystrophy, 166–167 calpains in myelodegenerative diseases, 163–165 involvement of calpains in development and differentiation, 157–158 molecular organisation of calpains, 154–155 regulation of physiological events by proteolytic function,155–157 Calponin calcium-dependent binding to, 209 homology (CH), 96, 122 myosin-ATPase inhibition by, 124 Calreticulin, 30, 35, 51 Calretinin, 32, 33, 131–134 alternatively spliced isoforms, 131–132 expression in cell proliferation and differentiation, 133 hormonal regulation of, 132 as marker for serous carcinoma of ovary, 134 possible neuroprotective property, 133 as potential tumour marker, 133–134 regulation of calretinin expression, 132–133 Calsequestrin, 30, 53 Caltropin, 200 Calumenin, 149 CaM, see Calmodulin family, of calcium binding proteins CAMPKs, see Calcium/calmodulindependent protein kinases cAMP response element binding protein (CREB), 23 cAMP response elements (CREs), 25 Cancer, see also specific types -associated retinopathy (CAR), 83 caveolin expression in, 28 gelsolin in, 42 involvement of fimbrin in, 100 markers, experimental, 233 prognosis, relationship between EGFr expression and, 232 progression osteonectin and, 196 potential role of thymosins in, 95 relation of actinin to, 108

352 S100A4 expression as marker of, 244 S100P in, 241 Capase-9, cytochrome c-mediated activation of, 170 Capping protein (CP), 97 CAR, see Cancer-associated retinopathy Carboxyamido-triazole (CAI), 9 Caspases, in apoptosis, cell migration, proliferation, and neoplasia, 169–179 caspase-mediated apoptosis and cell growth inhibition in tumour expansion, 173–176 caspase-mediated proteolysis of fodrin, 176–177 caspases in apoptotic cell death, 169–172 caspases and neuronal loss in Alzheimer’s disease, 177–179 poly (ADP-ribose) polymerase as marker of apoptosis, 172–173 Caveolin, 28, 98 CBD, see Calbindin D-28K CBD-9K, see Calbindin D-9K CBPs, see Calcium-binding proteins Cell–substratum adhesion, 58 Central nervous system (CNS), 1 Centrins, 145–147 cGMP, see Cyclic guanine monophosphate CH, see Calponin homology Chemoattractants, 210 Chinese hamster ovary (CHO) cell line, 161 Chlamydomonas, 145, 146 CHO cell line, see Chinese hamster ovary cell line Clotrimazole, 78 CNS, see Central nervous system Cofilin, 88 Collagen, binding of osteonectin to, 191 Colon cancer metastatic, 163 model, 43 Colorectal neoplasms, S100A4 expression in, 243 Contractile proteins, structure of, 87–124 actin component of contractile machinery of cell, 87–93 actin isoforms, 87–88

Calcium Signalling in Cancer cofilin in regulation of actin dynamics, 88–90 interaction of formin with profilin and Rhp GTPases, 92–93 profilin in regulation of actin dynamics, 90 regulation of actin dynamics, 88 Rho GTPases in actin dynamics and signal transduction, 90–92 fimbrin family of actin-binding proteins, 96–110 α-actinin, 102 α-actinin isoforms, 103 actinins in cell adhesion, motility, and signal transduction, 104 cadherin–catenin complex in signal transduction and cell adhesion, 104–110 function of α-actinin, 104 function of fimbrin in cytoskeletal organisation, 97–99 involvement of fimbrin in cancer, 100–101 modulation of actin dynamics and cancer cell dissemination, 101–102 molecular features of fimbrin, 96–97 molecular structure of α-actinin, 102–103 regulation of fimbrin expression, 99–100 myosin filaments, 110–117 actomyosin assembly, 112–115 myosin heavy chain isoforms, 111–112 myosin light chain phosphorylation and function, 115–117 role of thymosin family actin-binding proteins in actin dynamics, 93–96 effects of thymosins on cell proliferation, 93–94 expression of thymosins in embryonic development, 94–95 potential role of thymosins in cancer progression, 95–96 sequestration of actin by thymosins, 93 thymosins and cell motility and differentiation, 94

353

Index troponins and tropomyosins in regulation of muscle cell contraction, 118–124 calponin, 122–124 caltropin-mediated reversal of myosinATPase inhibition by caldesmon and calponin, 124 regulatory role of caldesmon, 121–122 regulatory role of troponins and tropomyosins in muscle contraction, 118–119 tropomyosin isoforms in benign and malignant cells, 119–121 Corticosterone, 127 CP, see Capping protein CREB, see cAMP response element binding protein CREs, see cAMP response elements Crocalbin, 149 Cyclic AMP, in calcium signalling, 21 Cyclic guanine monophosphate (cGMP), 16 Cyclosporin, 141 Cytochalasin D, 18, 211 Cytokinesis, 93, 217 Cytosolic fatty acids, 237

D DAG, see1,2-Diacylglycerol Darier’s disease (DD), 7 DD, see Darier’s disease Demyelination process, autoimmunemediated, 164 Dexamethasone, 18, 172 -inducible promoter, 206 S100A4 transfectants exposed to, 226 1,2-Diacylglycerol (DAG), 13, 14, 32 Dictyostelium discoideum, 22, 44, 96, 102 Dimethyl sulphoxide (DMSO), 19 DMD, see Duchenne muscular dystrophy DMSO, see Dimethyl sulphoxide DNA binding of transcription factors to, 67 damage, 72 fragmentation, 170, 174 hypermethylation, 73 methylation, in cancer, 72 Drosophila melanogaster, 42, 102, 150, 231 Drug–immunophilin complex, 141

Duchenne muscular dystrophy (DMD), 165

E EAE, see Experimental allergic encephalomyelitis EC, see Endothelial cell Ecdysone, 18 ECM, see Extracellular matrix EDC, see Epidermal differentiation complex EFABP, see Epidermal-type fatty acidbinding protein EF-hand calcium binding proteins, 63–74 alternatively spliced variants of S100A4, 68–69 calcium binding and molecular configuration of calcium binding proteins, 65–68 functional significance of alternatively spliced isoforms, 69–70 molecular organisation, 63–64 regulation of expression of S100 family genes, 71–74 DNA methylation in cancer, 72–73 regulation of gene expression by DNA methylation, 72 regulation of S100 gene transcription by methylation, 74 transcriptional regulation of S100 genes, 71 structure and organisation of S100 family genes, 68 EGF, see Epidermal growth factor EGFr, see Epidermal growth factor receptors Embryonic stem (ES) cells, 51 Endoplasmic reticulum, 5, 46 Endothelial cell (EC), 27 Endothelial nitric oxide synthase (eNOS), 26 Enhancer element, 74 eNOS, see Endothelial nitric oxide synthase Enzymes, activation of target, 75 Epidermal differentiation complex (EDC), 202 Epidermal growth factor (EGF), 15, 224

354 Epidermal growth factor receptors (EGFr), 76–77 Epidermal-type fatty acid-binding protein (EFABP), 237 Epithelial–mesenchymal transformation, 214, 226 ERK, see Extracellular signal-related receptor kinases ERs, see Oestrogen receptors ES cells, see Embryonic stem cells Experimental allergic encephalomyelitis (EAE), 164 Extracellular matrix (ECM), 186 protein synthesis, 197 remodelling, 214 Extracellular signal-related receptor kinases (ERK), 77 Ezrin, 108

F FH, see Formin homology Fibroblast(s) annexin in human foreskin, 37 -specific protein 1 (FSP1), 214 Fibronectin (FN), 57, 60 Fimbrins, 96, 100 FK506, 141 FLG, see Profilaggrin FN, see Fibronectin Fodrin calpain-specific degradation of, 164 influence of cell adhesion and migration by, 176 Follicle-stimulating hormone (FSH), 25 Follistatin, 183 Formin homology (FH), 92 FSH, see Follicle-stimulating hormone FSP1, see Fibroblast-specific protein 1

G GAP, see Guanosine triphosphatase activating proteins Gastrin-releasing peptide (GRP), 19 GDNF, see Glial cell-derived neurotropic factor GDP, see Guanosine diphosphate Gelation factor, 109 Gelsolin, 30, 40, 43

Calcium Signalling in Cancer in cancer, 42 expression, in amyloidosis, 42 Gene regulation, modes of, 71 therapy, with cytotoxic drugs, 62 Genetic activation, signal transduction and, 2 Genistein, 104 GFAP, see Glial fibrillary acidic protein Ginkgo biloba L., 46 GI tract, osteonection immunoreactivity in invasive tumours of, 193 Glial cell-derived neurotropic factor (GDNF), 132 Glial fibrillary acidic protein (GFAP), 207 Glutamate, 114, 206 Glycoprotein(s) adhesion mediating, 59 transmembrane, 246 G-protein -coupled receptors, 26 signalling pathway, 79 Growth hormone, 127 GRP, see Gastrin-releasing peptide GTP, see Guanosine triphosphate GTPase, see Guanosine triphosphatase Guanosine diphosphate (GDP), 80 Guanosine triphosphatase (GTPase), 80 Guanosine triphosphatase activating proteins (GAP), 80 Guanosine triphosphate (GTP), 77 Guanylate cyclase, 32, 81, 85

H Hailey–Hailey disease, 8 Heat shock protein (HSP), 84, 138, 216, 217 Heme oxygenase (HO), 229 HIV-1, see Human immunodeficiency virus HO, see Heme oxygenase HPV, see Human papilloma virus HSP, see Heat shock protein Human immunodeficiency virus (HIV-1), 137 Human papilloma virus (HPV), 156 Human umbilical vein endothelial cells (HUVEC), 175 HUVEC, see Human umbilical vein endothelial cells

355

Index Hyaluronic acid, 211 Hydra vulgaris, 35 Hyperthermia, 220, 221, 229 Hypomethylation, 72

I Ibandronate treatment, of metastatic breast cancer, 61 ICE, see Interleukin-1β-converting enzymes IF, see Intermediate filaments IGF, see Insulin-like growth factor Inflammatory diseases, S100A8 and S100A9 proteins in, 239 Inositol phosphates, in calcium signal transduction, 16 Insulin-like growth factor (IGF), 49 Integrin receptor, cell surface, 210 Interblastomere adhesion, 107 Interleukin, 229 Interleukin-1β-converting enzymes (ICE), 169 Intermediate filaments (IF), 87 Intracellular adhesion, 58 Invasion suppressor gene, 59

J Jurkat T lymphocytes, 171

K Kallikrein-1, 242 Keratin filament aggregation, 32 Keratinocyte differentiation, profilaggrin in, 202 Keutel syndrome, 55 Kidney, osteonectin immunoreactivity in invasive tumours of, 193

L LAK cells, see Lymphokine-activated killer cells Laminin, cooperative functioning of in osteoblast migration, 59 Leiomyosarcomas, expression of calponin in, 124 Leischmania donovani RNA, 47 LGMD, see Limb girdle muscular

dystrophy Limb girdle muscular dystrophy (LGMD), 28, 165, 166, 167 Liriodendron tulipifera L., 46 LOH, see Loss of heterozygosity Long terminal repeats (LTR), 137 Loss of heterozygosity (LOH), 12 LTR, see Long terminal repeats Lung, osteonection immunoreactivity in invasive tumours of, 193 Lymphocyte proliferation, inhibition of, 143 Lymphokine-activated killer (LAK) cells, 98 Lymphoma, B-cell diffuse large cell, 175

M Madin–Darby bovine kidney (MDBK), 127 Major histocompatibility complex genes, 25 Mammalian calpains, 153 Matrilysin promoter, 109 Matrix metalloproteinases, 10, 187 5-MC, see 5-Methylcytosine MCBK, see Madin–Darby bovine kidney Mechanoenzymes, 110 Melanocyte-stimulating hormone (MSH), 22 Melanomas, 234, 243 Merlin, 108 Mesotheliomas, cadherin and calretinin as marker for, 134 Metalloproteinases, tumour-associated, 195, 213 Metastasis gene concept, 199, 241 Metastatic dissemination, osteotropism of, 60 N-Methyl-D-aspartate (NMDA), 135, 206 5-Methylcytosine (5-MC), 72 N-Methyl-N-nitrosourea (NMU), 236 MHC, see Myosin heavy chain Microtubules, 91, 217 MLC, see Myosin light chain MLCK, see Myosin light chain kinase MMTV, see Murine mammary tumour virus Moesin, 108 Monocytes, transendothelial migration of, 239 Mooren’s ulcer, 200 mRNA(s)

356 expression, up-regulation of calcineurin, 143 ICE, 174 tropomyosin, 119 MS, see Multiple sclerosis MSH, see Melanocyte-stimulating hormone Multiple sclerosis (MS), 164 Murine mammary tumour virus (MMTV), 219 Muscular dystrophy, calpains in, 165 Myelin basic protein, 163 Myelodegenerative diseases, 163 Myocardial infarction, 222 Myosin association with cytoskeleton, 22 -ATPase enzyme, 240 heavy chain (MHC), 110, 111 light chain (MLC), 63, 64, 110 kinase (MLCK), 76 phosphorylation, 115, 116, 117

N Naegleria gruberi, 145 NCAM, see Neural cell adhesion molecules NCBPs, see Neural calcium-binding proteins N-Desmethyltamoxifen, 79 NDP, see Nucleoside diphosphate Nebulin, 114 Nemaline myopathy, 115 Neovascularisation, 191 Nerve growth factor (NGF), 5 Neural calcium-binding proteins (NCBPs), 79 Neural cell adhesion molecules (NCAM), 206 Neurite extension factor, 206 Neurodegenerative disease, implication of calpains in, 158 Neurofibrillary tangles (NFTs), 140 Neurofibromatosis type 2 (NF2), 155 Neuronal calcium-binding protein, 131 Neurones, calcium homeostasis regulation in, 125 Neurotransmission, calcineurin and, 136 Neurotropin-3 (NT3), 126 NF2, see Neurofibromatosis type 2

Calcium Signalling in Cancer NFTs, see Neurofibrillary tangles NGF, see Nerve growth factor Nitric oxide inhibition of focal adhesion by, 27 synthase (NOS), 10, 27, 81 Nitrogen-activated protein kinase, 11 NMDA, see N-Methyl-D-aspartate NMU, see N-Methyl-N-nitrosourea Non-EF-hand calcium binding proteins, 30–31, 35–62 annexins, 35–40 annexins in cancer growth and progression, 38–39 annexins in morphogenesis and differentiation, 39–40 biological functions, 36–38 structure, 35–36 calreticulin and functional diversity, 46–53 calreticulin and calnexin as molecular chaperones, 49–50 calreticulin in cell adhesion, 50–51 calreticulin in cell proliferation and differentiation, 50 calreticulin in intracellular calcium storage, 48–49 calreticulin in neoplasia, 51–52 immunological implications of calreticulin function, 52–53 intracellular distribution of calreticulin, 48 phosphorylation of calreticulin, 47–48 regulation of calreticulin expression, 46–47 structure and molecular features of calreticulin, 46 calsequestrin and intracellular calcium storage, 53–54 gelsolin family of calcium-binding proteins, 40–45 gelsolin in cancer, 42–43 galsolin in embryonic development and morphogenesis, 41–42 gelsolin expression in amyloidosis, 42 gelsolin in severing and capping of actin filaments, 40–41 severin and cytoskeletal reorganisation, 44

Index villin in differentiation and neoplasia, 44–45 osteocalcin in bone metabolism and osteotropism of cancer, 54–62 biology of osteocalcin, 54–55 calcium-binding properties of osteocalcin, 55 osteocalcin in cell proliferation and differentiation, 57–60 osteocalcin gene structure and function, 55–56 osteotropism of metastatic dissemination, 60–62 regulation of osteocalcin by vitamin D3, 56 Non-small cell lung carcinoma (NSCLC), 198 NOS, see Nitric oxide synthase NSCLC, see Non-small cell lung carcinoma Nt3, see Neurotropin-3 Nucleoside diphosphate (NDP), 230 Nucleotide metabolism, 1

O OA, see Osteoarthritis Odontoblast differentiation, 185 17β-Oestradiol, 79 Oestrogen receptors (ERs), 79, 101, 159 OM, see Oncomodulin Oncogenes, overexpression of, 73 Oncogenic retroviral genes, 120 Oncomodulin (OM), 182 Open reading frame (ORF), 68, 150 ORF, see Open reading frame Ornithogalum virens, 122 OSE, see Osteonectin silencer element Osteoarthritis (OA), 196 Osteoblast migration, cooperative functioning of laminin and FN in, 59 Osteocalcin, 30, 35 in bone metabolism, 54 in cell proliferation and differentiation, 57 functions of, 60 gene transcription, 55 Osteoclasts, localisation of fimbrin in, 97

357 Osteonectin, 184, 247 cancer progression and, 196 expression, correlation between angiogenesis and modulation of, 192 immunoreactivity, 193 inhibition of cell spreading by, 190 molecule, functional domains of, 185 silencer element (OSE), 55 Osteonectin, in cell function and behaviour, 183–198 effects of osteonectin on angiogenesis, 191–192 functions and functional domains of osteonectin, 184 modulation of cell proliferation by osteonectin, 190–191 modulation of cellular adhesion, cell shape, and motility by osteonectin, 188–190 molecular structure of osteonectin, 183–184 osteonectin in embryonic development and differentiation, 187–188 osteonectin expression in cancer development and progression, 193–196 osteonetcin homologues and putative tumour suppressor properties, 197–198 osteonetcin involvement in other disease states, 196–197 osteonetcin in remodelling of extracellular matrix, 186–187 regulation of osteonectin expression, 184–186 Osteopontin-transfectant cells, 194 OT, see Oxytocin Ovariectomy, expression of CBD gene transcripts and, 128 Ovary calretinin as marker for serous carcinoma of, 134 carcinomas, nm23-H1 in epithelial, 231 osteonection immunoreactivity in invasive tumours of, 193 Oxytocin (OT), 132

358

P PA, see Plasminogen activator Paired helical filaments (PHF), 140 Paracentrotus lividus, 216 PARP, see Poly (ADP-ribose) polymerase Parvalbumin (PV), 64, 125, 181–182 Paxillin, 162 PCNA, see Proliferating cell nuclear antigen PDGF, see Platelet-derived growth factor PGE, see Prostaglandin E PgR, see Progesterone receptors Phenylephrine, 123 PHF, see Paired helical filaments Phorbol 12-myristate 13-acetate (PMA), 99 Phosphatidylinositol (PI), 10 Phosphoglucomutase, 208 Phosphoinositide-3 kinase (PI3K), 11 Phospholipase C (PLC), 10 Phospholipid signalling, 10 Phototransduction, 81 Physarum polycephalum, 40 PI, see Phosphatidylinositol PI3K, see Phosphoinositide-3 kinase PKA, see Protein kinase A PKC, see Protein kinase C Plasma membrane Ca2+-ATPase extrusion pump (PMCA), 5 Plasminogen activator (PA), 186 Platelet -derived growth factor (PDGF), 18 integrin, 178 PLC, see Phospholipase C PMA, see Phorbol 12-myristate 13-acetate PMCA, see Plasma membrane Ca2+-ATPase extrusion pump PMN, see Polymorphonuclear leukocytes Poly (ADP-ribose) polymerase (PARP), 171, 172 Polymorphonuclear leukocytes (PMN), 99, 115 Profilaggrin (FLG), 199, 201 abnormal expression of, 204 genomic and molecular features of, 203 in keratinocyte differentiation, 202 monomers, 204 Profilin, 90, 92 Progesterone receptors (PgR), 101 Programmed cell death, 159

Calcium Signalling in Cancer Proliferating cell nuclear antigen (PCNA), 38 Proline, 114 Prostaglandin, 229, 230 Prostaglandin E (PGE), 25 Prostate adenocarcinoma cells, apoptosis of by calpain inhibitors, 159 cancer cell lines, protection of, 52 carcinomas, Gleeson grade in, 95 ductal carcinoma of, 98 Proteases, activation of by calcium signalling, 159 Proteasome, 166 Protein(s) actin-binding, 88, 92, 93, 176 adenomatous polyposis coli, 104 adhesion-mediating, 94 amyloid precursor, 178, 179 β-amyloid precursor, 141 armadillo, 105 CaM binding domains of target, 75 capping, 97 EF-hand, 67 folding, 84 glial fibrillary acidic, 207 guanosine triphosphatase activating, 80 guanylate cyclase-activating, 85 heat shock, 84, 216 intracellular, 36 linking plasma membrane with actin cytoskeleton, 108 mobilisation, requirement for, 166 myelin basic, 163 myofibrillar, 165 ras family, 26 receptor, 22 retroviral, 68 Rho, 107 S100, 214, 217 stress-induced, 138 tau, 179 Proteinases, 10 Protein kinase A (PKA), 13, 99 Protein kinase C (PKC), 13, 14 -mediated regulation, of annexin function, 36 pathway, 13 Prothoracicotropic hormone (PTTH), 18

359

Index Psoriasin, 200, 236 PTEN abnormalities, 12 germline mutations, 12 overexpression, 13 PTTH, see Prothoracicotropic hormone PV, see Parvalbumin

R RA, see Rheumatoid arthritis Radixin, 108 Rana rugosa, 46 Rapamycin, 141 Ras family proteins, 26 RBP, see Retinol-binding protein RCN, see Recoverin Receptor proteins, 22 tyrosine kinases (RTKs), 26 Recoverin (RCN), 79, 80 cancer-associated retinopathy and, 83 mode of action of, 82 –rhodopsin kinase complex, 83 Repetin, 201, 203 Reticulocalbin family, of EF-hand proteins, 149–151 molecular features of reticulocalbin homologues, 150 putative functions of reticulocalbin and homologues, 150–151 Retinitis pigmentosa (RP), 81, 85 Retinoblastoma gene product, 17 susceptibility regulatory element, 50 Retinoic acid, 60, 209 Retinoid X receptor (RXR), 56 Retinol-binding protein (RBP), 21 Retroviral proteins, 68 Reverse transcriptase polymerase chain reaction (RT-PCR), 69 Rheumatoid arthritis (RA), 84, 196, 204 Rhodopsin kinase, 81 Rho protein, 107 Rod domain, 103 RP, see Retinitis pigmentosa RTKs, see Receptor tyrosine kinases RT-PCR, see Reverse transcriptase polymerase chain reaction Rubella virus (RV), 47

RV, see Rubella virus RXR, see Retinoid X receptor Ryanodine receptor (RyR), 20, 53 RyR, see Ryanodine receptor

S S100 family genes, structure and organisation of, 68 S100 proteins, 199–244 biological properties of S100A7, 236–239 S100A7 in neoplastic disease, 238–239 S100A7 in skin pathology, 237–238 structure and molecular properties of S100A7, 236–237 cell cycle-related expression of, 217 effects of S100 proteins on cell deformity and cellular morphology, 205–222 cell adhesion and invasive potential of cancer cells, 210–212 cell cycle-related expression of S100 proteins, 217–219 postulated mechanism of cell cycle control by S100A4, 219–222 S100 proteins in cell proliferation, 214–217 S100 proteins in remodelling of extracellular matrix, 213–214 potential value of S100 proteins as markers of cancer progression and prognosis, 243–244 S100 proteins in cell differentiation, motility, and cancer invasion, 202–205 profilaggrin in keratinocyte differentiation, 202–205 trichohyalin, 205 S100A isoforms, 222 S100A2 as putative tumour suppressor, 223–224 S100A3 expression in cell differentiation and neoplasia, 224–225 molecular features of S100A3, 224–225 S100A3 expression in cell differentiation and human gliomas, 225 S100A4 in cancer development and

360 progression, 225–235 clinical potential of S100A4 as marker for cancer prognosis, 230 S100A4 expression and metastatic potential of cancers, 225–230 S100A4 in human breast cancer, 230–234 S100A4 in other forms of human cancer, 234–235 S100A6 in cancer, 235–236 S100A8 and S100A9 proteins in inflammatory diseases, 239 S100A11 and possible modes of function, 239–240 S100P in cancer progression, 241–243 Saccharomyces cerevisiae, 38, 90, 97, 145 pombe, 136 Sarcoplasmic–endoplasmic reticulum Ca2+–ATPase pump (SERCA), 5 SCCA, see Squamous cell lung carcinoma antigen Schizossaccharomyces pombe, 112 Sciatic nerve regeneration, stimulation of by S100B, 215 SCLC, see Small cell lung carcinoma SDKs, see Sphingosine-dependent kinases Secreted protein acidic, rich in cysteine (SPARC), 183 SERCA, see Sarcoplasmic–endoplasmic reticulum Ca2+–ATPase pump Serine protease, kallikrein-like, 242 /threonine kinase, 11 Severin, 30 Shigella flexneri, 100, 109 Signal transduction genetic activation and, 2 machinery, architectural aspects of, 25 Skin neoplasms, 7 pathology, S100A7 in, 237 tumours, trichohyalin expression in, 205 SLE, see Systemic lupus erythematosus Small cell lung carcinoma (SCLC), 83 Smith–Magenis syndrome (SMS), 1, 41 SMS, see Smith–Magenis syndrome SPARC, see Secreted protein acidic, rich in cysteine

Calcium Signalling in Cancer Sphingosine-dependent kinases (SDKs), 47–48 Squamous cell carcinoma, bladder, 238 Squamous cell lung carcinoma antigen (SCCA), 162 Stathmin expression, up-regulation of, 221 promoter, down-regulation of, 220 Steroid(s) calbindin expression and, 129 hormone, 18 Systemic lupus erythematosus (SLE), 52, 144

T Talin, 51 Tau protein, 179 T-cell antigen receptor (TCR), 49, 141 signal transduction cascade, in SLE, 144 TCR, see T-cell antigen receptor Testicular hormones, 101 Thapsigargin, 172 THH, see Trichohyalin Thioridazine, 78 Threonine phosphatase, 137 Thrombospondin, 192 Thymidine kinase (TK), 55 Thymosins effects of on cell proliferation, 93 potential role of in cancer progression, 95 Thyroid carcinoma, human anaplastic, 107 hormones, 57, 67 Tissue plasminogen activator (tPA), 186 Titin, 114 TK, see Thymidine kinase T-lymphocytes, IP3R1-deficient, 17 TNF, see Tumour necrosis factor Topoisomerase II, 150 tPA, see Tissue plasminogen activator Transcription factors, activator of, 156 Transfection studies, 11 Trichohyalin (THH), 201 expression, in skin tumours, 205 genomic and molecular features of, 203 Triticum aestivum, 96

361

Index Tropolyosin, down-regulation of, 42 Tropomyosins, 118 Troponin, 114, 118 Troponin C, 63 Tubulin monomers, 208 Tumour necrosis factor (TNF), 24 progression, expression of gelsolin and severin in, 44 suppressor gene, 73 S100A2 a putative, 223 Tyrosine kinase inhibitors, 137

U UMUC-2, human bladder cancer cell line, 43 Urokinase-type plasminogen activator, 10

V Valine, 114 Vascular cell adhesion molecule (VCAM), 139 Vascular endothelial growth factor (VEGF), 27, 28 Vasodilator-stimulated phosphoprotein (VASP), 101 Vasopressin (VP), 132 VASP, see Vasodilator-stimulated phosphoprotein VCAM, see Vascular cell adhesion molecule VD3, see Vitamin D3 VDR, see VD3 receptor

VDRE, see VD response element VD3 receptor (VDR), 56 VD response element (VDRE), 56 VEGF, see Vascular endothelial growth factor Verapamil, 9, 10, 22, 23 VGCCs, see Voltage-gated calcium channels Villin, in differentiation and neoplasia, 44 Vinculin, 51 Vitamin D3 (VD3), 56 Vitamin D3 receptor, 67 Voltage-gated calcium channels (VGCCs), 6, 8 VP, see Vasopressin

W WAS, see Wiskott-Aldrich syndrome WASP, see Wiskott-Aldrich syndrome protein WHO, see World Health Organization Wiskott-Aldrich syndrome (WAS), 91 gene mutation, 92 protein (WASP), 91 World Health Organization (WHO), 235

Y Yeast budding process, 92

Z Zinc finger transcription factors, 225