Mechanisms of carcinogenesis 9780898389913, 0898389917

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MECHANISMS OF CARCINOGENESIS

Cancer Growth and Progression SERIES EDITOR: HANS E. KAISER Department of Pathology, University of Maryland, Baltimore, Md, U.S.A. Scientific Advisors: Kenneth W. Brunson / Harvey A. Gilbert / Ronald H. Goldfarb / Alfred L. Goldson / Elizier Gorelik / Anton Gregl / Ronald B. Herberman / James F. Holland / Ernst H. Krokowski t / Arthur S. Levine / Annabel G. Liebelt / Lance A. Liotta / Seoras D. Morrison / Takao Ohnuma / Richard L. Schilsky / Harold L. Stewart / Jerome A. Urban / Elizabeth K. Weisburger / Paul V. Woolley Volume 1:

Fundamental Aspects of Cancer Volume Editor: Ronald H. Goldfarb ISBN 0-89838-990-9

Volume 2:

Mechanisms of Carcinogenesis Volume Editor: Elizabeth K. Weisburger ISBN 0-89838-991-7

Volume 3:

Influence of Tumor Development on the Host Volume Editor: Lance A. Liotta ISBN 0-89838-992-5

Volume 4:

Influence of the Host on Tumor Development Volume Editor: Ronald B. Herberman ISBN 0-89838-993-3

Volume 5:

Comparative Aspects of Tumor Development Volume Editor: Hans E. Kaiser ISBN 0-89838-994-1

Volume 6:

Etiology of Cancer in Man Volume Editor: Arthur S. Levine ISBN 0-89838-995-X

Volume 7:

Local Invasion and Spread of Cancer Volume Editor: Kenneth W. Brunson ISBN 0-89838-996-8

Volume 8:

Metastasis / Dissemination Volume Editor: Elizier L. Gorelik ISBN 0-89838-997-6

Volume 9:

Cancer Management in Man: Detection, Diagnosis, Surgery, Radiology, Chronobiology, Endocrine Therapy Volume Editor: Alfred L. Goldson ISBN 0-89838-998-4

Volume 10:

Cancer Management in Man: Biological Response Modifiers, Chemotherapy, Antibiotics, Hyperthermia, Supporting Measures Volume Editor: Paul V. Woolley ISBN 0-89838-999-2

Complete set: ISBN 0-89838-989-5

Mechanisms of Carcinogenesis Edited by ELIZABETH K. WEISBURGER National Cancer Institute, National Institutes of Health, Bethesda, Md., U.S.A.

Kluwer Academic Publishers DORDRECHT / BOSTON / LONDON

Library of Congress Cataloging in Publication Data Mechanisms of carcinogenesis.

(Cancer growth and progression; v. 2) Includes index. 1. Carcinogenesis. I. Weisburger, Elizabeth K. II. Series. [DNLM: 1. Carcinogens. 2. Carcinogens, Environmental. 3. Neoplasms--etiology.

QZ 200 C2151518 v.2] RC268.5.M425 1988

616.99'4071

ISBN-13: 978-94-010-7641-8

87-24664

e-ISBN-13: 978-94-009-2526-7

DOl: 10.1 007/978-94-009-2526-7

Published by Kluwer Academic Publishers, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. Kluwer Academic Publishers incorporates the publishing programmes of Martinus Nijhoff, Dr W. Junk, D. Reidel, and MTP Press. Sold and distributed in the U.S.A. and Canada by Kluwer Academic Publishers, 101 Philip Drive, Norwell, MA 02061, U.S.A. In all other countries, sold and distributed by Kluwer Academic Publishers Group, P.O. Box 322, 3300 AH Dordrecht, The Netherlands.

Cover design by Jos Vrolijk. All rights reserved © 1989 by Kluwer Academic Publishers Softcover reprint of the hardcover 1st edition 1989 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission from the copyright owners.

TABLE OF CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . List of contributors . . . . . . . . . . . . . . . . . . .

VII IX

1. The influence of human behavior on neoplastic progression H.E. KAISER . . . . . . . . . . . . . . . . . 2. The influence of stressors on the progression of neoplastic change H. ANISMAN, J. IRWIN and L.S. SKLAR . . . . . . .

7

3. The seven types of causes of neoplastic growth - an organismic view H.E. KAISER . . . . . . . . . . . . . . . . . . . . . .

19

4. Chemoprevention research W.F. MALONE . .

31

5. Premalignant and non-invasive lesions of the urinary bladder S.L. JOHANSSON and S.M. COHEN. . . . . . .

43

6. Species-specific aspects of the carcinogenicity of chloroform E.K. WEISBURGER . . . . . . . . . . . . . .

51

7. Carcinogenic effects of ionizing radiation A.c. UPTON . . . . . . . . .

54

8. Selected aspects of viral carcinogenesis P.S. SARIN . . . . . . . . .

71

9. The quantum theory of carcinogenesis - a short review J. LADIK and P. OTTO . . . . . . . . . . .

84

10. Cellular oncogene activation by chromosomal translocation K.R. HAREWOOD. . . . . . . . . . . . . . .

87

11. Amplification of the oncogene N-Myc as a correlate to advanced stage in human neuroblastomas M. SCHWAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

92

12. Potential roles of activated proto-oncogenes in malignant progression G.E. GALLICK . . . . . . . . . . . . . . . . . . . .

98

13. Oncogenes and their encoded products as targets for cancer therapy D.L. SLATE . . . . . . . . . . . . . . . . . . . . . .

106

14. Methods to study RAS oncogene-mediated induction of the metastatic phenotype UP. THORGEIRSSON, T. TURPEENNIEMI-HUJANEN, M. BALLIN and L.A. LIOTTA. 15. Oncogene products as potential therapeutic targets for control of established metastatic disease R.H. GOLDFARB and K.W. BRUNSON . . . . . . . . . . . . . . . . . . . . . . .

113 119

16. Neoplastic cell stages and progression in experimental hepatocarcinogenesis E. SCHERER . . . . . . . . . . . . . . . . . . . . . . . .

128

17. Characterization of eleven important transplantable murine tumors from the standpoint of morphology, pyrimidine biosynthesis and responsiveness to pyrimidine antimetabolites T.W. KENSLER, H.M. SCHULLER, H.N. JAYARAM and D.A. COONEY. . . . . . . . . . .

145

18. Use of organ explant and cell culture in cancer research J.H. RESAU and J.R. COTTRELL . . . . . . . . . . . . . . . . . . .

157

v

VI

Contents

19. Season of operation and differential long-term progression: clinical, statistical, and psychological issues E.H. KROKOWSKI, H.w. WENDT and P.A. HOMYAK. . . . . . . . . . . . .

160

20. Endogenous factors affecting the progression of carcinogen-induced rat mammary carcinomas C.W. WELSCH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169

21. Age factors in the presence of selected neoplasms H.E. KAISER . . . . . . . . . . . . .

179

22. Extremely low frequency electromagnetic fields as possible promotors of carcinogenesis N. WERTHEIMER . . . . . . . . . . . . . . . . . . . . . . . . . . . 23. Medical geography and neoplasms J.P. FULTON . . . . . Index of subjects . . . . . . . . .

188

191 194

INTRODUCTION

but also the possibility of intervention in specific stages. In addition, variables which affect cancer development as well as some endogenous factors can be better delineated through such investigations. The topics of this volume encompass premalignant noninvasive lesions, species-specific aspects of carcinogenicity, radiation, viruses, a quantum theory of carinogenesis, oncogenes, and selected environmental carcinogens.

Human behavior, including stress and other factors, plays an important role in neoplasia, although too little is known on the reasons for such development. Carcinogens, which help initiate the neoplastic process, may be either synthetic or naturally-occurring. Cancer causation may be ascribed to certain chemicals, physical agents, radioactive materials, viruses, parasites, the genetic make-up of the organism, and bacteria. Humans, eumetazoan animals and vascular plants are susceptible to the first six groups of cancer causes, whereas the last group, bacteria, seems to affect only vascular plants. Neoplastic development may begin with impairment ofJmdy defenses by a toxic material (carcinogen) which acts as an initiator, followed by promotion and progression to an overt neoplastic state. Investigation of these processes allows not only a better insight into the mechanism of action

Series Editor Hans E. Kaiser

vii

Volume Editor Elizabeth K. Weisburger

ACKNOWLEDGEMENT

Inspiration and encouragement for this wide ranging project on cancer distribution and dissemination from a comparative biological and clinical point of view, was given by my late friend E. H. Krokowski. Those engaged on the project included 252 scientists, listed as contributors, volume editors and scientific advisors, and a dedicated staff. Special assistence was furnished by J. P. Dickson, J. A. Feulner, and I. Theloe. 1. Bauer, D. L. Fisher, S. Fleishman, K. Joshi, A. M. Lewis, J. Taylor and K. E. Yinug have provided additional assistence. The firm support of the publisher, especially B. F. Commandeur, is deeply appreciated. The support of the University of Maryland throughout the preparation of the series is acknowledged. To the completion of this undertaking my wife, Charlotte Kaiser, has devoted her unslagging energy and invaluable support.

CONTRIBUTORS

Gary E. GALLICK, Ph.D. Department of Tumor Biology Virology Section Box 79 University of Texas M.D. Anderson Hospital and Tumor Institute 6723 Bertner Ave. Houston Texas 77030, USA

Hymnie ANISMAN, Ph.D. Department of Psychology Carleton University Ottawa, Ontario, KLS 5B6 Canada Marina BALLIN, PhD. Laboratory of Pathology National Cancer Institute/ National Institutes of Health Bethesda Maryland 20892, USA

Ronald H. GOLDFARB, Ph.D. Pittsburgh Cancer Institute 230 Lothrop St. Pittsburgh Pennsylvania 15213-2592 USA and Department of Pathology University of Pittsburg School of Medicine

K.W. BRUNSON, Ph.D. Department of Immunology & Infectious Diseases Pfizer Central Research Eastern Point Road Croton Connecticut 06340, USA

K.R. HAREWOOD, Ph.D. Department of Immunology and Infectious Diseases Pfizer Central Research Eastern Point Road Croton Connecticut 06340, USA

Samuel M. COHEN, M.D., Ph.D. Department of Pathology and Microbiology University of Nebraska Medical Center 42nd and Dewey Avenue Omaha Nebraska 68105, USA

P.A. HOMYAK, MHA Department of Psychology MacAlester College 1600 Grand Ave. St. Paul Minnesota 55105, USA

David A. COONEY, MD. Laboratory of Medicinal Chemistry and Pharmacology National Cancer Institute/ National Institutes of Bethesda Maryland 20892, USA

J. IRWIN, Ph.D. Department of Psychology Queens University Kingston, Ontario K7L3N6 Canada

J.R. COTTRELL, M.S. Department of Pathology School of Medicine University of Maryland 10 S. Pine Street Baltimore Maryland 21201, USA

Hiremagalur N. JAY ARAM Division of Cancer Treatment National Institutes of Health Bethesda Maryland 20892, USA

John P. FULTON, Ph.D. Rhode Island Cancer Registry Rhode Island Department of Health Office of Health Statistics Cannon Building - 75 Davis Street Providence, RI 02908-509 and Department of Community Health Brown University Providence Rhode Island 02902, USA

Sonny L. JOHANSSON, M.D., Ph.D. Department of Pathology and Microbiology University of Nebraska Medical Center 42nd and Dewey Avenue Omaha Nebraska 68105, USA ix

X

Contributors

Hans E. KAISER, D.Sc. Department of Pathology School of Medicine University of Maryland 10 S. Pine Street Baltimore Maryland, 21201, USA

Manfred SCHWAB, D.Sc. Department of Experimental Pathology DKFZ 1m Neuenheimer Feld 280 6900 Heidelberg I FRG

Thomas W. KENSLER, Dr. Department of Toxicology The Johns Hopkins School of Public Health The Johns Hopkins University Baltimore Maryland 21218, USA

L.S. SKLAR, M.D., Ph.D. Department of Psychology University of Toronto School of Medicine Toronto, Ontario, Canada

E.H. KROKOWSKI, M.D., Ph.D.,* Central Radiological Institute with Radiologic Hospital Municipal Hospitals of Kassel Moenchebergstr. 41/43 35 Kassel, FRG *deceased November 5, 1985

1. LADIK, Ph.D., Dr.math. h.c. Chair for Theoretical Chemistry University of Erlangen-Nuremberg 852 Erlangen Egerlandstrass 3, FRG Lance A. LIOTTA, M.D., Ph.D. Department of Pathology National Cancer Institute/ National Institutes of Health Bethesda Maryland 20892, USA Winfred F. MALONE, Ph.D., M.P.H. National Cancer Institute National Institutes of Health Bethesda Maryland 20892, USA Peter OTTO, Ph.D. Chair for Theoretical Chemistry University Erlangen-Nuremberg 852 Erlangen Egerlandstr. 3, FRG

D.L. SLATE, Ph.D. Syntex Inc. Syntex Research R-I-215 3401 Hillview Avenue Palo Alfo California 94303, USA Unnur P. THORGEIRSSON, M.D. Department of Pathology National Cancer Institute/ National Institutes of Health Bethesda Maryland 20892, USA Taina TURPEENNIEMI-HUJANEN, M.D., Ph.D. Department of Biochemistry University of Oulo Oulo Finland Arthur C. UPTON, M.D. Department of Environmental Medicine New York University Medical Center New York New York 10016, USA

James H. RESAU, Ph.D. Department of Pathology University of Maryland School of Medicine 10 S. Pine Street Baltimore Maryland 21201, USA

E.K. WEISBURGER, Ph.D. Chemical Carcinogenesis National Cancer Institute National Institutes of Health Bethesda Maryland 20892, USA

Prem S. SARIN, Ph.D. Laboratory of Tumor Cell Biology National Cancer Institure/ National Institutes of Health Bethesda Maryland 20892, USA

Clifford W. WELSCH, Ph.D. Department of Anatomy State University of Michigan East Lansing Michigan 48892, USA

E. SCHERER, M.D. Antoni van Leeuwenhoek Hospital at the Netherlands Cancer Institute Plasmanlaan 121 Amsterdam The Netherlands H.M. SCHULLER, DVM Laboratory of Experimental Therapeutic Metabolism National Cancer Institute/ National Institutes of Health Bethesda Maryland 20892, USA

H.W. WENDT, Ph.D. Department of Psychology MacA lester College 1600 Grand Ave. S!. Paul Minnesota 55105, USA Nancy WERTHEIMER, Ph.D. 1330 Fifth Street Boulder Colorado 80302, USA

1 THE INFLUENCE OF HUMAN BEHAVIOR ON NEOPLASTIC PROGRESSION H.E. KAISER

INTRODUCTION The incidence of common cancers in humans is determined by various potentially controllable external factors, because the incidence of cancer varies from region to region, depending on the habits, diet, and customs of people. Cancer is, to some extent, a preventable disease (4). Human behavior can change the enviroment and its connections with carcinogens. The mind can distinguish between its own actions, and the influence of the minds of others on behavior. The actions of people can interfer drastically with the initiation and progression of neoplasms in humans and other creatures. Eating fish from a lake contaminated with waste substances or carcinogens leading to their deposition in the fish may reflect on one's health in the long run, and produce cancer even though the fish itself appeared healthy. This may be an example of the influence of other's behavior on health. We know the different cancer-causing agents such as particular chemicals or certain types of radiation, viruses, genetic traits, etc. They do not act alone, and by their behavior people expose themselves to these agents. Other actions may initiate, promote or otherwise change the carcinogenic manifestation. In nature, a number of factors generally react together to produce a carcinogenic environment, or a carcinogenic chain reaction in which human beings are involved. In general, animal studies on possible carcinogens use much higher doses than are usually present in the human environment. Many parameters are involved in human cancer risk which are difficult to identify properly (6, 11, 18,20,23,34). Many sides of the issue must be considered if the evaluation is to be correct (35). EMOTIONAL ASPECTS Continuous repetition of the psychological impacts caused by excitement may lead to changes in body functions, thus producing a continuous stimulus. Chronic irritation leads to a neoplastic state of tissues. Continued aggravation and embarrassment can significantly contribute to the formation of stones and inflammation in the gallbladder, which if not treated, may lead to cancer in this organ. Other examples are known in the literature. STRESS Lasting stress can force hormonal reactions and other functions to go astray and to produce carcinogenic hazards. E. K. Weisburger (ed.), Mechanisms of carcinogenesis.

Certain cancers in the affected population, as are vascular diseases, are the result. For review and experimental evidence see Chapter 2, Vol. II. LIFELONG HABITS Lifelong habits can cause cancer. People in some regions carry little stoves under their clothes to keep warm but this leads to a constant burning and irritation of their abdominal skin with resultant development of cancer. ETHNIC ASPECTS The influence of ethnic aspects in human neoplastic progression in contrast to racial aspects can be seen in the differences of neoplasms in Japanese immigrants to the United States, compared with those living in Hawaii. As long as they maintained their native habits, especially those pertaining to food, the high rate of stomach cancer found in the Japanese persisted. When they adapted their diet to the US standards, the rate of stomach cancer fell to the rate of other US citizens. Stomach cancer in Japan is the most common cancer and in 1977 it was responsible for 45% of male and 20% of female cancer deaths. (9, 8). Among Koreans in Osaka, the mortality rate from liver cancer was about twice that of Japanese, and in the Koreans in Japan the mortality rate due to stomach cancer declined more rapidly than it has among Japanese (27). For several cancers, striking differences in mortality are found between single counties or clusters of county groups in adjacent areas of mainland China (Table 1) (22)). RELIGIOUS ASPECTS Religious aspects influencing the believers' lives include rituals, housing, food, sex and education. Nuns have a lower incidence of cancer of the cervix but a higher risk of breast cancer. Cervical cancer is low among Jewish women, probably due to circumcision of the males. Seventh-Day Adventists experience fewer cancers of several types such as squamous cell (epidermoid) lung cancer or esophageal and other cancers of the alimentary canal and its glands because they are not permitted to smoke or drink. Because of the psychologic impact of religion, a patient's belief may playa role during the terminal phase of the

© 1989, Kluwer Academic Publishers, Dordrecht. ISBN 978-94-010-7641-8

2

H.E. Kaiser

cancer patient's life, which is the last step of neoplastic progression (see Chapters 23jVIII and 19jX). IMMEDIATE SURROUNDINGS (ENVIRONMENT) The home itself may harbor carcinogenic hazards. Television screens of certain types might cause dangerous radiation; ultraviolet lamps for tanning the skin if used improperly may cause melanoma (see Chapter 7jII). Asbestos in the walls of older buildings can be inhaled, a process which may lead to mesothelioma. Some paints and solvents used in the home may contain carcinogenic substances. NUTRITION Nutrition is one of the most important behavioral activities of man with regard to his interference and production of carcinogenic dangers, as shown by many epidemiological investigations offood habits in different groups and parts of the world. The problem can be broken down into three aspects: (1) food as such, (2) preparation and preservation of food, (3) food contaminants. It is well-known that obesity is accompanied by a cancer risk, especially for colon cancer or breast cancer in women. On the other hand, a somewhat restricted food intake, measured in form of total calories, lowers the risk of cancer in people as well as in research animals and prolongs the life span. It is not known whether this is due to differences in the total caloric intake or to special classes of compounds contained in it. "Cancers of most major sites are influenced by dietary patterns - but in the free world at least, people eat and are not fed, they choose." (4). Preparation of food Two examples can be provided. The grilling of meat over a charcoal fire leads to polycyclic aromatic hydrocarbons in the food, and the cooking of proteinaceous foods (meat, fish, eggs) produces mutagenic heterocyclic amines which are also carcinogens. Preservation of food There are many preservatives, which were or still are in food. In this respect the laws of different countries vary widely. BHT and BHA, are allowed in the USA, nordihydroguaiaretic acid, etc. are not allowed in the USA, but allowed in the USSR and other countries. Food as such Total caloric intake is important because research on animals has shown that they live longer and develop fewer neoplasms when on a somewhat restricted diet. Obesity is unhealthy also in relation to the development of neoplasms. Lipids: Total fat and saturated fats have been correlated with the development of neoplasms, especially cancer of the

breast, prostate and large intestine. Polyunsaturated fats have a more carcinogenic effect than saturated fats. Fat cells convert to estrogen. Protein: The studies on protein are less numerous than those on fat. Protein intake, two or three times the normal amount, may enhance cancer, but too little is also unwise. Epidemiologic and laboratory studies seem to indicate that high protein intake may be connected with an increased risk of certain cancers. Carbohydrates. Studies with laboratory animals are even more restricted than those of the proteins, but one can assume that caloric excess due to carbohydrates may modify carcinogenesis. Dietary fiber. It cannot be said with assurance that the fiber content of the food has a cancer-inhibiting effect. Vitamins. Laboratory and epidemiological data suggest that foods rich in carotenes or vitamin A are associated with a reduced risk of cancer. Vitamin C. Based on several studies, vitamin C is able to inhibit the formation of some carcinogens and its consumption leads to a lower risk of cancers of the esophagus and stomach. Vitamin E. This vitamin inhibits the formation of nitrosamines in vivo and in vitro, perhaps also tumorigenesis by other chemicals. It is impossible to draw exact conclusions on the effects of human consumption. Minerals. Epidemiological and laboratory studies suggest that selenium may offer some protection against the risk of cancer. The studies concerning other minerals such as iron, copper, zinc, molybdenum, iodine, arsenic, cadmium and lead in food are inconclusive. The consumption of dark green and yellow vegetables and cruciferous vegetables such as cabbage, broccoli, cauliflower, and brussel sprouts is associated with inhibition of cancer. Naturally occurring carcinogens in food: hydrazines are in mushrooms, and mycotoxins including the potent carcinogen aflatoxin B2 occur in food, generally in very small amounts. Aflatoxin is produced by mold which grows on food. MS Masri (24) discussed various means of defense: interfacing of the farm animal as a biologic filter between contaminated crop (feed) and human food derived from animal products; chemical detoxification with ammonia or of contaminated milk by ultraviolet light; prevention of mold growth and aflatoxin elaboration by application of ammonium carbonate during storage and drying of agricultural products; development of ultrasensitive analytical methods for aflatoxins; and natural defenses related to the role of mammalian metabolism. There are any numbers of mutagens in food - grape juice, etc. Food additives. In the United States, nearly 3,000 substances may be used during processing of food. But they do not, as far as we know, contribute to the risk of cancer. Environmental contaminants. Organochlorine pesticides, polychlorinated biphenyls and polycyclic aromatic hydro-

1.' The influence of human behavior on neoplastic progresssion

carbons and industrial chemicals, as well as other contaminants, may also occur in food. Some have been carcinogenic in laboratory animals. Some of these compounds counteract the effect of each other in laboratory experiments. The aspects of food consumption and recommended diet, which may decrease the risk of cancer can be summarized as follows (4). A diet which would protect everyone from cancer is not realistic; but certain suggestions made by the National Research Council should be helpful: (1) high fat consumption is connected with an increased incidence of breast and colon cancer. The intake of calories by fat is now 40% in the US population and should not be more than 30%; (2) the diet should contain fruits (citrus fruits), vegetables (carotene rich and cruciferous) and whole - grain cereals; (3) esophageal and stomach cancers are increased in some countries such as China, Japan, and Iceland, where more salt-cured and smoked foods are consumed. The preparation of these foods seems to produce higher levels of polycyclic aromatic hydrocarbons and N-nitroso compounds; (4) certain non-nutritive constituents of foods, whether occurring naturally or introduced inadvertently (as contaminants) during production, processing and storage may pose a potential risk of cancer to humans. Even vitamin C can alter cells.

DRINKS: ALCOHOL CONSUMPTION-PROMOTERS IN SOFT DRINKS Excessive beer drinking has been associated with an increased risk of colorectal and especially rectal cancer. These findings could not be confirmed in other studies, however. The same holds true for excessive consumption of other alcoholic beverages - hepatic injury, cirrhosis and liver cancer as a chain reaction. Alcohol is the only fluid at least partially absorbed immediately in the mouth (oral) cavity. When combined with tobacco (drinking and smoking), a carcinogenic effect may result. When consumed in large quantities, alcoholic beverages appear to act synergistically with inhaled cigarette smoke to increase the risk of cancer in the mouth, larynx, esophagus, and the respiratory tract. Hard liquor has itself an irritating effect on such organs as the esophagus, stomach and liver. Other beverages as tea, cola drinks and coffee, contain phenols which may be cancer-promoting agents (14), but under some conditions they inhibit cancer (25). The only additive found to be carcinogenic in animal experiments and 110t banned as an additive, is the sweetener saccharin which is still present in certain beverages (see also (3)).

TOBACCO AND LUXURY ARTICLES AND HABITS Cigarette smoking is one of the major causes of lung cancer, one of the most widespread and most frequent types of cancer. Not all types of lung cancer are caused by tobacco smoking. The cancer from cigarette smoking is the squamous cell cancer (epidermoid cancer) of the lung, but adenocarcinoma of the lung is never influenced by smoking. For

3

the two other types of cancer of the lung (large cell and small or oat cell), the situation is questionable. Pipe smoking is not as dangerous because the smoke is generally not inhaled, and the mouth cavity is covered by stratified squamous cell epithelium, in contrast to the pseudo stratified ciliated epithelium in trachea and bronchus which is more sensitive. Tobacco chewing or use of snuff produces cancer in the oral cavity. Potent carcinogens derived from nicotine have been identified in these products (19). Creams, hair dyes, lipsticks, makeup, and soaps, to name a few, have occasionally contained carcinogenic ingredients.

SEXUAL ACTIVITY, MARRIAGE, RITUALS, HYGIENE, CIRCUMCISION, AIDS, IMMUNOLOGIC DEFENSE, HERPES VIRUS Sexual activity is important to bisexual creatures for the preservation of the species. Thus, certain marriage practices and rituals have developed. Sexual hygiene is often observed less in poorer population groups; deposited smegma under the foreskin of the male may result in penile cancer, and through coitus, in cervical cancer in the female partner. This is less frequent in the Jewish population due to circumcision. A special problem is the phenomenon of AIDS (see Chapter 18/VI). This is a variation of Kaposi's sarcoma (multiple idiopathic hemorrhagic sarcoma) detected by Kaposi (18371902). It was in those years a vascular sarcoma, especially in the lower legs of males over 50 years of age. It was a rather benign, slowly progressing disease in the small blood vessels. The disease appearing today as AIDS, especially in homosexual males because of a constant changing of partners, is a very aggressive, fast-progressing and often fatal disease found especially in males below 40 years of age. It is believed that during the process of coitus, the rectum becomes injured. When this happens repeatedly with varying partners, a number of infections develop which destroy the immunologic defense of the person. Latent viruses are able to act easily. However, AIDS is also a concern among heterosexuals, users of intravenous drugs and hemophiliacs. Certain herpes viruses are also believed to playa role in cervical cancer.

MEDICAL TREATMENT: CARCINOGENS IN THE FIGHT AGAINST OTHER DISEASES, IMMUNOLOGY AND TRANSPLANTATION, RADIATION AND CHEMOTHERAPY-INDUCED NEOPLASMS Carcinogens in the fight against other diseases: certain infectious or parasitic diseases are treated with drugs, which are carcinogenic, because no others are available. This is the case with Chagas disease (Chagas Cruz disease, South American Trypanosomiasis) for which the only drug in long-run use today is carcinogenic (personal communication Dr. Perreira). Another example is the use of the (itself carcinogenic) cytostatic agent cyclophosphamide in the treatment of multiple sclerosis (see Chapter 15/VI; (17».

4

H.E. Kaiser

EXPOSURE OF HEALTH CARE PERSONNEL TO ANTICANCER DRUGS A number of findings have demonstrated a risk to healthcare personnel when they work with anticancer drugs. Hirst (12) reported of two nurses and five volunteers who had skin absorption of cyclophosphamide. The urine samples from the two nurses working with cyclophosphamide (CP) in a cancer clinic showed the presence of CPo The five volunteers who received CP typically applied to the cubital fossa area showed that the drug was most evident in urine samples taken more than six hours after application. CP appeared sooner in the urine samples taken from the nurses than in those from the volunteers. The faster absorption may perhaps be effected through inhalation of aerosols generated during dissolution of the drug. The collapse of the immune system in organ transplant patients has also resulted in the development of neoplasms generally transmitted with the transplant (see Chapter 16/ VI) or the freeing of dormant neoplastic cells of the recipient's body. Chemotherapy-induced neoplasms resulted from the treatment of other neoplasms by cytostatica such as melphalan, chlorambucil, cyclophosphamide and others, including combined cancer chemotherapy (see Chapter 14/ VI). Diagnostic x-rays and radiotherapy for other diseases, often benign ones, have resulted in neoplasms after many years, even decades. The treatment of the childhood tumor nephroblastoma with surgery and follow-up radiation gave rise to such lymphomas as Hodgkin's disease when the child was full-grown (see Chapter 13/VI). RECREATION: SUNBATHING, SPORT, DIVING, AND OTHER BEHAVIOR Sunbathing, if prolonged and too frequent may lead to skin cancer and especially initiation of melanoma. Judicious selection and use of an appropriate sun-screen preparation according to individual skin type and life-style, as well as the introduction of strict testing and labeling regulations, are essential measures for reducing the risk of sun-induced skin cancers and malignant melanomas (I). A puzzling question is the use of anabolic steroids by athletes for better performance (10) since this practice increases the risk of cancer (31). Many cancer patients can return successfully to normal life, to work and, in some cases, to the practice of sports. Exercise programs must be simple, their effectiveness must be explained, and their benefits must be felt. As has been the case with the coronary sport groups, the establishment of local cancer sport groups needs to be actively pursued (32). OCCUPATIONAL ACTIVITIES: HISTORIC BACKGROUND, MINING, INDUSTRIAL ACTIVITIES, AND OTHERS Paracelsus (1493-1541) considered arsenic as a cause of cancer. In 1775, the English physician P. Pott attributed scrotal cancer of chimney sweeps to the influence of soot. In

1875, R. Volkmann correlated skin cancer in German laborers to the work in the coal, tar and paraffin industry. In 1895, L. Rehn attributed bladder cancer of workers in the dye-stuff industry to use of aromatic amines such as aniline. Radium was contained in paints with which workers used to paint the numbers of the faces of clocks. Licking the tip of the paint brush to sharpen it resulted years later in the appearance of cancers. Maintaining an awareness that within the workplace there may be factors which can contribute to occupational cancer is an important objective for our time (29). The IARC defined at least 7 industrial processes and occupational exposures which are causally associated with human cancers. These are auramine manufacturing, boot and shoe manufacturing and repair (certain occupations), furniture manufacturing, isopropyl alcohol manufacturing (strong acid process), nickel refining, the rubber industry (certain occupations), and underground hematite mining (exposure to radon). The possibility that excess cancers result from occupational exposures in oil refineries has generated a great deal of interest. The results are very inconsistent, but there is some suggestion of excess risks for melanoma and for brain, stomach, kidney, and pancreatic cancers (30). In 1979 workers on an ethanol unit which used sulfuric acid in high concentrations at a large refinery and chemical plant in Baton Rouge, Louisiana, were reported to be at excess risk for upper-respiratory cancer. Among workers classified as potentially highly exposed, four-fold relative risks for all upper-respiratory cancer sites combined, were exceeded by the relative risk for laryngeal cancer. Exposureresponse and consistency across various comparisons after controlling statistically for tobacco use, alcoholism and other previously implicated risk factors, suggest increased cancer risk with higher exposure (33). Patients with bone infarction who had worked in a hyperbaric atmosphere, such as divers experience, are at greater risk of developing malignant fibrous histiocytoma. The prolonged reparative process following bone infarction seems to be an important factor in the pathogenesis of this type of tumor (16). Formaldehyde as a possible factor in carcinogenesis has come into the limelight. There has been criticism of most of the epidemiologic studies reported but there is no definitive evidence for excess cancer from formaldehyde in the workplace ((2); (26); see Chapter 6/II; 28). TRAFFIC The exhaust from motor cars and other vehicles contains some carcinogenic polycyclic aromatic hydrocarbons. Compared to cigarette smoke, the effect of automobile exhaust is much lower. Other factors are the abrasion of concrete particles from the highways, the rubber abrasion from tires, and the exhaust from asphalt. POLLUTION This topic is covered in Volumes II, V, VI. A few examples can be given here. There has been a great deal of controversy

1: The influence of human behavior on neoplastic progresssion

about the effects of dioxin on human health due to the incidents at Sevesco and at Times Beach, Missouri. Dioxin is a contaminant of widely-used chemicals. Chloracene is the most sensitive indicator of significant dioxin exposure. Porphyrin cutanea tarda and hyperpigmentation are other known cutaneous effects, and possibly malignant fibrous histiocyomas of the skin which remain questionable. Dermatologists should be aware of the problem and may discover early cases of previously unsuspected exposure to this group of chemicals (7). One ppb of 2,3, 7,8-tetrachlorodibenzodioxin in soil is a reasonable level at which to begin consideration of action to limit human exposure to contaminated soil (15). The contamination of farms and people in Michigan from polybrominated biphenyls is another case where continued observation of the population is needed. CHAIN REACTION WITH OTHER SPECIES

7. 8. 9.

10. I!.

12. 13.

See Chapter 22/IV and Volume V. 14.

WAR

Pieces of metal in the body of veterans due to gun-shot wounds can act as physical carcinogens producing sarcoma. Leukemias in atomic-bomb survivors of Hiroshima, for example, were well-known and often described. Due to wartime conditions, moldy grain may sometimes be consumed with devastating effects (13). SUMMARY AND CONCLUSIONS

This foregoing chapter gives only a few examples of the influence human behavior plays on neoplastic progression by performing of unnaturals cultural, technical, industrial, and other processes. An interaction of many factors occurs in reality. It is justified to consider these factors and activities as effects of human behavior because man does perform in such ways in his attempts to reach certain goals. The explained neoplastic progressions cited here are interferences in man's own health by activities which he himself has created'.

15.

16. 17. 18. 19. 20. 21.

22.

23.

REFERENCES 1. 2. 3. 4. 5. 6.

Azizi E, Kushelevsky AP, Echewach-Miller M: Efficacy of topical sunscreen preparations on the human skin: combined indoor-outdoor study. Isr J Med Sci 20(7):569-77, 1934 Blair A, Stewart P, O'Berg M et al: Mortality among industrial workers exposed to formaldehyde. JNCI 76(6): 1071-84, 1986 Caffeine labeling. Council on Scientific Affairs. JAMA 25 2( 6): 603-6 Committee on Diet. Nutrition, and Cancer National Research Council. Diet, Nutrition, and Cancer. National Academy Press, Washington, DC, 1982 Dempsey, DT, Feurer ID, Knox LS et al: Energy expenditure in malnourished gastrointestinal cancer patients. Cancer 53(6):1265-73, 1984 Derdiarian AK: An instrument for theory and research de-

24. 25.

26. 27.

28. 29.

5

velopment using the behavioral systems model for nursing: the cancer patient. Kango Kenkyu 17(2): 134-42, 1984 Ounagin WG: Cutaneous signs of systemic toxicity due to dioxins and related chemicals. JAm Acad DermatoII0(4):688700, 1984 Fourth Symposium on Epidemiology and Cancer Registries in the Pacific Basin. Proc. Symp Kona, Hawaii. Washington, DC.:U.S. Government Printing Office, 1984 Hanai A, Fujimoto I: Cancer incidence in Japan in 1975 and changes of epidemiological features for cancer in Osaka. In: Third Symp on Epidemiology and Cancer Registries in the Pacific Basin Monogr 62. U.S. Dept of Health and Human Services Natl Inst of Health, NCI, Bethesda, Maryland, USA. pp. 3-8, 1982 Haupt HA, Rovere GD: Anabolic steroids: a review of the literature. Am J Sports Med 12(6):469-84, 1984 Higginson J: Cancer risk factors in human studies. NaIl Cancer Insl Monogr 67:187-92, 1985 Hirst M, Tse S, Mills DG et al: Occupational exposure to cyclophosphamide. Lancet 1(8370): 186-8, 1984 Joffe AZ, Ungar H: Cutaneous lesions produced by topical application of aflatoxin to rabbit skin. J Invest Dermato! 52(6):504-7, 1969 Kaiser HE: Cancer promoting effects of phenols in tea. Cancer 20(5):614-16, 1967 Kimbrough RO, Falk H, Stehr P, Fries G: Health implications of 2,3,7,8-tetrachlorodibenzodioxin (TCOD) contamination of residential soil. J Toxicol Environ Health 14(1):47-93, 1984 Kitano M, Iwasaki H, Yoh SS et al: Malignant fibrous histiocytoma at site of bone infarction in association with DCS. Undersea Biomed Res II (3):305-14, 1984 Kornhuber HH, Mauch E, Petra E et al: Risk of cancer in cyclophosphamide therapy of mUltiple sclerosis (letter). Dtsch Med Wochenschr 112(13):530, 1987 Krantz OS, Grunger NE, Baum A: Health psychology. Annu Rev Psychol 36:349-83, 1985 Kuller LH, Garfinkel L, Correa P et al: Contribution of passive smoking to respiratory cancer. Environ Health Perspect 70:57-69, 1986 Larson PJ: Important nurse caring behaviors perceived by patients with cancer. Oncol Nurs Forum 11(6):46-50, 1984 Li FP: Second cancers. In: DeVita VT Jr, Hellman S, Rosenberg SA (eds) Cancer Principles & Practice of Oncology. Philadelphia: JB Lippincott, pp. 1717-29, 1982 Li J -Y: Investigation of geographic patterns of cancer mortality in China. In: Third Symposium on Epidemiology and Cancer Registries in the Pacific Basin. Natl Cancer Inst Monogr 62: 1742, 1982 Lindsey AM: Cancer cachexia: effects of the disease and its treatment. Semin On col Nurs 2(1):19-29, 1986 Masri MS: Defenses against aflatoxin carcinogenesis in humans. Adv Exp Med BioI 177:115-46, 1984 Namiki M, Osawa T: Antioxidants/antimutagens in food. In: Antimutagenesis and Anticarcinogenesis Mechanisms, Shankal DE, Hartman PE, Kada T, Hollaender (eds) pp. 131-192. Plenum Press, New York, 1986 Nelson N, Levine RJ, Albert RE et al: Contribution offormaldehyde to respiratory cancer. Environ Health Perspect 70:2335, 1986 Oshima A, Hanai A, Fujimoto I, Song K: Cancer mortality among Koreans in Osaka, Japan. In: Third Symp on Epidemiology and Cancer Registries in the Pacific Basin. NCI Monograph. 62, pp. 13-16, 1982 Report on the Consensus Workshop on Formaldehyde. Environ Health Perspect 58:323-81, 1984 Rosenstock L: Occupational cancer: clinical interpretation and application of scientific evidence. J Toxieol Clin Toxicol 22(3):261-82, 1984

6 30. 31. 32.

H.E. Kaiser Savitz DA, Moure R: Cancer risk among oil refinery workers. A review of epidemiologic studies. J Occup M 26(9):662-70, 1984 Schmitt HP: Sports accident or natural death? Hematocephalus internus caused by rupture of a choroid plexus angioma. Z Rechtsmed 91(2):129-33, 1983 Schule K, Hamacher F, Booz S: Sports and exercise therapy in the framework ofa cancer aftercare course. OffGesundheitswes 45(4):191-6, 1983

33. 34. 35.

Soskolne CL, Zeighami EA, Hanis NM et al: Laryngeal cancer and occupational exposure to sulfuric acid. Am J Epidemial 120(3):358-69, 1984 Spinette JJ: Methodology in behavioral and psychosocial cancer research. Development of psychometric assessment methods by life cycle stages. Cancer 53(10 Suppl):2222-7, 1984 Wortman CB: Social support and the cancer patient. Conceptual and methodologic issues. Cancer 53(10 Suppl):2339-62, 1984

2 THE INFLUENCE OF STRESSORS ON THE PROGRESSION OF NEOPLASTIC CHANGE HYMIE ANISMAN, JILL IRWIN and LAWRENCE S. SKLAR

the number of splenocytes, thymocytes and lymphocytes, and reduced proliferation in response to the T cell mitogen stimulation with concanavalin A (Con A). There is evidence that in addition to the anterior hypothalamus, the hippocampus may also regulate immunological functioning. Lesions of this structure increased the number ofthymocytes and enhanced the response of thymus and spleen cells to Con A (25). Furthermore, the enhanced mitogen response of thymic lymphocytes noted following lesioning of the hippocampus and amygdala could be reduced by hypophysectomy (34). It was suggested that these immunological alterations resulted from neuroendocrine changes produced by the central nervous system manipulations. Additionally, it appears that immunological activity may come to modify central neuronal processes. In particular, it was demonstrated that in rats immunized with sheep red blood cells (SRBC), electrical activity in the hypothalamus was comparable to baseline levels when assessed at a time preceding the appearance of plaque forming cells. However during the peak phase of plaque formation increased electrical activity was observed (19). Subsequently it was reported that in animals that mounted a strong plaque forming response to SRBC, hypothalamic NE turnover was appreciably reduced at the time of maximum immunological responsivity (18). The effects of hypothalamic manipulations on immune functioning may be attributable to the influence of any number of hormones or steroids. For example, corticotropin releasing factor (CRF), secreted in the median eminence of the hypothalamus, is transported to the pituitary through the hypophyseal portal system. There the releasing factor stimulates the secretion of adrenocorticotrophic hormone (ACTH) which ultimately provokes the release of corticosteroids from the adrenal. In addition to CRF, numerous other releasing factors, which may engender either an excitatory or inhibitory effect upon secretion of pituitary hormones, have been identified, including thyroid hormone (TH), leutinizing hormone (LH), prolactin, and growth hormone (GH) (see reviews in 54, 85). It is well recognized that exogenously administered corticosteroids will depress the proliferation of lymphocytes, macrophages and other accessory cells, and consequently these steroids are used clinically in the treatment of inflammatory conditions and for the prevention of graft rejection (46). Predictably, when corticoid secretion is inhibited through adrenalectomy, immunofacilitation is induced (4, 117). In contrast to' corticosteroids, growth hormone apparently enhances immune functioning (45), as does thyroid

It has long been considered that life stressors may have a

considerable impact on an individual's physical well being. To a great extent, this supposition has been based on anecdotal reports that traumatic events or affective changes are frequently associated with illness. Despite its intuitive appeal, experimental data consistent with this position have only recently become available. However, at present, limited information is available concerning the mechanisms subserving the relationship between stressful events and pathology. Evidence from both human and infrahuman research suggests that stressful events may have a profound impact on central neurochemical processes, immune functioning, and the progress of tumor development. The present report will provide a brief review of this literature, with particular emphasis devoted to some of the limiting conditions which influence vulnerability to central neurochemical alterations, immunocompetence and tumor development. Central nervous system influences on immune system

Although variations of immunological functioning have traditionally been considered to be independent of central nervous system activity, it has become increasingly more evident that such a view should be reconsidered. Numerous reports are available which indicate that manipulations of central mechanisms wiIl alter various aspects of immune system activity. Lesions of the hypothalamus, for instance, have been shown to inhibit anaphylactic responses (79, 80), reduce circulating levels of antibodies in response to antigenic challenge (80), and suppress cell-mediated processes (25, 126). Conversely, electrical stimulation of brain sites has been shown to enhance both humoral and cell mediated immune responses (63, 73). It is thought that the anterior hypothalamus may be fundamental in determining the immunological variations. Lesions of this portion of the hypothalamus have been shown to diminish the delayed cutaneous response to antigen challenge induced by tuberculin purified protein derivative, as well as the lymphocyte responses to phytohemagglutinin (PHA), (69, 126). Lesions of the medial and posterior hypothalamus, in contrast, were without effect (80); see also (112). In addition, bilateral lesions of the anterior hypothalamus were found to decrease Supported by Grants A9845 and MT-6486 from the Natural Sciences and Engineering Research Council of Canada and the Medical Research Council of Canada, respectively. 7

E. K. Weisburger (ed.), Mechanisms of carcinogenesis. © 1989, Kluwer Academic Publishers, Dordrecht. ISBN 978-94-010-7641-8

8

Hymie Anisman, Jill Irwin and Lawrence S. Sklar

hormone (44). Additionally, recent data have implicated a role for estrogen, testosterone and prostaglandins in the regulation of the immune response. These hormonal responses are themselves influenced by variations in central neurotransmitters, and it has been demonstrated that transmitter manipulations will influence immune functioning. Treatments which reduce central serotonin (5-HT) concentrations may increase antibody production (23, 37), and conversely, the latent phase for antibody production following immunization is associated with altered 5-HT levels (37). There is also reason to believe that stimulation of dopamine (DA) receptors results in immunofacilitation, while reduction ofDA has the opposite effect (33, 40). Moreover, catecholamine stimulants, such as L-DOPA and amphetamine were found to inhibit DMBAinduced tumors and transplanted syngeneic tumors (39, 96), provisionally suggesting that these drugs produced changes in the immune system. Indeed, direct acting NE receptor

blockers have been shown to reduce the lymphocyte response to Con A, and B-adrenergic agonists and antagonists can alter such responses (55, 65). Finally, it seems that immune functioning may be influenced by endogenous opioids, since morphine administration depresses rosette formation of T-Iymphocytes in vitro (130), and suppresses the activity of Natural Killer cells (76). Stress-provoked neurochemical change It appears that immunological changes may be provoked by

alterations of central neuronal activity or by the secretion of hormones, and conversely, immunological changes may come to affect central neuronal activity. Accordingly, the possibility should be entertained that variations of central neurotransmitters may be responsible for pathological states which are provoked or exacerbated by aversive events. Accordingly, it would be appropriate to consider the consequences of stressful events on the activity of central neurotransmitters. When an organism is confronted with a stressor it makes behavioral attempts to eliminate or reduce the impact of the aversive stimulation. Concurrently, several neurochemical changes occur which are thought to be of adaptive significance. For instance, they may induce an antinociceptive effect, increase the organism's preparedness to deal with the stressor through behavioral means, and may limit the emotional reactivity of the organism to permit optimal levels of arousal. We have reviewed in some detail the varied neurochemical consequences associated with stressors in earlier reports (e.g., 8, 13), and hence only a cursory overview of this literature will be presented at this time. Upon inception of a stressor the utilization of norepinephrine (NE) increases in several brain regions, including the hypothalamus, hippocampus, cortex, amygdala and locus coeruleus. The increased release of the transmitter is accompanied by a compensatory increase of synthesis, thereby assuring that concentrations of the amine remain relatively stable (see 114). If the stressor is sufficiently severe and protracted, the release ofNE increases appreciably, and may come to exceed synthesis, ultimately resulting in a net reduction of the transmitter concentrations (8, 114, 119). It appears that some brain regions are more vulnerable to the

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NE reductions than are others, such that hypothalamic NE reductions are noted more readily than are cortical or hippocampal alterations (86). Irrespective of the brain region examined, however, it seems that the NE reduction is fairly transient, persisting for only a matter of a few hours (9), unless the stressor is a particularly severe one in which case amine reductions may persist as long as 72 hr (128). The NE reductions provoked by stressors are influenced by a number of experiential, organismic and environmental factors. In this respect, it appears that the organism's ability to exert control over stressor termination is fundamental in determining whether or not depletion of the transmitter will be observed. As seen in Figure I, exposure to 60 foot-shocks (150 jlA, 6 sec duration) from which escape is possible, does not appreciably influence concentrations ofNE. In contrast,. in mice that received precisely the same amount of shock and at the same time, but could not influence shock offset through their responses (i.e., yoked inescapable shock), a marked reduction of NE concentrations was observed (10, 127). Likewise, it was reported that the NE depletion was not only induced more readily in animals that received the inescapable shock, but was also more persistent than in rats that received escapable shock (118). These data suggest that shock per se was not the critical variable in determining the NE reductions, but rather the psychological dimension of being unable to control shock offset (i.e., behavioral coping) was responsible for the amine alterations. In accounting for these findings, it has been our contention that animals deal with stressors through both behavioral and neurochemical processes. Under conditions where the organism is unable to influence environmental events through its behavioral responses, the burden of coping with the stressor rests primarily upon neurochemical mechanisms. Consequently, these neurochemical processes may be overly taxed, hence increasing the possibility of amine reductions. We have argued that under such conditions ani-

2: The influence of stressors on the progression of neoplastic change

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relative to control values (dashed line) in mice that received either 1, 3 or 15 days of shock. During each session mice received either 60 shocks of 6 sec duration, 150 J1A (open bars), or 180 shocks (striped bars). Additional groups of mice received 180 shock trials on each day but the last, when only 60 shocks were administered (hatched bars). Note that the decline of NE induced in hypothalamus following a single session of shock was absent after repeated shock, and actually exceeded control values. (From (61)). mals may be less well prepared to cope effectively should other stressors be introduced, and additionally animals may be more vulnerable to pathologies that require adequate NE stores or activity (8, 12, 109). Although utilization of brain NE may exceed its synthesis following acute exposure to a stressor, repeated application of a stressor may engender a series of neurochemical events which may be of adaptive significance. For instance, in animals that receive a sufficiently protracted stress session or repeated exposure to a stressor on successive days, a further increase of amine synthesis is induced (see 74, 127) and hence depletion of the transmitter is not incurred (74, 101, 127). Additionally, it seems that following a chronic stress regimen the rate of NE reuptake is reduced, thus increasing the functional efficiency of the transmitter that is released (127). It has been postulated that in the absence of such adaptive changes vulnerability to pathology is enhanced (13, 109). It seems that the changes of NE turnover and concentra-

9

tions provoked by repeated exposure to a stressor vary with time following stressor termination. Immediately after the last session of a chronic regimen, the utilization of NE is increased relative to nonstressed animals, but owing to a compensatory increase of synthesis, levels of the transmitter remain relatively stable. However, it appears that over the course of the ensuing 24 hr the rate of utilization declines appreciably (61), although the increased synthesis may be sustained for relatively long periods (120). As a result, levels of the amine may substantially exceed those of nonstressed animals (see Figure 2). It is likely that these variations of turnover and levels reflect a highly efficient means of dealing with stressful events. In particular, the increased utilization seen upon stressor termination (presumably reflecting NE activity during stressor presentation) may be necessary in order for the organism to meet immediate environmental demands. The subsequent decline of utilization rates may represent a method of conserving amine stores in order to deal with impending stressful events (see 115). Accordingly, in considering the effects of stressor on the induction or provocation of pathology, it is not only important to assess the potential impact of the immediate neurochemical alterations, but also the variations of neuronal activity that occur at various intervals following a stress session. In addition to the alterations of synthesis and utilization ofNE that are evident with repeated exposure to a stressor, it seems that variations of receptor sensitivity are induced following a chronic stress regimen. In particular, it has been demonstrated that among rats that received repeated exposure to a stressor the sensitivity of f3-NE receptors is reduced, and this may be accompanied by a decline in NE sensitive cyclic AMP activity (89,115,116,123). The sugestion has been offered that the down regulation of f3-NE receptors may be an adaptive mechanism to offset the adverse influence of excessive receptor stimulation otherwise engendered by repeated exposure to a stressor (115). The effects of stressors on NE neuronal activity have been assessed more extensively than that of other transmitters. Nevertheless, data are available concerning stress-induced variations of dopamine (DA), serotonin (5-HT), endorphins, and to a lesser extent, some attention has been devoted to stressor provoked variations of epinephrine, acetylcholine, and gamma amino butyric acid. In the case of DA, most of the available data suggest that variations of this transmitter in brain are not as widespread as that of NE. While the NE variations are seen in a substantial number of brain areas, the DA alterations appear to be restricted to only a few regions. Thus, while DA reductions are typically not observed when concentrations are assessed within the entire hypothalamus, considerable reductions have been detected in the arcuate nucleus of the hypothalamus (72,74). Moreover, while stressors typically do not affect DA turnover in the substantia nigra and caudate nucleus (see, however, 41), it has been demonstrated that concentrations of the DA metabolite, DOPAC, as well as the DOPAC/DA ratio are increased in the nucleus accumbens and the mesolimbic frontal cortex (21, 47, 121), and concentrations of the amine have also been shown to decline in the lateral septum (102). The arcuate nucleus plays an integral role in determining the secretion of pituitary hormones, and the nucleus accumbens and mesolimbic frontal cortex may be fundamental in affecting emotional state or lability. Accord-

10

Hymie Anisman, Jill Irwin and Lawrence S. Sklar

quences of stressful events on immunological alterations. These include variations in disease susceptibility, as well as assessment of the competence of phagocytic, humoral and cell-mediated immune processes. Retrospective analyses conducted in humans have revealed that infectious diseases such as tuberculosis and upper respiratory illness are associated with antecedent life event changes (see review in 64). Likewise, increased incidence of life stress events has been associated with the onset and exacerbation of allergic and autoimmune disorders (see reviews in 60, 111). Unfortunately, there is considerable variation in the results of studies concerning the impact of life stressors on disease susceptibility. In part, this variability may derive from the fact that researchers frequently failed to consider personality characteristics which may affect the impact of stressors (64), and secondly, from the undue reliance placed upon retrospective analyses. That is, when subjects who have contracted a particular illness are required to recall previously encountered life stressors, their reports may be influenced by the effective state promoted by the illness. Moreover, there is ample evidence indicating that a subject's recall of past events may not be particularly accurate (see discussions in 30, 97; see also the discussion later in this paper). These caveats notwithstanding, prospective studies (i.e., where life events are monitored prior to illness onset) have generally supported the view that promotion or exacerbation of pathology may be associated with aversive life stress events. It might be noted that such stressors not only include major traumas, but also the day-to-day minor inconveniences that the individual may encounter (64). Consistent with the findings concerning disease susceptibility, more direct measures of immune functioning have demonstrated that stressful events may compromise the competence of the immune system. Stressors such as sleep deprivation and surgery were shown to suppress the activity of phagocytic cells (90), and examination stress may reduce the efficiency of B lymphoblast transformation (38). Similarly, major trauma, such as bereavement was found to reduce the mitogen induced proliferation of T lymphocytes, although the actual number of T -cells was not affected by the stressful experience (104). Additionally, it was observed that stressful life events were associated with variations in Natural Killer (NK) cell activity following virus inoculation (77). Interestingly, it seemed that the reduction of the NK cell activity was related to life-stress events that had occurred during the preceding 2 weeks and was unrelated to major life changes that subjects had experienced during the preceding year. It was also suggested that the individual's ability to contend with the stressor through behavioral means (i.e. the effectiveness of coping style) was a major determinant of whether a reduction of NK cells would be evident (64). Studies conducted with animals have yielded results consistent with those of the human experiments. Furthermore, these experiments have been instrumental in delineating some of the conditions which determine whether or not disease susceptibility would be exacerbated, and whether immunological alterations would be provoked by stressors. In particular, it has been demonstrated that acute application of stressors such as footshock, restraint, and overcrowding will increase morbidity and mortality following: exposure to viruses, including Coxsackie B, Poliomyelitis, Stressor-provoked immunological changes and herpes simplex, and will increase susceptibility to adSeveral approaches have been employed to assess the conse- juvant-induced arthritis (6, 50, 66, 67). Consistent with such

ingly, the contribution of the DA variations, as limited as they are, may have a great impact on the behavior of organisms in stress situations, as well as on their ability to cope effectively with the stressors. While there is inferential evidence suggesting that the. variations ofDA activity, at least in the nucleus accumbens, may be influenced by the animal's ability to control stressor termination (131), direct evidence in favor of this supposition has yet to be reported. It has, however, been demonstrated that sensitivity of DA receptors, particularly in the nucleus accumbens, may be influenced by the organism's ability to contend with the stressor through behavioral means (29). Variations of 5-HT concentration have been shown to occur following stressor application, although the severity of the stressor needed to induce such an effect is greater than that required to induce the NE variations (see 119). Although limited data are available, it has been reported that the reduction of this amine, at least in the lateral septum, is readily induced by uncontrollable shock, but is not appreciably influenced by controllable shock (95). Likewise, it has been shown that central variations of endogenous opioids as well as acetylcholine may be influenced by stressors (15, 32, 53, 83, 100, 132) and there is reason to believe that these alterations are subject to the animal's ability to cope with the stressor through behavioral means (68, 81). The effectiveness of stressors in modifying neurochemical activity is not limited to that period immediately following stressor application. In this respect two independent factors should be considered. First, it appears that the neurochemical variations induced by stressors may be subject to conditioning or sensitization processes, such that upon reexposure to a stressor the organism may be more vulnerable to neurochemical variations. It has been demonstrated, for instance, that neutral cues that had been paired with a stressor may subsequently come to increase the utilization of brain NE (27), DA (58), acetylcholine (59) as well as endorphins (28). Moreover, in animals that were previously exposed to an uncontrollable stressor, reexposure to a limited amount of stress may reinduce reductions ofNE concentrations (11). In effect, when assessing the impact of aversive events on pathology it would be appropriate to consider the immediate effects of the stressor and also the effects attributable to environmental cues that had been associated with that stressor. The second issue to be considered is that stressors may induce several time-dependent neurochemical variations. It has been argued (7) that in addition to the immediate neurochemical consequences engendered by aversive stimulation, delayed compensatory changes of neurochemical activity may be provoked. Such rebound effects may take the form of biphasic changes in the activity or concentrations of a single transmitter system, or they may be reflected by secondary changes in other transmitter systems. Accordingly, the possibility should be considered that the critical factor in affecting pathology is not the immediate neurochemical consequence of the aversive stimulation, but rather the compensatory changes which occur subsequent to the initial insult.

2: The influence of stressors on the progression of neoplastic change

findings, direct measures of immunocompetence have revealed that acute application of aversive stimuli results in suppression of various immune functions. For instance, a variety of stressors were shown to impair phagocytic functioning (16), depress antibody levels (43, 56) and reduce Tand B-cell proliferation in response to a mitogen It seems that the effects of stressors on immune functioning may be dependent upon a number of experimental variables. Among other things, there is reason to believe that the chronicity of the stressor application may be fundamental in determining the nature of the immunological changes observed. It was suggested that while acute stressors will result in immunosuppression, repeated application of a stressor may actually result in facilitation of immune system functions (84). Thus, it might be expected that disease susceptibility would likewise vary as a function of stressor chronicity. It will be recalled from the results of investigations with human subjects that there is reason to assume that coping factors are important in determining the effects of stressors on immune processes. Animal experimentation has indeed supported this contention. In rats that were exposed to footshock which was escapable, appreciable changes of Tcell proliferation in response to a mitogen were not evident. In contrast, in rats that received precisely the same amount of shock at exactly the same time, but were prohibited from terminating the stressor themselves, the response to a mitogen was significantly reduced (75). Along the same line, it was recently shown that escapable and inescapable shock differentially influenced Natural Killer cell activity (106). It seems that the effects of the stressors on immune functioning, as on central catecholamines, were related to the organism's ability to contend with the stressor through behavioral means. The effects of stressors on immune system functioning are not restricted to those involving physical insults, but are also evident after the application of a variety of social stressors or modifications of the social environment. Following social isolation or overcrowding, immune activity is suppressed and susceptibility to infectious disorders is increased (17, 52). Moreover, it appears that the effects of such manipulations will vary as a function of the strain of animal employed (51). It should be underscored that social factors may not only have immediate consequences on immune functioning, but may also provoke long term alterations. For instance, it was demonstrated that handling of mice prior to weaning, or early nutritional deprivation, resulted in increased disease susceptibility and reduced cell mediated responses during adulthood (42, 105). Further evidence that immunological processes may be subject to behavioral and neural control is derived from studies employing classical conditioning procedures (1-3). In particular, it was shown that if the immunosuppressant drug, cyclophosphamide, was administered to animals following consumption of a novel tasting solution (the conditioned stimulus), this stimulus, when subsequently presented alone, reduced the antibody response to sheep red blood cells (1). Moreover, this procedure was also effective in inhibiting a graft-versus-host response (24) and was found to retard the symptoms associated with lupus erythematosus in New Zealand mice (3). Together, these data suggest that environmental factors will not only influence immunological functioning, but that the cues associated with environmental

11

events may serve in a similar capacity. As discussed earlier, various attributes of central neuronal functioning also appear to be subject to conditioning processes in stress paradigms. Accordingly, in assessing the long-term effects of stressors on immune functioning it might be appropriate to consider the potential contribution of altered neurochemical functioning. Stress and neoplasia

The contribution of stressful events to neoplastic disease has been evaluated in both humans and infrahumans. This literature has been detailed in several recent reports (49, 60, 109) and hence an extensive review of the literature will not be presented at this time. Rather, a general overview will be provided, together with several caveats that should be considered concerning this literature. Evaluating the stress-cancer topography in humans has generally supported the contention that stressful events may exacerbate tumor growth; however, numerous problems are associated with many of the experiments and hence the data must be considered cautiously. Several research approaches were adopted to determine the relationship between stressful events and neoplasia, the most commonly used being a retrospective approach. This strategy, as indicated earlier, involves evaluation of stress histories of patients with cancer relative to those who are suffering either some other illness or non affected individuals. Such studies have frequently revealed that life stresses occurred more frequently among cancer patients than control subjects, or that the cancer patients were less well adjusted or coped more poorly with stressful events (see review in 22). To be sure, however, not all investigations have yielded results consistent with these findings, and the retrospective type of strategy appears to be a methodologically unsuitable approach to employ (see reviews in 13, 22, 49, 109). As discussed previously, when subjects are asked to recall events that had occurred, even over the course of several months, their retrieval appears to be limited. Moreover, it is likely that the recall of past events or the subjective interpretation of these events may be colored by their current affective state. Even if it were to be assumed that the subjects' recall was accurate and unbiased, the approach would still not be a particularly useful one. Clinical diagnosis of cancer may not be made until years after the appearance of the first malignant cells. Hence, the value of assessing life-stress events during the 6-12 month period prior to clinical diagnosis, as is often the case, is questionable. Moreover, in most studies life stress events are considered to have an equivalent impact in all individuals' scores. Clearly, however, subjects do not necessarily interpret life-event changes in a comparable fashion. That which is stressful to one individual may not be to a second (divorce, for example, may be stressful to one individual but a blessing to another), and other things being equal, subjects may not be equally adept at coping with a stressor (see further discusssion in 13, 49). Prospective studies, in which subjects' life histories are evaluated on an ongoing basis and related to the occurrence of subsequent pathology, avoid some of the problems inherent in the retrospective approach. While such studies have lent credence to the suggestion that stressful events or coping styles are related to neoplasia (99, 122; see review in

12

Hymie Anisman, Jill Irwin and Lawrence S. Sklar

22), this procedure is not without its own problems. Typically, such studies consider only major life changes, with relative disregard to the day-to-day hassles subjects encounter. Yet, as indicated earlier, these daily inconveniences may have a major impact with respect to endogenous steroids (31) and may influence pathology (64). A problem inherent in many studies has been the failure to differentiate between the potential contributions of various forms of stressors on tumor development. In this respect, there has also been a tendency to generalize across tumor types, rather than distinguishing between different forms of neoplasia. To be sure, there is no a priori reason to believe that stressors will similarly influence all forms of cancer, and likewise, neurochemical or immunological changes may be dependent on several different characteristics associated with the stressor (8). Indeed, on the basis of a comprehensive review of the human literature, it was argued that stressors may differentially influence the course of various types of neoplasia, making it difficult to evaluate the stress-cancer topography unless consideration is devoted to the impact of stressors on diverse forms of neoplasia (22). Specifically, it was indicated that the data concerning the contribution of psychological factors and stressful events to pancreatic, abdominal, colorectal, prostatic and cervical cancer was inconsistent. However, lung cancer appeared to be associated with increased early parental loss and denial of personal distress, while breast cancer was associated with suppression of anger (or conversely, excessively explosive behavior) or the perception of being a self-sacrificing individual (see 22). This proposition is certainly a highly provisional one, but if nothing else it emphasizes the importance of more detailed analyses of the stress - cancer topography. Prognostic studies, where life-stress histories are documented prior to biopsy of suspected tumors, revealed that malignant tumors were more frequent among women that had encountered greater life stress events (36). Inasmuch as all women in these studies reported their life histories under similar conditions (i.e., prior to the tumor being diagnosed as malignant or benign), it is unlikely that the reported life histories were confounded by differential biases in the interpretation of past events. However, as in the case of the prospective studies, the issue of minor daily life stresses has been largely ignored, and it is not clear whether the effects observed are restricted to only some forms of neoplasia (22). Additionally, each of the approaches typically does not consider a wide range of epidemiological variables which might interact with life stress-events in provoking tumor changes (variations of coping strategy with age, sex, rural vs urban living, diet and so forth; see 49), or the possibility that variations of tumor development may be secondary to a variety of factors promoted by stressors (e.g., smoking frequency, dietary changes, drug intake etc). It is clear that the data gathered in human investigations have provided some support in favor of a stress-cancer relationship; however, the great number of procedural and interpretive problems associated with these studies permits this conclusion to be drawn only with very great reservation. At the same time it should be underscored that investigators have often adopted the inappropriate view that because these studies are frequently flawed, the conclusion can be derived that the relationship between stress and cancer is a

spurious one. At the very least, these data should be taken to imply that stressful events may influence tumor development (or perhaps some types of tumors) in a subset of individuals, given appropriate environmental or genetic histories. Indeed, experiments conducted using infrahuman animals have provided some evidence suggesting that stressful events may influence the course of neoplasia under conditions which are relatively uncontaminated by those factors which limit the validity of the human studies. Although the animal studies are not fraught with many of the difficulties inherent in the human investigations, this does not imply that they are without problems. (a) Experiments between laboratories have employed different types of tumors (e.g., viral, transplanted, carcinogen-induced, hormone-dependent or - independent, possibly regulated by the immune system or independent of immunological mechanisms) and there is the growing belief that stressors may not have uniform effects on these various tumor types. (b) Many different types of stressors (or stressor severities) have been employed, and the endogenous chemical variations induced by one type of stressor may be considerably different, both qualitatively and quantitatively, from that provoked by a second form of stressor. (c) Along the same line, in some investigations the stressors employed have been of an uncontrollable nature, while in other studies the stressors could be terminated through behavioral responses. This variable, it will be recalled, has a major impact on the nature and magnitude of the neurochemical and steroid changes induced, and hence it would not be surprising to find a similar dichotomy with respect to tumor development. (d) Furthermore, the social or environmental backdrop upon which these stressors are applied varies between studies, and such variables are also known to influence the neurochemical alterations ordinarily provoked by stressors. (e) Stressors are often applied at different times relative to the phase of tumor development (i.e., before tumor inoculation or at various times afterward), and it may well be the case that the effects of stressors vary with the developmental stage of the tumor. (f) Frequently, stressors are applied on only a single occasion, whereas in other instances stressors are repeatedly applied. It will be recalled that such procedures will differentially influence endogenous neurochemical and hormonal activity, and hence it would not be unreasonable to expect that these procedures will have differential effects on tumor development. In light of these considerations, it should come as no surprise to find inconsistent results across studies. On the one hand these numerous factors may lead the reader to dismiss the available data, since there are too many variables present to permit an accurate interpretation. On the other hand, the between laboratory differences in procedure and outcomes may provide the investigator with potent clues in defining those variables which are sufficient or necessary in order that particular outcomes be observed. Indeed, although there are still more unknowns about the stress-cancer relationship than there are known factors, it seems that some advances have been made in disentangling the contribution of several variables to the exacerbation of tumor development. Several investigators have demonstrated that social stressors will influence the development of transplanted tumors (35, 110). For instance, housing mice in isolation has been shown to increase the growth rate of a transplanted P8l5

2: The influence of stressors on the progression of neoplastic change

13

increase the probability of secondary tumor formation once cells have separated from the primary tumor mass. o Gil E 300 / Inasmuch as metastasis represents one of the major prob6___6 E "GIG lems in cancer treatment, the practical importance of delin1/ Q) • IIG eating the potential effects of stressors on the metastatic g250 o III 0process should be emphasized. It has been reported, for ~ instance, that various forms of systemic stress (tumbling, 300 200 «w .j;! restraint, seizure, and intraperitoneal injection of adrenalin 0: « and other {:I-adrenergic agonists) increase the colony form! 150 0: ing efficiency of intravenously injected Walker 256 tumor 0 cells (124). Likewise, surgical stress has been shown to in::1: ::J 100 crease the frequency of pulmonary (103) and hepatic (4S) ~ tumors following intravenous cell inoculation. More recentz «W 50 ly it was demonstrated that thoracotomy and laparothora'ef'; 9~X} ::1: cotomy increased the number and area of metastatic nodules on lungs following inoculation of Saito lung cancer (57; see 15 3 5 7 11 13 9 also 78). Moreover, it appeared that the area of metastatic nodules in the right lobes and the left lobe of the rat lungs DAYS was comparable, despite the fact that thoracotomy was Figure 3. Mean (± S.E.M.) tumor area at various days following 6 transplantation of approximately 0.0625 x 10 PSIS cells in DBA/2 performed in the right thorax. Thus, these data suggest that mice. Mice were housed either in groups of 5 or individually from the increase of metastatic nodules was due to a decline of the time of weaning until tumor transplantation. At that time mice host resistance, rather than to local factors associated with were maintained in either the grouped (G/G) or isolated (1/1) con- the surgical procedure (57). These data clearly indicate the dition, or were transferred from the grouped to isolated (G/I) or potential impact that surgical stress may have on tumor from the isolated to grouped (I/G) condition. The inset displays the progression and metastasis, and points to the importance of tumor growth in mice transferred from the isolation to grouped minimizing operative stress in the treatment of cancer. condition comparing those mice that engaged in fighting with those As in the case of the neurochemical and immunological that did not. (From (108)). alterations, there is reason to believe that the organism's ability to cope with the stressor through behavioral means mastocytoma (110) as well as a Krebs-2 ascites tumor (35). may be fundamental in determining the stressor-elicited Even the daily procedures of cleaning vivaria or cages, odors variations of tumor growth. In particular, it has been demin the animal colony, background noise conditions, as well onstrated that escapable shock did not affect the growth of as the stress of shipping animals from the breeders have been a PSI5 mastocytoma or rejection following application of shown to influence the development of spontaneous mam- Walker 256 cells (107, 125). In contrast, however, as shown mary tumors (9S). Likewise, it was demonstrated that dis- in Figure 4, an identical amount of inescapable shock was turbances in social conditions, such as transferring mice shown to enhance the growth of the syngeneic tumor and from isolated to grouped conditions, or vice versa, would also decrease rejection of the non syngeneic tumor. Inasinfluence the course of tumor development (35, 110). In- much as the same stress manipulations were shown to interestingly, these studies also revealed that such effects fluence central neurotransmitter activity, most prominently would be dependent upon the behavioral styles animals biogenic amines, it was suggested that the effects of the exhibited following the social disturbance. In particular, the stressor may be due to the altered transmitter activity. It has increased tumor growth observed following transfer from since been demonstrated that those stressor parameters isolated to grouped conditions was not evident among mice that engaged in fighting (see Figure 3). It was suggested that since fighting may offset some of the consequences ordinarily elicited by other stressors (e.g., stress-provoked central E300 neurochemical changes, as well as stressor-induced gastric E ulceration; see 113, 129), fighting in these studies also acted ~ 250 ]" as a coping factor to offset the adverse consequences engen-

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Compound

Mouse, rat

Man

Diethylstilbestrol

Oral contraceptives

Catechol TPA Merezein Teleocidin A + B (Lyngbyatoxin) Aplysiatoxin

Promoters:

Drugs Sera

Cancer in pituitary, mamma and vagina Benign & malignant hemorrhagic liver tumors

Man

Nickel

Bronchial cancer, especially when combined with smoking, Mesothelioma Sarcoma

Bladder cancer Liver tumors Hepatocellular carcinoma Colon cancer Bladder cancer, liver cancer

Organ specificity Injection site

Bronchiogenic cancer Upper respiratory system

Rat, mouse

Man

Cattle Rat Rat Rat Mice, rats, dogs

Species in which tumor occurred

Man

Formula

Chromates

Iron-carbohydrate complexes

Asbestos

Natural products, food contaminants and additives, and drugs mold toxins, drugs, antibiotics

Groups of compounds

Cocarcinogens:

Immunosuppressors:

Hormones:

Inorganic chemicals:

Solid state carcinogens:

Type of carcinogen

Table 1. Continued.

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3: The seven types of causes of neoplastic growth ing and they playa role in air, soil, and water pollution. Certain chemical carcinogens are also in use of chemotherapeutica. This is possible, because as with x-rays the shortterm effect is therapeutic, the long-term effect carcinogenic. For selected details see Table 1.

Table 3. Radioactive emissions.

Physical carcinogens (16, 18) The second most important group of carcinogens is that of physical causes. Two types of carcinogens can be distinguished: (1) solid physical products operating through pressure or by other ways, continued irritation of the tissues, such as structures interrupting the metabolism if implanted, e.g. foils, or inhaled fibers and particles from asbestos; and (2) rays such as x-rays or ultraviolet light (sunlight), or heat. ad 1. Continued pressure of a needle on a tailor's thumb or the pressure of the hammer on the hand of a shoemaker may lead to cancer after many years. The same is the case with the bridle in the mouth of the horse. If we implant small metal foils under the skin of a rat it may lead to neoplastic development. The best-known carcinogenic agent of this kind is the cancer-producing activity of crumbling asbestos fibers; if inhaled they produce mesothelioma. Foils of metal and plastics have been widely used in experimental animals; other comparable structures such as remnants of bullet shells, where metal pieces remained in different organ sites of man (soldiers) especially through war should not be forgotten. Other physical carcinogens are dust particles like concrete particles from the surface of highways or rubber particles from the tires of cars distributed by abrasion. ad 2. Radiation. The effect of rays includes also the effect of heat, which may cause cancer, especially when continuously applied or applied in combination with other carcinogens,

23

Carcinogen

Site or effect

Radium Emission Radiothorium Mesothorium Thorium X Radioactive isotopes

Bone, leukemias Lung Skin, bone and leukemias Skin, bone and leukemias Skin, bone and leukemias Skin, bone, glands, leukemias

Uranium & atomic bombs Leukemias

'such as tobacco smoke, as in the case of the burning of a pipe. Pierre Masson regarded the melanogenic cells as a special histologic system in our body. It is responsible for the dark skin of the black and other noncaucasian races; examples in causasians are the dark skin of the nipple, the anus and the sex organs. Other portions of the body include parts of the eye and parts of the brain (nucleus niger). But melanogenic cells do occur in the whole skin (sun tent) of caucasians. Extensive sunlight, through its ultraviolet portion, is able to produce malignant melanoma, the most dangerous neoplasm of the skin and one of the most aggressive neoplasms to be found. (Ippen). Malignant melanoma caused by ultraviolet light is one of the most hazardous of neoplasms involving radiation. X-rays were detected in 1896 by Conrad Wilhelm ~oent­ gen. In the early years of radiology physicians dealing with the developing speciality of radiology suffered from neoplastic development produced by these rays. Today, x-rays are a therapeuticum against different neoplastic diseases because neoplastic cells are more sensitive to radiation than are normal cells. But very often radiologic treatment of such childhood tumors leads to the development of certain leukemias, or such lymphomas as Hodgkin's disease in the adult (see Chapter 7III and 14/VI). The average person receives

Table 2. Physical carcinogens. Irritant

Species

Neoplasms

A. Solid physical irritants Bridle

Equus cabal/us, horse

Needles

Homo sapiens, man (tailor)

Metal foils Plastic foils Asbestos

Rattus norvegicus, rat Rattus norvegicus, rat Homo sapiens, man

squamous cell cancer of corner of the mouth thumb, squamous cell cancer implant: sarcoma implant: sarcoma mesothelioma

Homo sapiens, man

cutaneous melanoma

Bos sp., cattle in Australia

cutaneous melanoma

Equus cabal/us, white horse

cutaneous melanoma

Homo sapiens, man

cutaneous melanoma

Homo sapiens, man different sites Laboratory animals Various mammals

various neoplasms

B. Rays

Ultraviolet rays (sunlight) Ultraviolet rays (sunlight) Ultraviolet rays (sunlight) Ultraviolet rays (ultraviolet lamp) X-rays

X-rays Heat

various neoplasms mainly squamous cell cancer

Vertebrates (also Eumetazoans)

Platyhelminthes Arthropoda

Monocots

Vascular plants

Class Amphibia

Hepatic carcinoma Leukemia, leucocytosis Lymphosarcoma Lymphosarcoma Plasma cell leukemia Undifferentiated malignant tumor Skin sarcoma Frog kidney carcinoma Frog kidney carcinoma Frog kidney carcinoma Frog kidney carcinoma Lymphosarcoma

Virus? Virus? Virus Virus Virus Virus Virus Herpes virus Herpes virus Herpes virus Herpes virus Virus

Baltic pike Rana pipiens (Leopard frog) Rana calmitans (Green frog) Rana catesbiana (Bull frog) Rana palustris Xaenopus laevis (South African clawed toad)

Salmo gairdneri (rainbow trout) Mugil curema Valenciennis (salt water mullet) Esox masquinongy (muskellunge) Esox lucius (northern pike) Ictalurus nebulosus (Brown bullhead) Pristella riddlei

Drosophila melanogaster Bombyx mori Agrotis segetum

0.: Lepidoptera

RSV, SV40 Tipula virus Granulosis virus I and 2 (expeL applied)

"Intestinal tumors" Cell proliferation in esophagus Cell proliferation in esophagus Proliferations at midintestinal epithelium Melanotic changes Epidermal proliferations

Oncornavirus Western-X -disease virus W estern-X -disease virus Polyhedrosis

Locusta migratoria Leucophaea maderae Callodomus montanus

Bending of tail

Virus

Gilpinia hercyniae

Class Pisces (fishes)

Fiji disease

Virus of Fiji disease Wallaby ear Club root Enations disease Wound tumor Wallaby ear disease Club root Enations virus Wound tumor

Disease

Virus

Dugesia dorotocephala

Zea mays Nicotiana sp. Nicotiana pomiculata Sweet clover

Saccharum sp.

Host. species

0.: Diptera

Class Planaria Class Insecta 0.: Orthoptera

Dicots

Lower systemic unit

Higher systemic unit

Table 4. Oncogenic viruses.

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M armonata monax

Hylobates sp. (Gibbon) Oryctolagus cuniculus (Old world rabbit)

Herpesvirus saimiri

Callitrichidae, marmosets Callitrichidae, marmosets Lagothrix sp. (woolly monkey) Macaca mulatta (rhesus monkey) Papio sp. (Baboon)

Leukemia Lymphosarcomas Transmissible Gibbon lymphosarcoma Myxomatosis

C-type virus particles

Rabbit fibroma virus Rabbit oral papilloma virus Virus of transmissible fibroma (Syrian hamster)

Myxomatosis virus

Transmissible fibroma of grey squirrels Virus

Shope rabbit fibroma Oral papillomatosis

Spontaneous mammary tumor

Virus particles

C-type virus particles

Fibrosarcoma, spontaneous

Burkitt's lymphoma Kuru Creutzfeldt-Jakob disease (presenile dementia) Malignant lymphoma

?

Warts, papillomas

Lymphoid leucosis Ocular lymphomatosis Osteopetrosis Neurolymphomatosis (Marek's disease)

Avian erythroblastosis

Avian myeloblastosis

Rous chicken sarcoma

C-type virus particles

Herpesvirus ateles

Papilloma virus HTLV C type virus of placenta Epstein-Barr virus

Rous sarcoma virus

Homo sapiens and the mammals

Gallus gallus

Skin papillomas in Syrian hamsters.

Rabbit species vary considerably in the susceptibility to infection with this virus.

Virus isolated from healthy squirrel monkey Virus isolated from black spider monkeys (Ateles sp.)

Transmission to chimpanzees, New world monkeys, cats, mice and rats

This virus exhibits different strains (Carr-Zilber strain, Prague-strain, Schmidt-Ruppin strain, Bryanstrain, Harris-strain and Mill Hillstrain) and can induce tumors in ducks, pheasants, turkeys, pigeons, beside chickens but also in mice, rats, hamsters, guinea pigs, rabbits, and monkeys. Avian myeloblastosis and erythroblastosis are the true leukemias of the chicken leucosis complex, whereas the following diseases belong to the extravascular group of the chicken leucosis complex.

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INITIATION

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Figure 1. Multi-hit model of chemical carcinogenesis, illustrated by a 3-hit process. a) normal cells; b) initiated cells; c) more advanced precancerous cells; d) tumor cells. Thunderbold: (genetic) rare event, hit.

presupposes resting cells (Figure I b). Proliferation of intermediate cells, having acquired already one or more critical alterations, would however increase the intermediate cell population (Figure Ic), and thus markedly enhance with time (age) the probability of further conversion (45, 46, 166, 167). It can be shown (5, 45, 53) that according to the rate of proliferation of the latter cells assumed for the calculations the number r of "apparent" critical events becomes reduced to 2-3 "real" rare events. They may be brought about by carcinogen or occur spontaneously. The critical components to be altered are considered to be DNA molecules, and the discrete changes-hits-may thus represent somatic mutations as originally proposed by Boveri (22) and by Bauer (16). The somatic mutation hypothesis has been the subject of recent critical reviews (157, 188). The arguments against this hypothesis are mainly based on the differentiational potential of i.a. the teratocarcinoma in mice, and on the (high) transformation probability as observed in certain in vitro

129

systems. It is supported by the mutagenic potency of most if not all carcinogens, the human hereditary syndromes predisposing for cancer and by recent evidence accumulated in the field of oncogene research. Pathways of activation of (cellular) proto-oncogenes were found to involve i.a. alterations of DNA, such as adjacent insertion of a provirus or a point mutation at a critical site of the structural gene (99, 190). The step from normal cells to cells of stage I is generally called initiation, and only a small fraction of cells of an exposed organ - in the order of I per million (164) - becomes initiated by the treatment with carcinogen. It is strongly indicated that this step involves a specific somatic mutation caused by the applied carcinogen, and that these initiated cells represent the precursors - the cells at risk - for the second specific change. Therefore their number is of prime importance for the overall probability that cells with two (and more) specific changes, and thus that cancer will develop. The number of initiated cells increases with the doserate of carcinogen and the duration of exposure. More important, however, may be the proliferation in time of the initiated cells themselves. This process of clonal expansion can be influenced by factors modifying carcinogenesis such as promoters and anticarcinogens. Efficient clonal expansion should dramatically increase the probability that any one of the initiated cells undergoes a second specific change. The resulting new cell, if it has a selective advantage such as its precursor population, will again develop into a cell clone which eventually gives rise to a cell with a third specific change. In this way properties as independence of specific growth factors and metastasizing capacity may develop step by step. The multi-hit model of neoplastic evolution (Figure lc) thus appears as the most appropriate one for the discussion of cancer formation either under conditions of prolonged exposure to carcinogens, or after short exposure which is followed by promotion. The one-step model (Figure la) is inconsistent with the steep time-tumor incidence kinetics observed generally in human and experimental carcinogenesis (23, 45). The multistep model with nonproliferative intermediate cells seems improbable as long as relevant cells with proliferative advantage are induced together with the non-proliferative intermediate cells. A multistep model intermediate between the models band c, could apply to rapidly renewing epithelia, where precancerous alterations eventually lead to a steady-state situation after some early clonal expansion. A case in point may be the epithelia of the intestines.

NEOPLASTIC STAGES IN EXPERIMENTAL LIVER CARCINOGENESIS The summativity of even smallest doses of carcinogen during the "latency" period preceding the appearance of cancer «43, 44), previous section) implies that each single dose - even if subcarcinogenic - must induce relevant alterations which mayor may not manifest themselves by an altered cell phenotype, allowing their demonstration by suitable methods. The summativity also implies that relevant alterations must persist after their induction; this makes it possible to differentiate between the potentially relevant cellular alterations and the non-relevant ones (toxicity),

130

Ewalt Scherer

which generally will disappear after discontinuation of the carcinogenic treatment. The discussion of neoplastic progression will be limited to the rat liver system. For mouse liver, an experimental system which has received increasing attention during the last few years, and which exhibits many parallels to the rat liver system, some recent references are added: Frith et al. (60), Moore et al. (114), Becker (18), Tennekes et al. (195), Goldfarb et al. (64), Koen et al. (93), Vesselinovitch and Mihailovich (202), Vesselinovitch et al. (201,203,204). Early focal alterations

Persistent focal alterations ofliver parenchyma are observed in carcinogen treated rats long before the occurrence ofliver cancer, by enzyme-histochemical and routine histological methods (46). These lesions and liver carcinoma were classified by a recent workshop (185) and an expert committee (83), mainly on the basis of routine histology. Foci and areas distinguished were: clear cell foci. exhibiting an empty appearance of the cell cytoplasm due to the excessive storage of (nonstaining) glycogen; acidophilic foci. standing out against the surrounding, normal liver because of intense acidophilic staining (eosinophilia); basophilic foci. exhibiting a diffusely basophilic staining of the cell cytoplasm. Enzyme histochemical techniques which allow the very early recognition of even single altered cells and their characterization with respect of specific functional markers, have not or only

marginally been covered. This classification may therefore fail for the differentiation of the early precancerous stages of rat liver carcinogenesis. The earliest focal alteration of rat liver parenchyma as induced in high number by (subcarcinogenic doses of) hepatocarcinogenic agents, are mainly of the clear cell type, i.e. at least part of the focus cells exhibit a changed carbohydrate metabolism leading to the excessive storage of glycogen. The changed carbohydrate metabolism has been emphasized especially by Bannasch and coworkers (13, 72). The basic properties of these foci are best studied after a single low dose of a liver carcinogen exhibiting low toxicity, such as DEN (46, 171). This avoids as far as possible the modifying influences of the carcinogen itself and the formation of more advanced cell stages. Focus cells are arranged in plates of one cell thick. The focus cells and their nuclei are increased in size especially at the central venous part of the focus which deviates more from the often slightly compressed surrounding liver tissue than its periportal part (Figure 2). There the focus cells blend into the normal liver, structurally as well as in respect of staining properties (Table I). This staining gradient develops as foci induced in the periportal part of the liver lobule reaches the central venous area (phenotypic change from type IA to IB, (46). It is obvious for the markers ATPase, G6Pase, GGT, glycogen, epoxide hydrolase and others. At first sight this staining heterogeneity of the foci seems to be in contrast with their well-documented clonal origin (143, 163), but it becomes reasonable if the functional

Figure 2. Precancerous foci induced in rat liver by a single. subcarcinogenic dose of DEN (10 mg/kg), 24 hours after two-thirds hepatectomy. The foci exhibit a gradient of marker expression. (a), kryostat section, ATPase reaction; (b), paraffin-embedded section, hematoxylin and eosin. The ATPase reaction is most decreased at the central venous, peripheral part of the focus, where most glycogen is stored. The glycogen content confers the clear cell character in paraffin sections (b). Bar, 100,um; P, portal triad; C, central vein.

131

16: Neoplastic cell stages and progression in experimental hepatocarcinogenesis Table 1. Histochemical markers of precancerous foci of rat liver. Staining reaction

Marker expression

Change during progression

Normal rat liver

Precancerous foci

ATPase G6Pase

+++,H + + to + + +, G

o to o to

++, G ++, G

GGT

o to (+), G

o to

+ + +, G

Glycogen fed animal starved Iron accumulation

+ + to + + +, G o to +, G

+ + + to + + + +, G o to + + +, G

decrease decrease

++

o to

+

?

PHO

+ to ++, G

o to

+, G

G6PDH GAPDH

0 ++

+ to + +, G ++ to +++

increase increase

Cytochrome P-450 EH L-PK GST-P

o to

++, H

increase?, later decrease decrease ? ?

+

+ +,G 0

+ to + ++, G decreased ++

decrease decrease, later increase increase, later decrease?

Ref

50

} 72, 112

174 94 152 160, 161

H, homogeneous staining; G, staining gradient. Abbreviations: PHO, glycogen phosphorylase; G6PDG, glucose-6-phosphate dehydrogenase; L-PK, pyruvate kinase, liver type; G6Pase, glucose-6-phosphatase; GGT, y-glutamyltranspeptidase; EH, epoxide-hydrolase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST-P, glutathione S-transferase, placental type. heterogeneity of the liver lobule itself, exhibiting activity gradients for many liver cell functions, is considered (66, 210). Redifferentiation of focus cell clones after discontinuation of promoting (selecting) regimens (47, 192), leads to a similar type of heterogenous focus (46). This type is by far the most prevalent one after subcarcinogenic doses of alkylating carcinogens. As these foci exhibit some of the functional gradients of normal liver it seems that they are still under firm differentiational control. It has to be stressed that this staining gradient of individual foci is not always obvious in the focus trans sections as observed in microscopical slides. Depending on the orientation of the focus with respect of the section plane either a homogenous weak or strong deviation from the normal surrounding liver tissue, or a gradient type of focus will be observed. As staining gradients can be of opposite direction (ATPase vs. GGT) this will lead to an apparently increased staining heterogeneity of foci. The latter may apply to some of the many phenotypes described by Pitot and coworkers (131). The biochemical and enzymatic properties used as marker for the identification of precancerous foci were compiled by Farber (50), and updated recently (162); the most commonly used properties are given in Table I. The homogenously positive ATPase reaction of the cytoplasm of normal hepatocytes, combined with the most obvious decrease of ATPase reation being at the periphery of the foci, makes the ATPase staining especially useful for the recognition and quantitation of even slightly aberrant foci. The cytoplasmic and bile canalicular reactions are both decreased. GGT (73) has been advocated as a universal marker of precancerous foci; however, as the staining of focus cells - if at all present

- is most intense at their central and periportal part of the focus, peripheral transsections may not be detected and the size of the foci underestimated. After treatment with PB the GGT is induced and foci are best recognized by this marker. Foci induced by hypolipidemic drugs were found to exhibit no increase of GGT activity (147, 187). ATPase deficient foci persist after their induction and increase slowly in size up to nearly macroscopic dimension (171). Under nontoxic conditions the rate of proliferation of these foci is independent of the inducing carcinogen (46, 165). It is indicated that this slow proliferation is due to an increased response of focus cells to endogenous growth stimuli (14, 89, 140). Many hepatocytic functions are changed coincidentally in focus cells; since a one-hit mechanism underlies focus formation (164), it is probable that the target site of precancerous transformation is a gene which influences or regulates the expression of a whole program of structural genes. Neoplastic stages Primary neoplasms distinguished according to recommended nomenclature (83, 185) are neoplastic nodule and the various patterns of hepatocellular carcinoma: trabecular, adenocarcinoma, poorly differentiated and hepatoblastoma. These are more advanced stages than the early foci from which they are believed to be derived. Neoplastic nodules are characterized by acidophilic and basophilic cell cytoplasm. The acidophilic reaction - intense staining by eosin - results from the proliferation of agranular endoplasmic reticulum (13), the diffusely basophilic staining of the cyto-

132

Ewalt Scherer

plasm corresponding to free ribosomes combined with a relative paucity of parallel-arrayed, rough endoplasmic reticulum (80). Cells and ,nuclei are enlarged or small (constant within a nodule or somewhat pleomorphic) forming cell plates of more than one cell thick. The neoplastic nodules may still store glycogen in excess, whereas the hepatocellular carcinoma is characterized by a progressive loss of glycogen correlated with a shift of the carbohydrate metabolism towards glycolysis and the pentose phosphate pathway (72). Recommended nomenclature seems inappropriate for the classification of more subtile differences, as observed in cell populations which may form intermediate stages between the early, non-uniform staining focal lesions and the obviously neoplastic stages. It has been argued (46) that the large enzyme-altered areas observed about 1 year after a single dose of 10-20mg DEN/ kg applied to partially hepatectomized rats, may represent a more advanced cell stage (type II island). A quite uniform, pronounced deficiency of ATPase and G6Pase reactions and homogenous glycogen storage histochemically characterize these nodules which seem to be less dependent on the local tissue environment for the expression of their markers than the early enzyme altered foci. The number of these small nodules was increased in a dose-dependent way by DEN applied for one month, 13 months after initiation by the above procedure (46). Persistent hyperplastic nodules as obtained by the cyclic feeding of AAF or ethionine (48), or by the also quite toxic Solt-Farber protocol for the selection of initiated cells (183,184), may correspond to these type II islands. Attempts to transplant cells of hyperplastic nodules were unsuccessful (49), which is, however, in contrast to

more recent reports on the transplantation of normal and altered liver cells (52, 101) and to the ease of transplantation of the neoplastic focus lesions as obtained by the initiationpromotion-initiation protocol «(172), section Induced progression). Ultrastructurally persistent hyperplastic foci, as identified by the resistance to iron accumulation, exhibited increased rough and smooth endoplasmic reticulum (80) whereas the neoplastic (basophilic) foci which resembled ultrastructurally the transplants obtained from focus in focus lesions (172) were characterized by the presence of numerous free polyribosomes diffusely scattered throughout the cytoplasm and a distended rough endoplasmic reticulum lacking parallel-stacked areas (80, 196) NEOPLASTIC PROGRESSION

Experimental systems Various experimental systems are commonly used for the study of neoplastic progression in rat hepatocarcinogenesis, viz. continuous application or special feeding cycles of potent hepatocarcinogens, a single high dose of carcinogen in combination with partial hepatectomy or subcarcinogenic initiation followed by tumor promoting regimens. Some of these experimental systems were recently discussed by Emmelot and Scherer (46). New promotion protocols comprising widely different groups of compounds (Table 2) were developed during the last years. The tumor endopoints reached by the different protocols

Table 2. Modulators of rat liver carcinogenesis, and their effects on the size of precancerous foci and on the incidence of neoplastic nodules and hepatocellular carcinoma. Class

Compound

Focus tissue

Tumor incidence

Ref

Carcinogens

AAF IPH, AAF ICC 14 DMN, DEN, Aflatoxin BJ 3'-Me-DAB, Ethionine ENU PB

+

++

102, 183, 184, 198, 199

+ +

++ ++

76, 85, 198, 214 15,65,76,91,96,102,113,119, 125-127,173,175,198,205

PCB (Arochlor, Clofene) DDT HCH TCCD ethinyl estradiol oral contraceptives progesterone cyproterone acetate

+ + + + + + + +

+ +

38, 88, 116, 120, 121, 132, 138 85,92, 126 173 132 27,85 211 173 175

Nafenopin, Clofibrate orotic acid fumaric acid ZAMI1305 choline-deficient diet BHA, BHT, ethoxyquin

+1-

+1-

+

+

+

+

Sedatives Chlorinated hydrocarbons

Steroids

Hypolipidemic drugs Miscellaneous

Antioxidants

+

+ +

+

89,119,151,173 31, 100 98 137, 145, 216 177,178,191 61, 198

Abbreviations: PH, partial hepatectomy; PB, phenobarbital; PCB, polychlorinated biphenyl; DDT, I, I, l-trichloro-2,2-bis (4-chlorophenyl) ethane; HCH, hexachlorocyclohexane; TCCD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; BHA, butylated hydroxyaniso1e; BHT, butylated hydroxy toluene.

16: Neoplastic cell stages and progression in experimental hepatocarcinogenesis

within life span of the animals are not necessarily the same. In the case of continuous DEN application, the rats die from internal bleeding of a small anaplastic area which can sometimes be located within a differentiated hepatocarcinoma (164). After limited treatment with DEN the rats end up with large benign-appearing hepatomas (168), which are equivalent to neoplastic nodules, and highly differentiated hepatocarcinoma. Application of AAF at a not overly hepatotoxic dose level (0.02%) for up to 13 weeks (208) resulted in small macroscopic foci which mainly remodelled phenotypically after discontinuation of the treatment; by a cyclic feeding protocol (4 cycles, (79» small nodules of only about I mm diameter were obtained, with progression to basophilic foci within 24 weeks after termination of treatment. Depending on the number of cycles AAF is fed at a toxic dose level (0.06%), remodelling (3 cycles) or persistent (4 cycles) nodules appear. The latter nodules further progress to hepatocellular carcinoma (17, 194). A single high dose of carcinogen applied after partial hepatectomy (36, 164) leads to neoplastic nodules and - at high age - to mainly highly differentiated hepatocellular carcinoma. Initiation-promotion regimens give rise to increased amounts of early focus tissue and to neoplastic nodules ("hyperplastic nodules"), and hepatocellular carcinoma (for references see Table 2), sometimes with pulmonary metastases (100, 184). Several drugs exhibiting no overt genotoxic/mutagenic activity were shown to induce foci of cellular alteration and liver tumors if applied for prolonged periods of time. A case in point are the antihistaminic methapyrilene (54, 62, 70, 153), hypolipidaemic agents (e.g. clofibrate, nafenopin) casing peroxisome proliferation (67, 148, 150, 151) and the f3-blocker ZAMI 1305 (145). For the latter substance, however, DNA damage and initiating capacity was recently reported (136, 137, 216). Many drugs used as tumor promoter in rat and mouse liver were also found to be carcinogenic after prolonged application (201). This initiating/ syncarcinogenic potency of promoters is probably involved in the occurrence of more advanced neoplastic phenotypes during prolonged promoter treatment. Precursor-product relationships

The assumption that cancer develops through a progressive sequence of precancerous and neoplastic cell stages is commonly based on the comparison of the putative precursor cells and the tumor cell in respect to cytological criteria, the time of appearance and the location within the tissue affected (46). Precancerous cells in rat liver share common features with the tumor cells (59, 63, 72, 81,123), and there is a good correlation between induction of precancerous cell stages in rat liver and the hepatocarcinogenic potency of an agent. ENU, not a classical liver carcinogen, was shown to induce efficiently enzyme altered liver foci and to contribute - as expected - to the hepatocarcinogenic process completed by DEN (46). Foci of enzyme-altered cells precede enzymealtered nodules and neoplastic nodules, which again precede the occurrence of glycogen-free hepatocellular carcinoma. In addition the amount of precancerous liver tissue (enzymealtered cells) was shown to be statistically related to the probability of cancer formation (164). The lobular focus

133

distribution was correlated to the subsequent development of more advanced cell stages (208). The increased amount of precancerous tissue observed during liver cancer promotion, is likewise correlated with an increased incidence of neoplastic nodules and hepatocellular carcinoma (for references see Table 2). Regimens which decrease the amount of precancerous tissue (number and/or size of the lesions), such as the application of fumaric acid after initiation (98) or the feeding of carcinogen and certain drugs (phenobarbital, phenolic antioxidants) together (3, 97, 108, 124, 161) decrease the incidence of liver tumors (conflicting results (128». Phenobarbital - a good promoter if applied after the carcinogen - may interfere in this instance with the metabolic activation of the carcinogen and thus the initiation process. Likewise, the phenolic antioxidants may have reduced the availability of activated metabolites (AAF) by conjugation to glucuronic acid. Fumaric acid, a substance present in certain vegetables (Cruciferae), has been supposed to act by inhibition of the progression phase, either by slowing down the proliferation of initiated cells to precancerous foci, or by reduction of carcinogen activation (98). Direct precursor-product relationships as expected from the multi hit - multi stage model (see Kinetics of tumor formation) should be observed as areas of focal atypia within or in close association with the precancerous lesion. This principle is difficult to establish since more advanced cells may overgrow their precursor cell population and thus destroy a relationship existing previously, but also because unrelated cell populations may become topologically associated by proliferation. Lack of phenotypic diversity between precursor and product cell populations, may also counteract the detection of such relationships. Nevertheless, in quite a number of cases clear topological associations between precursor and product have been reported. Ideally, such a relationship appears as focus in focus (or nodule in nodule, depending on the size of the lesion), the proposed new cell popUlation being sharply demarcated from the surrounding precursor cells. Areas of focal atypia were observed by Epstein et al. (48) within hyperplastic nodules induced by ethionine or AAF. Kitagawa and Sugano (80), using combined enzyme histochemistry and CH)-thymidine labelling/autoradiography, demonstrated the occurrence of enzyme-deficient foci within remodelled, enzyme-rich areas of hyperplasia; clonal development of the new cell population was indicated. Scherer and Emmelot (164) presented examples of close association between glycogen-free hepatocellular carcinoma and glycogen storing focus tissue. The close association observed between anaplastic, hemorrhagic tumor tissue and differentiated hepatocellular carcinoma (164) suggested that the latter tumors served as precursor for the anaplastic tumor cells. A relationship between neoplastic nodule and hepatocellular carcinoma has been presented by Solt et al. (183). Hepatocellular carcinoma surrounded by benign hepatoma tissue was observed by Kuhlmann et al. (94) in immunochemically stained (epoxide hydrolase) sections of NNM treated rats. Hacker et al. (72) showed a neoplastic nodule containing a focus of cells with enhanced basophilia and glyceraldehyde-3-phosphate dehydrogenase reaction and decreased glycogen content. Similar observations were regularly reported without photographic documentation.

134

Ewalt Scherer

Related cell populations are often not as sharply demarcated as the cases cited above. Two such examples suggesting a step-like change between the early foci of non-uniform marker expression and the foci or nodules which exhibit a uniform staining pattern, have been presented by Emmelot and Scherer (46). The increasing irregularity of the enzymehistochemical picture generally observed during continuous low-dose treatment with DEN or other hepatocarcinogens long before the first well-differentiated hepatocellular carcinomata are observed, may reflect such obscure alterations of parts of enzyme altered foci, often segments, exhibiting slightly changed architecture, marker expression (ATPase, G6Pase, glycogen), fatty vacuolization, or increased eH)thymidine labelling index. Most of these alterations become obvious only by the direct close contact of the involved cell populations. The lobular architecture is maintained and the plates of hepatocytes that compose both cell populations merge imperceptibly. The highly differentiated trabecular nodules which generally store glycogen in excess may result from such a hidden second change. Induced progression by an initiation-promotion-initiation protocol

In view of the occasional observation only of precursorproduct relationships between cell populations involved in rat liver carcinogenesis, it is not surprising that the exact sequence of cellular stages involved has not been established. An experimental system for the reproducible induction of progressive changes is needed for a more close investigation. In order to fulfill this need we developed a new protocol (169, 170) which is based on the hypothesis that the phenotypic changes exhibited by more advanced cell populations are due to an additional rare genetic event, a specific somatic mutation. Accordingly, this rare event should be inducible in precursor cells by genotoxic (mutagenic) carcinogens, most efficiently by direct acting ones which do not need metabolic activation. A large precursor cell population would increase the chance that a specific event is scored, and advanced cell populations thus will be observed. The experimental protocol (Figure 3) consequently consisted of initiation by a single subcarcinogenic dose of DEN applied to female Sprague-Dawley rats after 2/3 hepatectomy (171), expansion of the initiated cell population by a strong growth selection stimulus (promotion, (183,184)), and a second dose of a strongly initiating carcinogen. ENU was used since it is directly active, leads to the same pattern of DNA modifications as does DEN and induces high numbers of enzyme altered foci in regenerating rat liver (46). Such an experi-

mental protocol is a logical consequence of the multi-hit concept of carcinogenesis (135). This experimental protocol resulted - without second initiator - in a high number of enzyme altered foci of the non-uniform staining type, and no or only a very few more advanced foci. This lack of promotion which contrasts to the original findings of Solt and coworkers (183, 184) can be explained by the much lower toxicity in female rats, as compared to the originally used males, of the AAF used as selecting agent. If ENU (25-100 mg/kg) had been applied as second initiator at the end of the selection regimen, some of these foci had developed a small secondary focus (0.30.5 mm in diameter) 10 weeks after ENU (Figure 4). These secondary foci were characterized by more pronounced and uniform marker deficiency (ATPase, G6Pase), and eosinophilic and homogenously, slightly basophilic cell cytoplasm. Plates were more than one cell thick and the surrounding focus tissue was slightly compressed and tangentially arranged. This architecture implies a centrifugal, probably clonal, growth of the secondary focus. About lout of 50 enzyme altered foci contained such a secondary focus, a figure which is consistent with a transformation frequency as expected for a somatic mutation with precancerous focus cells as the population at risk (170). With time the secondary foci enlarged and became visible at autopsy (100 days: 0.52 mm, 200 days: 1-15 mm in diameter). Even at this time a close association could regularly be obs'erved between the heavily eosinophilic neoplastic nodule and its putative precursor tissue, covering it as a thin rim of enzyme altered, heavily glycogen storing (clear) cells (Figure 5). The neoplastic cell population itself generally still stored glycogen, however, no longer highly concentrated in certain areas of the cytoplasm (clear cell character), but more homogeneously distributed; mixed cell character (13) was generally observed in these neoplastic nodules, Cells prepared from these neoplastic nodules by collagenase digestion could be successfully transplanted into partially hepatectomized and - to a lesser extent - intact isogenic rats by infusion into a mesenteric vein (172); this stresses the franc neoplastic character of the secondary foci, III defined local deviations of the phenotype of nonuniform enzyme altered foci suggesting a change to the more uniform trabecular type (type II, (46)) were observed regularly, but could not yet be evaluated quantitatively because of the lack of a specific, unequivocal marker for the proposed new cell population. Roughly, this seems to occur as frequently as that to the eosinophilic (neoplastic) secondary focus. Progressive sequence

PH DEN

.------...Ji(~ENU

~#~----~IAAF~--------

Figure 3. Initiation-promotion-initiation protocol consisting of a

single dose of DEN (lOmg/kg) given after two-thirds hepatectomy (initiation), a rest period, 1 week of AAF application (15 mg/kg daily), partial hepatectomy and another week of AAF (promotion), and a single dose ofENU (25-100mg/kg) (second initiation). Animals are killed 10 or more weeks after the second initiation.

The observation of direct precursor-product relationships can be summarized in a scheme of neoplastic evolution of liver cancer in the rat (Figure 6): Normal hepatocytes, probably of the proliferating peripheral compartment of the lobule, are transformed during the initiation step by a onehit mechanism into focus forming cells which grow out (more or less quickly) into foci, especially of the nonuniform staining type, Some uniformly staining foci present early in carcinogenesis may be part of the spectrum of different focus phenotypes (46) induced by the initiating hit.

16: Neoplastic cell stages and progression in experimental hepatocarcinogenesis

135

Figure 4. Two different precancerous foci containing a secondary focus (arrows), which exhibits a homogeneously low ATPase reaction. Initiation-promotion-initiation protocol, 10 weeks after a single dose of 50 mg/kg ENU. Bar, 100 Jim; C central vein.

Figure 5. Eosinophilic tumor nodule induced in rat liver by the initiation-promotion-initiation protocol, 200 days after a single dose of 50 mg/kg ENU. (a), paraffin section, hematoxylin and eosin; the nodule is surrounded by a thin layer of glycogen storing focus cells (clear cell character); (b), higher magnification of the nodule cell population. Apoptotic bodies (arrow) and mitoses are encountered regularly. Bar, 100 Jim (a) resp. 25/-lm (b).

136

Ewalt Scherer

The progression of these foci to the neoplastic, mixed cell stage is due to a second hit as strongly indicated by the initiation-promotion-initiation experiment. Less well established is the proposed change from the non-uniform focus to the uniform trabecular nodule (type IB --t type II, (46)), since its study is hampered by the lack of a suitable marker. The persistent nodules induced during the 4th cycle of AAF-feeding, by subsequent application of DMN (17) or in low frequency during promotion, comprise probably both these types of advanced lesions. The sequence of further progression is mainly speculative. Such steps are, however, indicated by the analysis of timetumor incidence kinetics and by occasional histologic observations (see Precursor-product relationship). Emmelot and Scherer (45) obtained for rat hepatocarcinogenesis roughly a power of7 for the dose - tumor incidence relationship by continuous application of DEN. This figure has been equated to 5-7 dose-dependent steps (45, 46) being instrumental in the induction of the lethal, hemorrhagic type of anaplastic liver tumor; a lower number of hits, 2-3, was derived for the induction of less malignant liver tumors as encountered in stop experiments. Burns and Albert (24), using a similar approach for the analysis of the kinetics of lethal liver tumor formation by multiple doses of AAF, calculated a power of 3, equivalent to 3 dose-dependent specific changes or hits. Accordingly, the highly differentiated trabecular carcinoma could either be the result of further proliferation of the uniformly staining trabecular nodule, or, since such nodules likewise show local phenotypic deviations, it could be the product of an additional step of clonal progression to a phenotypically similar tumor type. The further neoplastic progression of the eosinophilic secondary focus (neoplastic nodule) remains open for discussion too. It has been stressed (13) that the glycogen-free differentiated hepatocellular carcinoma develops from the mixed cell neoplastic nodule. This change to deficiency of glycogen storage relatively late in the carcinogenic process has since long been recognized. Further experiments performed in analogy to the initiation-promotion-initiation protocol, i.e. application of carcinogen to animals harboring transplanted neoplastic nodules in high number, may help to further elucidate the progressive sequence, provided that such a protocol leads to more advanced nodule in nodule stages. Preliminary results of such an experiment (Scherer, unpublished observations) showed that neoplastic nodules can give rise to various subpopulations, most of which seemed to have no increased neoplastic potential: the subpopulations as observed by changed marker expressions or trabecular organization, remained small and did not compress the surrounding nodule tissue. This is expected for a non-directed, stochastic process (46) where only a minute fraction of the possible (genetic) alterations should be relevant for the carcinogenic process. The observed progressive changes in neoplastic nodules were especially into the direction of more pronounced ATpase and G6Pase deficiency combined with broadened trabeculae. Most interesting was the change to an increase again of the G6Pase activity together with the loss of the capacity to store glycogen, and a pronounced increase in the proliferation rate. Cell pleomorphism and mitotic figures

Relati ve

Anaplastic

degree of

carcinoma

autonomy

I

-If

I

Less well

differentiated ;jrCinOma

I

... I I I

"

Differentiated

carcinoma

I I

(G6Pase + J

glycogen + /-) I I Trabecular

I

carcinoma (glycogen ++)

~

/ 1( /

Neoplastic

I

nodule

Eosinophilic". T(G6Pase 0, neoplasti c '"

glycogen ++) I

focus/nodule

I

Enzyme

(G6Pase +,

't:,

glycogen ++,

t //

"Highly

"persistent") .. differentiated

carcinoma

Hyperplastic

(G6Pase +/-,

nodule

glycogen +++)

(G6Pase +,

altered " / glycogen +++ foci

I

highly trabecular,

(G6Pase ++, glycogen

"perSistent!!)

+ ++

"remodell ingl!)

Normal

hepatocytes

Number of steps/hits

Figure 6. Proposed sequence of steps of neoplastic progression in rat

hepatocarcinogenesis. Bold arrows, experimentally well established; light arrows, strongly indicated; broken arrows, some indications.

were generally obvious in these new tumor cell populations. It is not yet known whether the change to the glycogen

deficient carcinoma is from the original neoplastic nodule or a more advanced cell population. The complete picture of the progressive sequence will probably become still more complex: within a relatively glycogen-deficient mixed cell nodule a proliferative sub population has been observed which exhibited strongly de'creased G6Pase and increased glycogen storage. It is thus conceivable that many different options for the phenotype of progressive stages are available once the mixed cell neoplastic nodule stage has been reached. Whether all the different trabecular forms of liver cancer can ultimately progress to the anaplastic stage, and whether they are parallel or sequential forms of neoplasia, awaits further experimentation.

16: Neoplastic cell stages and progression in experimental hepatocarcinogenesis

FACTORS INVOLVED IN NEOPLASTIC PROGRESSION It is evident from the discussion of the multi hit - multi stage

hypothesis (Kinetics of tumor formation) that for the occurrence of the relevant rare events underlying the progression from one cell stage to the next, three factors are especially important - the size of the (target) cell population at risk, the rate of proliferation of the target cell population, and the transformation probability per cell. The role of these factors during neoplastic progression will be discussed in the following. Size of the target cell population The process of neoplastic progression requires cell proliferation since participating cell populations that do not increase in size have an extremely small chance of receiving a further hit. The time factor operating in carcinogenesis (21, 43, 46) can thus be understood: time allows the multiplication of the participating cells which have acquired proliferative advantage by increased proliferation and/or decreased maturation/cell death rate, and thereby leads to the enlargement of the cell population at risk to be further converted into the direction of cancer. In rat liver carcinogenesis, putative tumor precursor cells induced by a single low dose of carcinogen are capable of intrinsic proliferation (79, 164, 165, 171). The growth rate of these cell populations can be enhanced by various experimental procedures. These include regimens which differently affect the normal cells (inhibited) and the precursor cells (stimulated), mitogens and other compounds or conditions which act to a similar effect (for references see Table 2). This leads to the selection of precursor cells and - as expected from the model - to an increased rate of neoplastic progression. Programmed cell death (apoptosis) may play an important role in these population kinetics (26, 32). More advanced cell stages - the neoplastic nodule and the differentiated hepatocarcinoma - fulfill this need of enlargement of population size even better, since their intrinsic growth rate is considerably higher than that of the first precancerous cell stage. Especially important, these lesions can become much larger than the early foci which indicates altered vascularization (1, 33, 56, 180, 182). The enhancement of the carcinogenic process - once started - by a number of altogether different means, which share the capacity to increase the population of intermediary precursor cells, demonstrates the essential role the population size plays in the progressive phase of carcinogenesis. The rate of cell proliferation The rate of proliferation of the precursor cell population comes into operation since the occurrence of mutations and the transforming events is cell cycle related (20, 30, 46, 87, 115, 139, 142, 171, 181,213), modifications of DNA by carcinogen becoming "fixed" during the S-phase where they may lead to erroneous base-pairing. Precancerous foci have been shown to proliferate quite rapidly during the first weeks after initiation; later on the

137

population size becomes more stationary and mitoses are encountered only occasionally (171). Promotion protocols were reported to increase the proliferation rate, an aspect which is especially evident for the Solt-Farber selection protocol (183, 184). Selected cell clones proliferate vigorously and in a synchronous way, to form visible nodules within 10 days (122, ISS). During the remodelling process after discontinuation of the selection pressure, the proliferation decreases (192), and programmed cell death (apoptosis) may decrease the size of the precancerous cell population (26, 32). Persistent nodules continue to proliferate (192). The increase of the proliferation rate of the precancerous lesions is less pronunced by other promoting protocols (102, 173), and it has been argued that besides increased cell proliferation, decreased cell death by apoptosis may be responsible for the observed increase in population size (26); however, apoptosis could also furnish a highly localized compensatory cell proliferation stimulus increasing the probability for further progression (32). Partial hepatectomy has repeatedly been assayed for promoting capacity since it leads to a wave of proliferation of especially the early precancerous cells (140). Mainly negative results were obtained (15, 146) which could be due to the fact that part of the relevant cell population is removed with the excised liver lobes; the decreased population size could thus counteract a promoting effect of the extra cell proliferation. More advanced cell populations exhibit clearly increased mitotic and eH)-thymidine labelling indices (15, 89, 139, 141). The occurrence of apoptosis - indicative for cell turnover - seems also to increase with progression, the highest incidence having been observed in hepatocellular carcinoma (32). New phenotypic properties It is essentially unknown whether the new cellular functions of precancerous foci, as used for their histochemical recognition, or as detectable by biochemical methods (12, 130, 154, 189), play any role in the progression of early foci to nodules and further to hepatocellular carcinoma. As pointed ouf earlier (early focal alterations) many properties are changed coincidentally by the one-hit process of focus induction. This indicated that an atypical gene program is switched on by the initiating hit. Since this program may represent a "normal" cell function, the observed changes may really be without any effect on the process of neoplastic progression. Some of the new properties, however, were discussed as possibly relevant for the carcinogenic process. The increased resistence of initiated cells against cytotoxic damage by xenobiotic substances - involving decreased levels of enzymes activating carcinogens (40) and enhanced levels of epoxide hydrolase, DT-diaphorase, glutathione, glutathione S-transferases, GGT (73, 154) - may give these cells a selective advantage over their normal counterparts leading to an increased cell population size. The change of the carbohydrate metabolism to glycolysis, involving increased levels (112) of glucose-6-phosphate dehydrogenase (pentose phosphate pathway) and, later, of glyceraldehyde3-phosphate dehydrogenase (anaerobic glycolysis) together with decreased liver type pyruvate kinase activity (152) may lead to an increased production of reactive oxygen and

138

Ewalt Scherer

an increased chance for the realization oflater hits by radical damage of the genetic material. The transformation probability: the role of promoters

The result of the initiation-promotion-initiation experiment and the analysis of time-tumor incidence kinetics (J ~ dID; m = 3 to 7) indicate that genotoxic carcinogen is not only involved in the initiation step, but also later during the phase of neoplastic progression. Similar conclusions can be drawn from various experiments where genotoxic carcinogens have been applied after an initiating treatment to test their activity as promoter. The strong (liver) carcinogens tested: DMN, DEN, ENU, 3-MC, Aflatoxin Bb 3'-Me-DAB, and AAF, proved to be highly efficient in the test systems in that they led to more nodules (77, 85), or to more and earlier tumors and to a higher degree of malignancy than the liver tumor promoter phenobarbital used as reference (209, 214). This points to genetic alterations - as postulated by the somatic mutation hypothesis of cancer - as the necessary events in the step by step progression to fully developed cancer. Besides by the above protocols tumors can be obtained by a single (carcinogenic) dose of carcinogen or a subcarcinogenic dose followed by (non-genotoxic) tumor promoter (see Experimental systems). In case of carcinogenesis by a single or short-term administration of a cancer causing dose, the tumor does not arise immediately, i.e. after one step, but its appearance is preceded by a protracted developmental phase after which a relatively steep increase of the timetumor incidence relationship is observed. For rat liver it has been calculated from such data that after initiation another 3 hits are indicated (45). The cell stages observed during this time-dependent development are similar to those arising during the repeated application of low doses of carcinogen (164). The same sequence of events may thus operate in both cases, i.e. precancerous cells progress to more advanced cell stages even spontaneously, in the absence of applied carcinogen. As promoters are considered to be not carcinogenic or mutagenic, the same argumentation applies for tumor formation by an initiation-promotion protocol. Taken that the relevant alterations underlying the progressive change from one cell stage to the next are at the genetic level, we have to ask the question which processes could lead to such spontaneous mutations. Several mechanisms have been proposed (167) such as: 1. Basic error proneness of the processes involved in the conservation of the genetic information. 2. Induced infidelity in precancerous cells of one of the functions operating in I), by the precancerous or a coinciding hit. 3. Late effects of persistent DNA-carcinogen adducts. 4. Background carcinogen. 5. Endogenous virus. In case of an initiation-promotion protocol: 6. The promoter could affect functions as metioned in 1) to 3). Point mutations, chromosome breaks and incorrect resealing, or chromosome translocations could result from the above mechanisms. Basic error proneness , I, and background carcinogens ,4, should play only a minor role in rat

liver carcinogenesis, seen the extremely low spontaneous liver tumor incidence in rats. Candidates for induced error proneness ,2, are infidel DNA polymerase as proposed by Loeb et al. (106, 107), and reported for aging fibroblasts (104), and during AAF carcinogenesis (28). The mechanism may be generalized to one which envisages a mutation to a less fidel or dysfunctional type of any protein or enzyme involved in the maintenance of the genome. Such genetic alterations would resemble mutator genes in their effect which confer genetic lability (82, 105, 118) by which the rate of new mutations is increased (42). Various lesions which could cause increased frequency of DNA alterations and thus occasionally may lead to a genetic change relevant to neoplastic progression may be conceived, e.g. altered cellular detoxification mechanisms that dispose of potential genotoxic agents generated by metabolic processes (H 2 0 2, free radicals, superoxide (0;», defects in DNA repair systems (78), such as in Xeroderma pigmentosum (29), or in the human hereditary chromosomal instability syndromes ataxia-telangiectasia, Bloom's syndrome, Franconi's anemia, e.a. (74, 133, 144, 176), alteration of structures involved in mitosis which could cause chromosomal instability, and unbalanced deoxyribonucleotide triphosphate pools (95). Inherent genetic instability may be a common property of advanced tumor stages (134). Examples of recent work in this area may be found in (84, 186, 206). These processes depend on cell proliferation/multiplication for their expression. Since they act at random, various different cellular alterations may result. Many of these would remain unnoticed because they do not afford growth advantage to the affected cells, others by lack of a suitable marker. Still others may be deleterious or lethal, and only seldomly a specific genetic alteration will result from these processes which confers a progressed neoplastic phenotype. Whether promoter influences in one way or another the genetic stability of the precancerous cells has not yet been elaborated. There are, however, some indications pointing into this direction. Choline-deficient diet has initiating and promoting properties (215), and its effect could be enhanced by unsaturated fat, but markedly inhibited by the antioxidants butylated hydroxy toluene (129). Lipid peroxidation by a choline-methionine-deficient diet has been demonstrated (158); DNA damage by free radicals could thus be involved in tumor promotion by a choline-deficient diet. The deficiency in methyl groups could lead to additional gene alterations as indicated by the co-initiating capacity of 5-azacytidine (39), a cytidine analogue which inhibits the physiological methylation of the newly fOf'11ed DNA strand. A similar conclusion has been drawn from the demonstration of single strand breaks of DNA by the hypolipidemic drug nafenopin (19), or by the f3-blocker ZAMI 1305 (136). In case of nafenopin indirect damage of DNA could have occurred as a result of elevation of the cellular H2 O2 content caused by the peroxisome proliferation. The pronounced proliferation of mitochondria by methapyrilene, leading eventually to an altered oxidative metabolism could work to the same end (86, 153). These results suggest that besides the increase in target cell population the perturbation of cellular defense mechanisms against reactive oxygen (or other harmful species) may be instrumental in tumor promotion and, similarly in spontaneous progression.

16: Neoplastic cell stages and progression in experimental hepatocarcinogenesis

Expression of oncogenes in hepatocarcinogenesis The demonstration by the group of Barbacid (190) that the expression of a point-mutated cellular oncogene (c-H-ras) is instrumental in the induction of rat mammary tumors by the direct-acting carcinogen MNU points to the possibility that similar mechanisms are involved in the induction of other chemically induced tumors as well. In addition the different neoplastic cell stages which can be distinguished in rat hepatocarcinogenesis raise the question whether each neoplastic phenotype can be related to the expression of a specific cellular oncogene. This aspect of the multistage carcinogenesis has been discussed recently by Balmain (9). Several publications have been dealing with the aspect of oncogene expression in rat hepatocarcinogenesis during the past few years. In regenerating rat liver a transient increase in the number of transcripts of the c-H-ras oncogene was observed at the onset of DNA synthesis (68). c-Ki-ras and c-myc were also increased (69), but the elevation of c-myc preceded that of c-ras; and c-src remained unchanged. During liver carcinogenesis by 3'-Me-DAB (109) c-H-ras expression was increased in non-tumorous parts of treated livers, in primary tumors and in hepatoma cell lines; c-myc expression was increased in tumor tissue only. This indicated that the expression of c-myc is associated with hepatocarcinogenesis, while the expression of c-ras reflects more the proliferative status of the cells, and thus is influenced by the cytotoxic action of carcinogen inducing regen era tive cell proliferation. The expression of these cellular oncogenes increased with the duration of 3'-Me-DAB treatment (35). During hepatocarcinogenesis by a choline-deficient diet, with or without additional ethionine (212), expression of c-H-ras was only transiently increased, while c-Ki-ras and c-myc were elevated during the whole period of feeding the carcinogenic diet. The oncogenes were expressed especially in oval cells, a cell type which is not involved in hepatocellular carcinogenesis in general, but proliferates extensively only under toxic regimens, such as carcinogenesis by choline-methionine-deficient diets and ethionine (179), or AAF (193). If direct toxic influences by the carcinogen were excluded by a long delay between the carcinogen application (DEN) and the measurement of oncogene expression (34) ras genes (c-H-ras, c-Ki-ras, N-ras) were, nevertheless, expressed 2-25 times more than in untreated controls. Nodular tissue and surrounding tissue exhibited both the increased expression which could partially be dissociated from cell proliferation. This may indicate that early focal alterations, as present in the perinodular tissue, have already an increased c-ras expression. It is interesting in this context that a transforming c-Ha-ras has been identified in skin papilloma (II), a precancerous cell population progressing to skin carcinoma (10, 75). No generally enhanced expression of c-ras genes

could, however, be detected in this system (197). Oncogenes with transforming properties in in vitro transfection assays were not isolated so far from chemically induced liver tumors.

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17 CHARACTERIZATION OF ELEVEN IMPORTANT TRANSPLANTABLE MURINE TUMORS FROM THE STANDPOINT OF MORPHOLOGY, PYRIMIDINE BIOSYNTHESIS AND RESPONSIVENESS TO PYRIMIDINE ANTIMETABOLITES THOMAS W. KENSLER, HILDEGARD M. SCHULLER, HIREMAGALUR N. JAYARAM and DAVID A. COONEY

INTRODUCTION

Morphology

Experimental cancer chemotherapy is generally assessed with tumor cell lines in which transplantation over many generations has promoted phenotypic and genotypic stability. However, like their spontaneous counterparts, transplantable tumors display heterogeneity in cell populations and can undergo a broad variety of transitions and transformations during the course of their growth. In the aggregate the behavior of these passaged tumors can mimic and sometimes presage that of cancers being treated in the clinic. For this reason, transplantable tumor lines have occupied and will continue to occupy a valuable place in the effort to detect agents capable of arresting progressive neoplastic growth in humans. In the present chapter we have endeavored to summarize the principal biological characteristics of the most important transplantable murine tumors in use in the drug screening program of the National Cancer Institute over the past decade. We will display the morphology of these tumors, their endowment with enzymes of the de novo pyrimidine biosynthetic and salvage pathways, along with their response to agents which are, at present, categorized as pyrimidine anti metabolites acting against the de novo pathway.

In order to present an up-to-date morphologic description of the eleven tumors treated in the present chapter, freshly excised nodules of the solid lines and freshly sampled ascites fluid from the leukemias were fixed in glutaraldehyde and examined by light and electron microscopy. What follows is a brief description of each neoplasm, along with photomicrographs representative of their morphology. Lewis lung carcinoma The Lewis lung carcinoma is a highly cellular neoplasm without detectable stroma and devoid of any particular growth pattern (Figure la). The proliferative component of this tumor is comprised of pale, small cells which exhibit frequent mitosis; in other areas of the tumor, cell degeneration is widespread. On electron microscopy, adjacent cells are seen to lie in close apposition but without the formation of junctional complexes (Figure 1b). The rough endoplasmic reticulum is well developed, and small, round mitochondria are numerous. Contrary to all other cell lines examined in this study, lysosomes and their derivatives are strikingly abundant. The cell membranes are generally smooth without cytoplasmic extensions, the nuclei oval, with occasional, slight indentations. Heterochromatin is abundant and nuclear pores are numerous. The nucleoli are condensed with a prominent granular component.

CHARACTERISTICS OF ELEVEN WIDELY USED TRANSPLANTABLE TUMORS Biology

In order to provide a background for the biochemical and therapeutic studies which follow, the biological and morphologic characteristics of the transplantable tumors treated in the present chapter will be presented at the outset. Table I recapitulates the routes of implantation and growth patterns of these lines and is generally self-explanatory. One feature of this compilation does, however, merit mention: the doubling times of the tumors constituting this panel cover an unusually broad range: from 6 hours in the case of the Ll210 leukemia to 7 days in the case of the colon carcinoma 26. Not shown in Table I is an additional biological feature of relevance to users of these tumors: certain of the solid lines (most notably the colon carcinoma 38) exhibit a lag after inoculation before their growth begins to manifest itself. The basis for this lag is unknown, but it may stem either from a kind of "transplantation shock" or from an inability of these tumors to elicit a prompt neovascularization.

Colon carcinoma 26 This tumor is comprised of pale epithelial cells which appear randomly arranged without any particular growth pattern (Figure 2a). Mitosis is frequent. The large nuclei vary in size and shape and exhibit multiple nucleoli and clumps of chromatin. On electron microscopy, the cytoplasm is generally electronlucent with scattered polyribosomes and well developed rough endoplasmic reticulum (Figure 2b). Adjacent cell membranes are closely apposed but desmosomes are rare. The nuclei have smooth outlines with infrequent slight indentations. Heterochromatin is found in clusters throughout the nucleoplasm and in marginal areas. The nucleoli are less condensed than in any other cell line included in the present study.

Colon carcinoma 38 Light microscopy of the Colon Carcinoma 38 reveals that large portions of the tumor are necrotic and that good preservation of morphology is generally restricted to perivascular areas (Figure 3a). The tall columnar to oval tumor cells demonstrate palisading growth around blood 145

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Table 1. Biological characteristics of eleven frequently used transplantable tumors.

Tumor

Host for propagation

Customary site of propagation

Doubling time in days

Metastatic potential

Lewis Lung Carcinoma

C57BL/6

Subcutaneously; or Intramuscularly

0.9-2.1

Lungs

Colon Carcinoma 26

BALB/c

Subcutaneously

2.0-7.0

Colon Carcinoma 38 Bl6 Melanoma

CS7BL/6

Subcutaneously

0.7-3.2

Lungs, ovaries adrenals, kidneys, heart, mesentery and diaphragm Lungs

CS7BL/6

Subcutaneously

0.8-1.6

Lungs

M5076 Ovarian Tumor

C57BL/6

Subcutaneously

I.S

Ll210

DBA/2

Intraperitoneally

0.24-0.34

Liver & spleen; uterus, kidney, and ovaries Omentum

Glioma 26

C57BL/6

Subcutaneously

~

{2-4}!

None observed

LSI78Y

DBA/2

Intraperitoneally

~

{O.S}!

Systemic

Ehrlich Ascites Carcinoma P388

BALB/C

Intraperitoneally

~{ldays}!

DBA/2

Intraperitoneally

0.39-0.S2

P815

DBA/2

Intraperitoneally

~

{0.5}!

Low metastatic potential Omentum Omentum, liver

Characteristics in screening: survival/tumor weight inhibition

Survival Average: 27 days Range: 19-35 days Survival Median: 17 days Range: 14-48 days Tumor weight inhibition Survival Average: 22 days Range: IS-30 days Survival Average: 29 days Range: 15-30 days Survival 8-11 days Survival Average: 60 days Range: 40-80 days Survival Average: 12 days Reduction in cell number Survival 9-14 days Survival Average: 11.5 days Range: 10-13.5 days

Legend: Most of the data in this table were extracted from NIH Publication No. 84-2635; In Vitro Cancer Models, February 1984. This brochure is available from the Superintendant of Documents of the U.S. Government Printing Office, Washington, D.C. !bracketed doubling times are approximate.

vessels and form acinar structures. Mitosis is common. Electron microscopy of the well preserved tumor areas shows that cytoplasmic differentiation is good. Acini are abundant, the acinar lumina are lined by numerous slender microvilli (Figure 3b), and junctional complexes connect the apical

portion of cells. Well developed short tubules of rough endoplasmic reticulum and round to oval mitochondria are common. There is no evidence of secretory activity. Nuclei are heterogeneous in size and shape and the heterochroma-

Figure lao Light Micrograph of Lewis Lung Tumor: Cells are closely packed; light cells demonstrate frequent mitosis while dark cells appear degenerated. x 160.

Figure lb. Electron Micrograph of Lewis Lung Tumor: Note numerous electron-dense lysosomal derivatives in cytoplasm. x 7,000.

17: Characterization of eleven important transplantable murine tumors

Figure 2a. Light Micrograph of Colon Carcinoma 26: Pale polymorph tumor cells (Lower right of picture) invade adjacent connective tissue of host animal. x 160.

tin is marginated. The nucleoli are condensed with a prominent granular component.

Melanoma B-16 Histopathologically, large areas of this tumor type are necrotic. The few tumor cells that remain intact are spindleshaped and exhibit a poor affinity for toluidine blue. Electron microscopically, abundant melanophages with phagocytized compound melanosomes are noticeable (Figure 4a) but true melanocytes with primary melanosomes are infrequent (Figure 4b). In many cases cytoplasmic processes containing melanosomes are sandwiched between connective tissue elements. The nuclei of tumor cells have little marginated heterochromatin and nucleoli are condensed. Glioma 26 Light microscopy of this transplantable glioma reveals tumor cells that are heterogenous in size and shape. Intercellular spaces are wide and no particular growth pattern is evident (Figure Sa). At higher magnification, the nuclei are seen to be positioned at one pole of the cell while the

Figure 3a. Light Micrograph of Mouse Colon Carcinoma 38: Well

preserved tumor cells are restricted to perivascular areas and exhibit a palisading growth pattern. x 160.

147

Figure 2b. Electron Micrograph of Mitotic Tumor Cell in Colon

Carcinoma 26: Note well developed rough endoplasmic reticulum and close apposition of cell membranes with lack of desmosomes. x 7,000.

cytoplasm of the opposite pole forms an extension of variable length (Figure Sb). The most prominent cytoplasmic features are the well developed cisternae of rough endoplasmic reticulum which are frequently distended. Adjacent cells are occasionally closely apposed but true junctional complexes are lacking as are glial filaments. Mitochondria are scanty and small. The nuclei are polymorphic and have deep indentations. The arrangement of heterochromatin varies.

Lymphoma P388 Histopathology of the subcutaneously grown P388 lymphoma reveals that the tumor is highly cellular without any particular growth pattern (Figure 6a). The relatively small cells are closely packed without stroma. The majority of cells stain intensely with toluidine blue and clear nuclear contours are difficult to distinguish. Occasional small areas are comprised of paler staining cells which demonstrate contrasting nuclear outlines. In the electron microscope none of

Figure 3b. Electron Micrograph of Mouse Colon Carcinoma 38: An acinar lumen (L) is lined by long slender microvilli. Note junctional complexes (arrow) and well-developed endoplasmic reticulum. x 10,000.

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Figure 4a. Electron Micrograph of melanophage in Melanoma B 16: Note numerous phagocytized compound melanosomes (arrow). x 10,000.

Figure 4b. Electron Micrograph of cell processes from melanocytes in melanoma B16: Note numerous melanosomes (arrow). x 13,000.

the tumor cells demonstrate desmosomes or any other cell junctions. The dark cells exhibit features of atypical lymphocytes while the few pale cells are lymphoblastic in nature (Figure 6b). Both cell types have irregular, sometimes deeply indented nuclei with marginated heterochromatin and clumps of heterochromatin throughout the nucleoplasm. The lymphocytic cell type exhibits abundant polyribosomes and mitochondria tend to be grouped in clusters. Microvillilike cytoplasmic extensions are common. The lymphoblastic cell type has an electronlucent matrix and fewer polyribosomes. A few short tubules of rough endoplasmic reticulum are noticeable and mitochondria are elongated but not aggregated in clusters.

sions. In ascites MS076 these pseudopods are especially numerous (Figure 7a). Ascites Ll210 and Ehrlich ascites (Figure 7b) demonstrate large cytoplasmic lipid droplets. The cytoplasmic matrix is very dense in all of the ascitic tumors while endoplasmic reticulum and mitochondria are scanty. Unlike the cells in solid tumors, the nuclei in ascites tumors exhibit a good amount of heterochromatin. The nucleoli are condensed with a prominent granular component. The foregoing morphologic characterization of the transplantable tumors treated in the present chapter serves to emphasize the substantial morphological diversity of these lines. In practical terms, of course, it is diversity which makes these tumor lines of such value in a drug-screening program. On this background it will not be surprising that the endowment of these eleven tumors with the enzymes of de novo pyrimidine biosynthesis - and, in fact, with all other components of intermediary metabolism - are comparably variable. In the sections which follow, this enzymic variability will be documented and discussed in greater detail.

Ascites tumors The six different ascites tumors (Ll210; P81S; L5178Y; M5076; P388 and Ehrlich ascites) investigated for this review are morphologically much alike. All of them consist of cells in suspension without any cell to cell connections. In certain respects these cells resemble macrophages in that they exhibit numerous pseudopodialtype cytoplasmic exten-

Figure 5a. Light Micrograph of Glioma 26: Heterogenous tumor

cells demonstrate wide intercellular spaces. x 160.

Figure 5b. Electron micrograph of Glioma 26: The cytoplasm opposite the nucleus (N) forms an extension: Note dilated cisternae of rough endoplasmic reticulum. x 7,000.

17: Characterization of eleven important transplantable murine tumors

Figure 6a. Light Micrographs of Mouse Lymphoma P388: Small

dark and light cells are packed without stroma. x 160.

CHARACTERISTICS OF PYRIMIDINE BIOSYNTHETIC ENZYMES IN THESE ELEVEN TRANSPLANTABLE TUMORS

149

Figure 6b. Electron Micrograph of Mouse Lymphoma P388: Cell-

to-cell junctions are lacking. Pale lymphoblast-like cells and more electron dense lymphocyte-type cells have irregular indented nuclei. x 7,000.

The requirement for pyrimidines is ubiquitously distributed throughout the spectrum of living organisms and can be fulfilled by two synthetic pathways: a de novo and a salvage pathway. Uridine-Y-monophosphate is a common product of these two pathways. The de novo pathway is generally considered to consist of Six enzymes: carbamyl phosphate synthetase II (CPS\ II; E'C 2.7.2.9), L-aspartate transcarbamylase (ATCase; BC 2.1.3.2), L-dihydroorotase (DHOase; EC 3.5.2.3), L-dihydroorotate dehydrogenase (DHO deHase; EC 3'.5.2.3), orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) and orotidine-S' -monophosphate decarboxylase (OMPdeCase; EC 4.1.1.23). Several excellent reviews ofthis\pathway appear in the recent literature (16, 35). The main components of the pyrimidine ring are derived from L-aspartic acid and L-glutamine while the ribosyl and phosphoryl moieties are transferred en bloc from phospho ribosyl pyrophosphate. So far as is known, this

"genealogy" of the pyrimidine ring is universal and serves to underscore the intimate interrelationship of the dicarboxylic amino acids and their amides with nucleic acid biosynthesis. The salvage pathway, by contrast, utilizes preformed nucleosides or bases in the biosynthesis of pyrimidine nucleotides (24). Two additional enzymes, namely, thymidylate and cytidylate synthetase, catalyze modification of the pyrimidine ring to yield the other pyrimidine nucleotides (3, 9, 40). The intracellular localization of the six catalytic activities of the de novo pyrimidine biosynthetic pathway appear to be optimized to ensure efficient flux or to permit braking of output in response to physiologic needs (36). CPSII, ATCase and DHOase exist as a large cytosilic multienzyme complex (pyr 1-3). The aggregation of consecutive catalysts serves to channel products from the antecedent to the subsequent catalytic centre without undue dilution or diffusion into the ambient cytoplasmic milieu. In essence, this complex is an enzymatic assembly line. The fourth enzyme of the pathway, DHO deHase, is particulate, sequestered on the

Figure 7a. Electron Micrograph of Tumor Cell in Ascites M5076: Note: macrophage-like appearance due to cytoplasmic pseudopods which are especially numerous in this tumor type. x 10,000.

Figure 7b. Electron Micrograph of Tumor Cells in Ehrlich Ascites: Note: macrophage-like morphology and prominent lipid droplets in cytoplasm (arrow). x 7,000.

150

Thomas W. Kensler, et al.

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i= u 90% tumor inhibition and/or> 75% increase in life span; +, 70-90% tumor inhibition and/or 40-75% increase in life span; and -. < 70% tumor inhibition and/or < 40% increase in life span. contributory - cause of the antitumor activity seen. The fact that the L-glutamine antagonists are (contrary to expectation) most active against tumors with comparatively rich endowments of CPS II (cf. Figure 8 and Table 3) might be taken to suggest, in fact, that these drugs are oncolytic principally because of activities at a site or sites other than the inaugural enzyme of pyrimidine biosynthesis. Azaserine, DON and acivicin have all been given phase I trials in man but with disappointing therapeutic results (23). Moreover, all produced severe gastrointestinal toxicities, ranging from glossitis to proctitis, and so were poorly tolerated in their clinical tests. Inhibitors of L-aspartate transcarbamylase

PALA (N-(phosphonacetyl)-L-aspartic acid) is a recently developed pyrimidine inhibitor and, in certain regards, the most distinctive. PALA was synthesized as a stable analog of the transition-state intermediate in the reaction catalyzed by ATCase (6) and, as such, combines the structural features of the two natural substrates, carbamyl phosphate and Laspartic acid. Transition-state analogs offer attractive

potentials as metabolic inhibitors because they can bind to their target enzymes with high affinity and specificity. In this instance, as best as is known, A TCase is the only enzyme directly affected by PALA. Because of its inhibitory potency (K j = 20 nanomolar in Lewis lung carcinoma), this agent has been subjected to uniquely intensive across-the-board screening. As Table 3 documents, PALA is highly active in the treatment of many solid trans!Jlantable rodent tumors, but is ineffective against the leukemias and most of the ascitic tumors. The observation that the specific activities of A TCase are lower in solid tumors than in the more rapidly developing leukemias provides support for the generalization that tumors deficient in this de novo biosynthetic capability are most susceptible to the cytotoxic actions of PALA (14). Despite its outstanding activity against solid transplantable murine tumors, the clinical performance of PALA has been disappointing. Although not melosuppressive, doselimiting toxicities in the skin and gastrointestinal tract are observed. Throughout intensive phase I and II trials the drug has failed to exhibit any material activity as a single agent against human neoplasia (39). Nevertheless, because PALA is an antimetabolite of well-defined specificity and

17: Characterization of eleven important transplantable murine tumors

153

Table 4. Properties of selected, clinically used inhibitors of the de novo pyrimidine biosynthetic pathway. Customary dose and route

Response rate at site of best chemotherapeutic activity

daily x 5, IV

ljl2 (8.3%)-NSC lung

Myelosuppression neurotoxicity mild gastrointestinal toxicity

single injection every 2 weeks; daily x 5, IV

6/52 (11.5%)-melanoma

Skin rash gastrointestinal toxicity stomatitis diarrhea mild neurotoxicity

weekly

ljl4 (7.1%) Multiple myeloma

Stomatitis skin rash mild myelosuppression

multiple routes and schedules

18/67 (10.9%) Colorectal carcinoma

Stomatitis myelosuppression gastroin testinal toxicity

3-Deazauridine

daily x 5 and by 24 hour continuous infusion

No activity

Acivicin

supra

supra

Myelosuppression nausea and vomiting mucositis fever supra

Enzyme target and drug Carbamyl Phosphate Synthetase II

Acivicin

L-Aspartate Transcarbamylase

PALA

Principal toxicities

L-Dihydroorotase

None

L-Dihydroorotic Acid Dehydrogenase

None

Oro tate Phosphoribosyl Transferase

None

Orotidylate Decarboxylase

Pyrazofurin

Thymidylate Synthetase

5-FU (and congeners)

CTP Synthetase

because of the striking oncolytic effects achieved when PALA is used in conjunction with acivicin in animal models (or, in other words, when concerted inhibition was inflicted on both the first and second steps of the de novo pathway) (19,26), it is anticipated that PALA will assume a role in combination chemotherapeutic strategies intended for control of cancer in man.

Inhibitors of L-dihydroorotase Since the first three enzymes of de novo pyrimidine biosynthesis coexist on a single megadalton polypeptide, it might be supposed that inhibition of one of the constituent steps of this complex would produce inhibitory reverberations on an adjacent step; this has not proven to be the case. For example, even when the activities of CPS II and ATCase are nearly fully extinguished by exposure to acivicin and PALA, the specific activity of the third enzyme on the pathway, DHOase, remains uneffected. Not only are allosteric repressants wanting in the case of DHOase, but there also prevails a paucity of specific or active-site directed inhibitors of this enzyme (18). From this narrow field only two

moderately potent substrate analogs have been identified as chemotherapeutic: 5-fluoroorotate (which is very active against L 121 0 leukemia) and 5-aminoorotate (which is comparatively active against P388 leukemia). The field is further narrowed when it is recalled that 5-fluoroorotate is metabolized in part to 5-fluorodeoxyuridine-Y-monophosphate, a metabolite which very probably makes a major contribution to the antileukemic effects which follow administration of this substrate analog. Plainly, L-dihydroorotase is a target awaiting attack by interested medicinal chemists.

Inhibitors of L-dihydroorotate dehydrogenase Inhibition of the mitochondrial enzyme involved in de novo pyrimidine biosynthesis, DHO deHase, is accomplished by two general classes of compounds. In common with most other enzymes in this pathway, DHO deHase is subject to product inhibition; orotic acid and some of its analogs are effective inhibitors, particularly dihydro-5-azaorotic acid. However, this compound is therapeutically inactive against several murine tumors. Additionally, naphthoquinones have been recently identified as potent inhibitors of DHO de-

154

Thomas W. Kensler, et al.

Hase. These drugs may act as analogs of the cofactor, ubiquinone, and serve as electron acceptors that alter electron flow. One such quinone, lapachol inhibits DHO deHase from mouse liver mitochondria uncompetitively with Ldihydroorotate as substrate, the apparent Ki being 2.1 x 10- 6 M (2). However, lapachol is without antitumor activity in humans, apparently because gastrointestinal toxicity becomes dose-limiting at subtherapeutic plasma concentrations (27). Dichloroallyllawsone, a close congener oflapachol, inhibits DHO deHase with a Ki of 2.7 x 10- 8 (2). Despite this two log augmentation in potency, lapachol is roughly 5 times more cytotoxic than dichloroallyllawsone to cultured LI2l0 cells and exhibits substantial activity in the LI210 and P388 leukemias, against which dichloroallyl lawsone is inactive. Conversely, against solid tumors dichloroallyl lawsone has greater activity and a better therapeutic index than lapachol. Dichloroallyl lawsone produces vastly more profound and persistent reduction of uridine nucleotides than its more cytotoxic congener. These disparate observations suggest that attributes of these agents other than inhibition of DHO deHase might playa role in their cytotoxicities. Two such attributes are their abilities to interrupt cellular respiration or to generate free radicals capable of damaging DNA and other biomolecules. It is relevant to add that dichloroallyl lawsone is not myelosuppressive in man and shows little gastrointestinal toxicity; however, high doses in primates induce acute cardiotoxicity, a feature which has sharply limited the clinical usefulness of the drug (28).

Inhibitors of orotate phosphoribosyl transferase and/or orotidylate decarboxylase

A number of orotate analogs have been described as inhibitors of OPRTase; many are also substrates for the enzyme and can form fraudulent nucleotides which, in turn, are potent inhibitors of OMP de Case, the second enzyme in the pyr5,6 complex. Potent inhibitors of OPRTase are scarce, which is regrettable because OPRTase is numerically rate-limiting in one of the eleven transplantable tumors whose enzymology was surveyed for the present chapter (M5076 ovarian carcinoma, Figure 8) and close to ratelimiting in the remainder. On this basis one would intuitively suppose it to be a prime target for inhibitory and therapeutic attack. Barbituric acid does inhibit the enzyme quite potentially in vivo, but wholly fails to achieve inhibition of the growth of the LI210 leukemia (the sole system for which test records are available). This negative result may be all the more salient when it is recalled that the specific activity of OPRTase is the lowest in LI210 of any of the leukemias studied, a feature which should render it susceptible to agents blockading this step. Since the ribotide of barbiturate is a potent inhibitor of purified OMP deCase (33), the biochemical features explaining the failure of barbituric acid to influence the growth of LI210 remain to be identified. Quite clearly, this is another case where the ingenuity of medicinal chemists must be recruited. Although 5-fiuorouracil is listed in Table 2 as an inhibitor of OPRTase, and although this pyrimidine base - in its

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Lewis Lung Colon Colon 816 M5076 Carcinoma Carcinoma Carcinoma Melanoma Ovarian 26 38 Carcinoma

-

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= = -

~

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Figure 9. Specific activities of pyrimidine salvage enzymes in transplantable murine tumors. Deoxycytidine kinase and uridine/cytidine

kinase (determined using either uridine or cytidine as substrate) activities were measured in tumor cytosols as detailed in the legend to Figure 8 and reference 14.

17: Characterization of eleven important transplantable murine tumors

capacity as an alternate substrate for this enzyme - can undoubtedly inhibit OPRTase under appropriate reaction conditions, such inhibition is ordinarily feeble (13). For this reason, the activity of 5-fiuorouracil should be viewed as supplementary to the drug's main locus of action, thymidylate synthetase (l). Two potent antimetabolites are available for the inhibition of OMP deCase, the last step in the assembly of the pyrimidine ring: 6-azauridine and pyrazofurin. Both agents are phosphorylated in vivo to 5' -monophosphate derivatives through the actions of uridine/cytidine and adenosine kinases, respectively, and it is these anabolites which strongly impede the decarboxylation of orotidine-5' -monophosphate (4, 8). The 5'-monophosphate derivative of pyrazofurin is a competitive inhibitor of purified OMP deCase with an apparent K j of 5 x 10- 9 M. Both 6-azauridine and pyrazofurin exhibit fairly good antileukemic activity in vivo and show modest activity against the two most widely used transplantable solid tumors, the Lewis lung carcinoma and the B 16 melanoma. Clinical trials have demonstrated that neither agent exhibits prominent antitumor activity in man, although 6-azauridine has been used clinically for the management of neoplasms such as chronic myelogenous and acute leukemias (4, 10-12,31). PROSPECTS The present generation of drugs described - by serendipity or design - as inhibitors of the enzymes of de novo pyrimidine biosynthesis is for the most part without significant therapeutic value in man. The therapeutic features of the principal drugs are summarized in Table IV. These present day inhibitors are not site-specific, with the notable exception of P ALA, and in no instances have been demonstrated to completely block precursor fiux through the pathway. It would appear that the use of pyrimidine pathway inhibitors as single agent therapies is a tactically improper approach. However, the judicious use of these drugs in combination chemotherapy offers encouraging possibilities, particularly when applied sequentially against the de novo pathway (19, 26) or when used in combination with agents that inhibit the salvage pathway, thus effecting a total pyrimidine deprivation (41). The presentation of the specific activities of several pyrimidine salvage pathway enzymes in Figure 9 namely uri dine/cytidine and deoxycytidine kinases, would encourage the suggestion that they are exploitable targets for inhibitor action. Towards this end, Karle et al. (17) have recently demonstrated that Ll210 cells preferentially utilize their pyrimidine salvage pathway over their de novo pathway, and that circulating plasma concentrations of uridine are sufficient to circumvent growth inhibition by inhibitors of de novo pyrimidine biosynthesis. These observations firmly support the need to develop inhibitors of uri dine/cytidine kinases as agents for use in combination with inhibitors of de novo pyrimidine biosynthesis in the treatment of cancer.

novo and salvage pathways enzymes, Mr. A Del Campo for the preparation of sections for light and electron microscopy, and Dr. S Marsoni for her compilation of the data presented in Table 4. Lastly, but most importantly, thanks are extended to Dr. RK Johnson at Smith, Kline, Beckman, Philadelphia, PA and many members of the Division of Cancer Treatment of the National Cancer Institute, Bethesda, MD who aided the authors in gaining access to the chemotherapeutic results tabulated in this chapter.

REFERENCES I.

2.

3. 4. 5.

6. 7. 8.

9. 10.

II. 12. 13. 14.

15.

ACKNOWLEDGEMENTS The authors thank Drs C Warren, H Milman and B Ardalan for their material assistance in the measurement of the de

155

16.

Ardalan B, Cooney DA, MacDonald JS: Physiological and pharmacological determinants of sensitivity and resistance to 5-fluorouracil in lower animals and man. Adv Pharmacal Therapeut 17:282-321, 1980 Bennett LL Jr, Smithers D, Rose LM, Adamson DJ, Thomas HJ: Inhibition of synthesis of pyrimidine nucleotides by 2-hydroxy-3(3, 3-dichloroallyl)-I, 4-naphthoquinone. Cancer Res 39:4868-4874, 1979 Buchanan JM: The amidotransferases. Adv Enzymol 39:91183, 1973 Cadman EC, Dix DE, Handschumacher RE: Clinical, biological and biochemical effects ofpyrazofurin. Cancer Res 38:682688, 1978 Christopherson RI, Jones ME: The effect of pH and inhibitors upon the catalytic activity of the dihydroorotase of multienzyme protein pyrl-3 from mouse Ehrlich ascites carcinoma. J Bioi Chem 255:3358-3370, 1980 Collins KD, Stark GR: Aspartate transcarbamylase interaction with the transition state analogue N-(phosphonacetyl)-Laspartate. J Bioi Chem 246:6599-6605, 1971 Dahl JL, Way JL, Parks RE Jr: The enzymatic synthesis of 5-fluorouridine 5'-phosphate. J Bioi Chem 234:2998-3002, 1959 Dix DE, Lehman CP, Jakubowski A, Moyer JD, Handschumacher RE: Pyrazofurin metabolism, enzyme inhibition and resistance in LSI78Y cells. Cancer Res 39:4485-4490, 1979 Dunlap RB: TMP synthetase from Lactobacillus casei. Methods Enzymol 51:90-97, 1978 Gutowski GE, Sweeney MJ, DeLong DC, Hamill RL, Gerzon K, Dyke RW: Biochemistry and biological effects of the pyrazofurins (pyrazomycins): initial clinical trials. Ann N Y Acad Sci 255:544-551, 1975 Handschumacher RE, Calabresi P, Welch AD, Bono V, Fallon H, Frei E III: Summary of current information on 6azauridine. Cancer Chemother Rep 21: 1-18, 1962 Hernandez K, Pinkel D, Lee S, Leone L: Chemotherapy with 6-azauridine (NSC-32074) for patients with leukemia. Cancer Chemother Rep 53:203-207, 1969 Holmes WL: Studies on the mode of action of analogues of orotic acid: 6-uracilsulfonic acid, 6-uracil sulfonamide, and 6-uracil methyl sulfone. J Bioi Chem 223:677-686, 1956 Jayaram HN, Cooney DA, Vistica DT, Kariya S, Johnson RK: Mechanisms of sensitivity or resistance of murine tumors to N-(phosphonacetyl)-L-aspartate (PALA). Cancer Treat Rep 63:1291-1302, 1979 Jayaram HN, Cooney DA, Ryan JA, Neil G, Dion RL, Bono VH: L-(aS,5S)-a-amino-3-chloro-4, 5, dihydro-5-isoxazolacetic acid (NSC-163501): a new amino acid antibiotic with the properties of an antagonist of L-glutamine. Cancer Treat Rep 59:481-491, 1975 Jones ME: Pyrimidine nucleotide biosynthesis in animals: genes, enzymes and regulation of UMP biosynthesis. Ann Rev Biochem 49:252-279, 1980

156 17. 18. 19.

20. 21. 22.

23.

24. 25. 26. 27.

28.

Thomas W. Kensler, et al. Karle JM, Anderson LW, Cysyk RL: Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultured LI210 cells. J Bioi Chem 259:67-72, 1984 Kensler TW, Cooney DA: Chemotherapeutic inhibitors of the enzymes of the de novo pyrimidine pathway. Adv Pharmacol Chemother 18:273-352, 1981 Kensler TW, Reck LJ, Cooney DA: Therapeutic effects of acivicin and N-(phosphonacetyl)-L-aspartic acid in a biochemically designed trial against a N-(phosphonacetyl)-Laspartic acid-resistant variant of the Lewis lung carcinoma. Cancer Res 41:905-909, 1981 Kensler TW, Jayaram HN, Cooney DA: Effects of acivicin and PALA, singly and in combination, on de novo pyrimidine biosynthesis. Adv Enzyme ReguI20:57-73, 1982 Kensler TW, Cooney DA, Jayaram HN, Schaeffer C, Choie DD: A facile tritium release assay for mammalian L-dihydroorotate dehydrogenase. Anal Biochem 117:315-319, 1981 Kensler TW, Mutter G, Hankerson JG, Reck LJ, Harley C, Han N, Ardalan B, Cysyk RJ, Johnson RK, Jayaram HN, Cooney DA: Mechanism of resistance of variants of the Lewis lung carcinoma to N-(phosphonacety1)-L-aspartic acid. Cancer Res 41:894-904, 1981 Kisner DL, Kuhn JG, Weiss GR, Dorr FA, Von Hoff DD: New anticancer drugs, pp. 120-160 in Cancer Chemotherapy. Annual 5 (Pinedo HM and Chabner BA eds.), Elsevier, New York, 1983 Levine RL, Hoogenraad NJ, Kretchmer N: A review: Biological and clinical aspects of pyrimidine metabolism. Pediatr Res 8:724-734, 1974 Livingston RB, Venditti JM, Cooney DA, Carter SK: Glutamine antagonists in chemotherapy. Adv Pharmacol Chemother 8:57-120, 1970 Loh E, Kufe DW: Synergistic effects with inhibitors of de novo pyrimidine synthesis, acivicin and N-(phosphonacetyl)-Laspartic acid. Cancer Res 41:3419-3423, 1981 Loo TL, Benjamin RS, Lu K, Benvenuto JA, Hall SW, McKelvey EM: Metabolism and disposition of Baker's antifolate (NSC-139105), ftorafur (NSC-148958) and dichloroallyllawsone (NSC-126771) in man. Drug Metab Rev 8:137-150, 1978 McKelvey EM, Lomedico M, Lu K, Chadwick M, Loo TL: Dichloroallyl lawsone. Clin Pharmacol Therap 25:586-590, 1979

29.

30.

31. 32. 33.

34. 35. 36.

37.

38.

39. 40. 41.

Mori M, Tatibana M: A multienzyme complex of carbamoylphosphate synthase (glutamine): aspartate transcarbamylase: dihydroorotase (rat ascites hepatoma and rat liver). Methods Enzymol 51: 11 1-121, 1978 Moyer JD, Handschumacher RE: Selective inhibition of pyrimidine synthesis and depletion of nucleotide pools by N(phosphonacetyl)-L-aspartate (PALA). Cancer Res 39:30893094, 1979 Ohnuma T, Roboz J, Shapiro ML, Holland JF: Pharmacological and biochemical effects of pyrazofurin in humans. Cancer Res 37:2043-2049, 1977 Porter RW, Modebe MO, Stark GR: Aspartate transcarbamylase: Kinetic studies of the catalytic subunit. J Bioi Chem 244:1846-1851, 1969 Potvin BW, Stern HJ, May SR, Lam GF, Krooth RS: Inhibition by barbituric acid and its derivatives of the enzymes in rat brain which participate in the synthesis of pyrimidine ribotides. Biochem Pharmacol 27:655-665, 1978 Pradham TK, Sander EG: Noncompetitive inhibition by substituted sulfonamides of dihydroorotase from Zymobacterium oroticum. Life Sci 13:1747-1752,1973 Shambaugh GE, III: Pyrimidine biosynthesis. Amer J Clin Nutr 32:1290-1297, 1979 Shoaf WT, Jones ME: Uridylic acid synthesis in Ehrlich ascites carcinoma. Properties, subcellular distribution, and nature of enzyme complexes of the six biosynthetic enzymes. Biochem 12:4039-4051, 1973 Traut TW, Jones ME: Kinetic and conformational studies of the oro tate phosphoribosyltransferase: orotidine-5'phosphate decarboxylase enzyme complex from mouse Ehrlich ascites cells. J Bioi Chem 252:8374-8381, 1977 Tso JY, Bower SG, Zalkin H: Mechanism of inactivation of glutamine amidotransferases by the anititumor drug L(as,5S)-a-amino-3-chloro-4,5-dihydro 5-isoxazoleacetic acid (AT-125). J Bioi Chem 255:6734-6738,1980 Von Hoff DD, Rosencweig M, Muggia FM: New anticancer drugs, pp. 126-148 in Cancer Chemotherapy Annual 1 (Pinedo HM, ed.), Elsevier, New York, 1979 Weinfeld H, Savage CR, McPartland RP: CTP synthetase of bovine calf liver. Methods Enzymol 51:84-90, 1978 Zhen YS, Lui MS, Weber G: Effects of acivicin and dipyridamole on hepatoma 3924A cells. Cancer Res 43:1616-1619, 1983

18 USE OF ORGAN EXPLANT AND CELL CULTURE IN CANCER RESEARCH JAMES H. RESAU and JOHN R. COTTRELL

Cell culture and organ culture are often combined into a more general term " tissue culture". Organ culture refers to the three-dimensional multicellular, multi-tissue in vitro growth of sections or pieces (explants) of organs which retains at least some of the histologic structural integrity of the tissue from which it was taken. Explant organ culture contains therefore the multiple cell types that are the components of the tissue from which the explants are resected. In the case of hamster pancreatic organ/explant culture (17) the explants in vitro are composed of differentiated pancreatic acinar, ductal, islet, endothelial, adipose, fibroblastic, and other connective tissue cells. Cell culture, however, refers to the growth of dispersed, disaggregated single cells of a single cell type (e.g. ductal cells, endothelial). These cells do not necessarily retain the histologic structural relationships of the cells and tissues from which they were removed. Removal and isolation is achieved by enzymatic, chemical, mechanical or physical separation of the cells. Such cells are initially isolated and identified as a primary cell culture later, in their in vitro life span, they may appear immortal and are then called cell lines. Cancer research has utilized both cell culture and organ explant culture methodologies in the study of cancer cell biology and the mechanisms of carcinogenesis (8). Human tumors were shown to be able to give rise to immortal cell lines (6). The most well known continuous cell line of human epithelial origin, HeLa cells, were obtained by Gey's group (1952) working in Baltimore, Maryland at the Johns Hopkins Hospital. The methods and techniques of tissue culture as it applies to cancer cell biology and carcinogenesis are explained quite well in the textbook of Freshney (4). Cells are usually removed by treatment with collagenase, trypsinization and/or mincing and grown in plastic culture vessels. They can be assessed morphologically and biochemically quite easily using standard techniques. He La cells and other carcinoma cells possess specific morphologic criteria which enable them to be identified as malignant cells (5, 15). There is angularity in the nuclear and cytoplasmic organelles and cytosolic material, lack of uniformity in the cellular characteristics or structures (e.g. lumens, nuclear membranes, membrane thickness), and accentuations of normal phenotypic features (e.g. clearing, amount of cytoplasm, clumping, size of cell). To these well established morphologic criteria based upon individual cytologic morphology and histopathologic patterns, tissue culture research has provided several additional criteria which enables researchers to identify malignant cells. Cancer tissues tend to have decreased adhesion between cells and

the isolated cells from the tumors are often described as being anchorage independent (11). Cancer cells are, therefore, logically more likely to grow in suspension and usually will grow in soft agar (22). Cancer cells do not follow contact inhibition restrictions and tend to grow differently on the cultured vessels as plaques or clusters of raised colonies. Cancer cells or tissues grown in culture often release specific marker substances into the medium. Such factors may include mucins (21) or tumor angiogenesis factor (TAF) - (16). Cultured carcinoma cells have been shown to pass through the basement membranes of human derived epithelium (10). Cytopathologic criteria has also been applied to explant organ cultures of mammalian tissue by Resau and Jones (18) and Resau and Albright (20). Cytologic techniques using imprint smears made from explants enable investigators to assess the morphology of the experimental explant tissue without "terminating the cultures" as occurs in histopathologic protocols because the tissue piece can continue in explant culture when the contact smears are done with sterile technique. These criteria are now being applied to human tissues in hopes of assessing the mechanisms and hallmarks of human carcinogenesis. Human tissues obtained from autopsy and surgery from nearly all epithelial tissues have been demonstrated to remain viable for extended periods of time in vitro (8, 9). Human tissues under experimental conditions in vitro can be exposed to carcinogenic compounds such as nitrosamines or polycyclic aromatic hydrocarbons and observed for morphologic (18), immunocytoskeletal keratin markers (1), and biological (1, 23) evidence of transformation. The use of human tissue studies (8) combined with the analytical models of metastasis (10) and growth of cells and explants in nude mice (23) offers significant advantages in understanding the mechanisms of carcinogenesis. When carcinogenic compounds are introduced into the culture media of mammalian tissue (19) specific cytologic changes are observed. Recently, Moyer and Aust (13) have shown that SV40 Tanegen is able to induce phenotypic changes in cell cultures consistent with transformation. Yoakum et al. (24) have been able to transform human bronchial epithelial cells with the transfection of H ras oncogene. The transformed cells produced tumors in nude mice. These dysplastic altered phenotypic tissues can be monitored over extended periods of time using cytologic imprint without requiring histologic sampling (18). The cells isolated from these explants can be assayed for metastatic potential according to the method of Liotta et al. (10). Large populations of these tumor cells and/or fragments can be grown in vivo in nude athymic mice

157 E. K. Weisburger (ed.), Mechanisms of carcinogenesis. © 1989, Kluwer Academic Publishers, Dordrecht. ISBN 978-94-010-7641-8

158

James H. Resau and John R. Cottrell

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Figure 1. Use of tissue culture to study human carcinogenesis. Cells and explants are cultured from tumor tissue to characterize their morphologic and biologic properties (e.g. metastatic potential, susceptibility to chemotherapeutic drugs). Normal cells and explants are cultured in protocols which expose these cells to carcinogens in order to induce transformation in vitro. The transformed tissues can then be assayed the same way as the tumor tissues are studied.

(23). The human nature of the heterotransplanted tissue can be confirmed with in situ hybridization probes (14). Once these methods for the serial propagation of human normal epithelial and carcinoma tissues become widespread studies such as those done by Fidler's group (2, 3) on metastases in the mouse can be routinely done with human tissues. Tissue culture methods offer significant advantages to research protocols studying the mechanisms of carcinogenesis and cell biology of the cancer cells. Explant culture has the advantage that the cells are maintained in a differentiated state arranged in the three-dimensional intercellular relationship of the tissue from which the tumor cells evolve. Cell culture protocols are used to study the cell biology of cancer cells (12), as well as the mechanism of metastasis (2), and the invasion of basement membrane (10). In the future it might be possible to explant cells and tissue fragments from the patient's tumor and expose those cells to specific treatment protocols (e.g. UV, heat, x-ray irradiation and cytotoxic drugs). Subsequently it may be possible to assess the cytotoxic effects the agents induce on the tumor cells much like what is now currently done with bacterial cultures in a clinical microbiology lab for identification, sensitivity, and resistance determinations prior to treatment of patients with antibiotics. Tissue culture currently represents a significant research tool which may become as well a clinically relevant and useful tool for the treatment of cancer. It may be possible to do a series of procedures clinically and experimentally (Figure I) which will greatly enhance our understanding of the cancer cell and its origins.

REFERENCES I.

Banks-Schlegel SP, McDowell EM, Wilson TS, Trump BF,

2. 3. 4. 5. 6. 7.

8. 9. 10. II. 12. 13.

Harris CC: Keratin proteins in human lung carcinoma. Combined use of morphology, keratin immunocytochemistry and keratin immunoprecipitation. Am J PathoII14:273-286, 1984 Fidler IJ: General considerations for studies of experimental cancer metastasis. Methods Cancer Res 15:399-439, 1978 Fidler IJ, Gersten OM, Kripke ML: Influence of immune status of metastasis of three murine fibrosarcomas of different immunogenicities. Cancer Res 39:3816-3821, 1979 Freshney RI: Culture of Animal Cells. Alan R. Liss, Inc., New York, NY, 1983 Frost JK: The Cell in Health and Disease. Williams and Wilkins, Baltimore, MD, 1969 Gey GO, Coffman WD, Kubicek MT: Tissue culture studies of the proliferative capacity of cervical cancer and normal epithelium. Cancer Res 12:364-365, 1952 Giovanella B.C, Stehlim JS, Williams 11: Heterotransplantation of human malignant tumors in nude thymusless mice. II. Malignant tumors induced by injection of cell cultures derived from human solid tumors. J Natl Cancer Inst 52:921-930,1974 Harris CC, Trump BF, Stoner GO: Normal human tissue and cell culture. In Prescott OM (series Ed.) Methods in Cell Biology, New York Academic Press, 1981 Jones RT, Hudson EA, Resau JH: A review of in vitro and in vivo culture techniques for the study of pancreatic carcinogenesis. Cancer 48:1490-1496, 1981 Liotta LA, Lee CW, Morakis OJ: New method for preparing large surfaces of intact human basement membrane for tumor invasion studies. Cancer Res 11:141-152, 1980 MacPherson I, Montagnier: Agar suspension culture for the selection assay of cells transformed by polyoma virus. Virology 23:291-294, 1964 Milo GE, Oldham IN, Zimmerman R, Hatch GG, Weisbrode ST: Characterization of human cells transformed by chemical and physical carcinogen in vitro. In Vitro 17:719-729, 1981 Moyer M, Aust J: Phenotypic changes and gene expression in human colon mucosal epithelial cells upon transfection of a SV40 DNA-GPT recombinant. In Vitro Cell Dev Bioi 23:1416, 1987

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tions of organ culture explant tissue. Virchows Arch (Cell Pathol) 52:15-24, 1986 Shamsuddin AK, Trump BF: Colon epithelium III. In vitro studies of colon carcinogenesis in Fisher 344 rats. N-methylN'-nitro-N-nitrosoguanidine-induced changes in colon epithelium in explant culture. J Natl Cancer Inst 66:403-411,

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19

SEASON OF OPERATION AND DIFFERENTIAL LONG-TERM PROGRESSION: CLINICAL, STATISTICAL, AND PSYCHOLOGICAL ISSUES E.H. KROKOWSKI*, H.W. WENDT and P.A. HOMYAK

INTRODUCTION Seasonal or more precisely, "circannual" rhythms (23) have been reported for neoplastic processes in various contexts, both in the living organism and in isolated organ systems (29). In human patients, for example, specific peaks have been noted for certain months of the year - and as one might expect, questioned as well- over several decades (10, 21, 27, 29, 34, 44, 49). Moreover, the partial resemblance to cyclic trends of infections or vector-borne diseases may have helped to keep some of the simpler models of cancer causation alive. More recently research has focused on associated circannual, biological and hormonal parameters, hematological variables, receptor concentration, immune responses, and many investigations bearing on bioavailability (9, 16,29, 31-33, 36, 46, 48, 51). The issues of immune capability in particular and other, sometimes obvious, overlap of risks, suggest that findings here would not be limited to cancers but would include other mechanisms or pathologies as well. AIDS could be a case in point. We would predict even general associations of a circannual nature (cf. 13), although the events intervening between first symptoms and clinical diagnosis are presumably more complex, and determined more often by idiosyncratic and personality characteristics than in the case of other immune centered pathologies. For example, the emotional status of the patient and the decisions made by him/ her, will necessarily influence the very interval whose seasonal end points we would study. The studies reviewed are concerned with the circannual timing in the contemporary system of the organism and its ecology. We are dealing with hypotheses and findings where the "here and now" intervention (in breast disease) appears to have long range consequences. Intervention is primarily surgery as regards this discussion; however, there could be analogous problems with other therapies such as radiation, chemotherapy, nutritional approaches (22) and others. In each case the objective is to explore the incidence (and other manifestations) of post-treatment - here, post-operative metastases; as a function or corollary of the seasonal circumstances at the time of intervention, etc.

* Empirical results and beginnings of a theoretical framework. Professor Dr. Dr. E.H. Krokowski died November 5, 1985. The editorial decision was made to leave the chapter essentially unchanged.

Actually there is somewhat of a parallel in studies involving long-term effects of seasonal/circannual interactions (if not usually deliberate "intervention") occurring close to the very formative stages of some organ system or another; or during higher order integrations of such systems, rather than in maturity. The following example attempts to highlight some of the differences and suggests that we should consider the potential linkages between the two domains of research. Indeed, some connections along these lines have been identified through phenomena sometimes labelled "anniversary effects". Numerous studies, not always well-controlled, noted phenomena akin to "imprinting", involving factors or conditions which are bound up with season. The incidence of certain anatomical defects, neurological or psychiatric contingencies, even immune parameters and malignancies, is associated with characteristics of the seasonal ecology within which the original formation of the relevant system(s) presumably occurred. Speculation and explanations in terms of critical stages have spanned the entire period from conception to the end of the first postnatal year or even later childhood. Since nearly the entire sequence of events and their duration bear fixed relationships to accessible markers such as birthdate, or sometimes other referents, this research has made use of such points in order to approximate the time of a specific stage, or to guide various other hypotheses. Among the results we find such examples as the overrepresentation of spring births among later psychotic suggesting critical events during winter; congenital heart defects are more frequent among summer to fall born; cleft palate has a slight association with December births (thus pointing to intervention of risk factors during summer); some malignancies are statistically associated with fall births; a variety of organic, physiological and behavioral characteristics are likewise associated with factors in the earliest seasonal environment. The literature includes positive and/or replicated results as well as critiques and criticisms (4,7, 11, 12, 14, 15, 25, 28, 35, 52, 53, 55, 56). As noted, on the other hand, there is an area of overlap between the studies of very early seasonal/circannual associations, and those centering on critical interventions in adult life. For example, the impact of a therapeutic measure sometimes differs from that expected when it is introduced at a time of high personal significance for the patient - certain holidays, birthdays, anniversaries of traumatic events all have been mentioned in this context. If validated further such phenomena would offer additional reasons for extending neuropsychoimmunological explanations (I, 2).

160 E. K. Weisburger (ed.), Mechanisms of carcinogenesis. © 1989. Kluwer Academic Publishers. Dordrecht. ISBN 978-94-010-7641-8

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161

environmental (and corresponding internal physiological and psychophysiological) changes, fewer reserves than norEarlier observations had suggested that the seasonal en- mal are available. Previous work suggests the particular vironment at the time of mastectomies was in some way importance of spring and fall periods, along with other associated with the likelihood of later progression (43). The special meteorobiological circumstances. When the immune phenomenon was of interest in the context of other potential system is already a partial casualty, additional stressors such risks of surgical intervention, especially in the absence of as surgery and post-surgical measures will be dealt with less antimetastatic prophylaxis (38, 39). Specific possiblities, e.g. effectively than at other times. The potential for specific further cell dissemination and colonization were as great a multiplier effects is obvious - all related, phenomenologiconcern as other consequences within the larger issue of the cally, to "season" whether or not the underlying mechacost-benefit equation in the treatment of breast disease (42). nisms are in any way apparent (27, 37). The issue is further At the time of the initial explorations few systematic complicated by the non-linear response dynamics (5, 30, 31): distinctions were made regarding diagnostic subclasses, age, low to moderate stress leads to activation and arousal of the and others. Even the limited observations were provocative, system: in a sense, its negentropic characteristics are mainhowever, in view of the lack of effort in this field. tained. Higher stress levels result in depletion of resources Further analyses have since alerted us to the importance and a negative balance; eventually, chaos follows. As a of such additional classifications, some of which are discus- consequence, tumor growth can proceed unchecked if other sed. Moreover, some observations seemed to implicate stress conditions favor such development. phenomena and associated psychological factors which evi(5) Incidental corollaries: The timing of operation may dently have to be taken into account wherever a more suggest a seasonal mechanism which manifests itself in complete understanding is intended (6, 47, 50). greater or lesser long-term risk, yet the interpretation is The finding that future progression or arrest of the patho- . misleading. The phenomena seen are mere proxies, as it logical process is associated with the seasonal or circannual were. Tapping such incidental corollaries is not uncommon situation, is open to several explanations. The initial think- in chronobiological research wherever powerful syning included the following - all stated in very general terms, chronizers strengthen (or actually "create") a set of phase and intended merely to indicate the direction of the system relationships among variables which are otherwise unrelated or networks which we might eventually explore. Virtually all in any functionally plausible sense. Indeed, the superthe components have been addressed elsewhere (29, 40-42, position, e.g., of circadian and circannual fluctuations can 58). result in spurious outcomes - such "aliasing" may suggest an (I) Cell characteristics, transport and fluid mechanics: annual periodicity which in fact, is accounted for by shifting Coagulation, adhesion, surface and associated features are, circadian peaks. Relationships, that is positive or negative without exception, subject to circadian and circannual correlations, are readily mimicked by unrecognized differperiodicities. It has been shown that the same dosages of ences of, or time dependent changes in, the central tendenanticoagulant, cytostatic, or other therapeutic substances, cies (means) of groups under study (20, 23, 56). While it is etc. have different effects depending upon the time of day or important to guard against such distortions and difficulties, time of year at which they are administered or become they need not be grossly misleading. It may be that a seaactivated (24, 28, 45). The attendant conditions for post- sonal parameter or finding is reduced, upon further scrutiny, operative metastatic progression would vary accordingly. to a "mere" predictor. Even so, however, our understanding (2) Hormones and receptors: Those have long been im- of one aspect or another of the functional matrix involved in plicated in the process and considered in some approaches disease progression, for example, might be enhanced. The to therapy. Hormone levels are among physiological charac- outcome will depend greatly on the specificity of the hypoteristics with the largest circadian and/or circannual am- theses pursued (and on the luck of the investigator. .. ) plitudes, ranging to several hundred percent (3, 19, 21, 23, None of the above ideas is fully testable with the informa31). Seasonal variations of estrogen and progesterone recep- tion at our disposal. These suggestions might, nevertheless, tor parameters are similarly large (33). Inasmuch as the be kept in mind, and some explanatory models might betiming of an operation is unavoidably bound up with season come more plausible than others. specific receptor activity, the overall risk functions would presumably differ at least for some classes of patients and/or DATA AND METHOD disease characteristics. (3) Tumor growth: Tumor processes sometimes become symptomatic or diagnostic during specific seasons. Differen- The chief data source for the study of postoperative protial growth rates of the tumor may be a partial explanation. gressions and the apparent role played by the timing of Further investigation might center on the determination of surgery consists in the records covering 503 mastectomies applicable sensing thresholds-the patients', the physicians', performed in Hesse, Germany. Nearly all initial operations as well as the decision outcomes of the diagnostic system as occurred between 1971 and 1974. With exceptions such as a whole. Complicating such studies would be the large in- those of recent immigrants to the region, the records come dividual differences in thresholds, alertness, distraction en- from approximately 50 hospitals of the Land, both public vironments, concern or lack thereof over symptoms, degree and semi-private. More than half of the operations were of sophistication and related logistic and psychological fac- performed in the approximately 10 surgical facilities of tors (6, 30). Kassel, a significant industrial and commercial centre (51, (4) Stress: Adaptation to circannually variable circum- 3°N, 9SE elevation 165 m) of 200,000 population. stances is differentially stressful. During periods of rapid The patients were classified by age (range, 29 to 83 years);

Scope of the study

162

E.H. Krokowski, H. W. Wendt and P.A. Homyak

lymph node status at admission (as node negative, No, or node positive, N +); menopause status if available; date of operation; tumor size; date of diagnosis of first postoperative metastasis if any; date of birth; date of death if deceased and information available. Except for the lymph node involvement the few cases having other metastases at the time of initial diagnosis or operation were excluded from this phase of the study. Generally this classification did not contribute appreciable information. The major exception is node status which proved extremely important. As one would expect trends emerged with other variables as well, but these were difficult to evaluate statistically. This is partly due to the small numbers in the relevant subgroups. In any case, the current treatment is limited to the results of the node breakdown in the context of the circannual variations. Of the other classifications age is apparently the most important, but the patterns are complex (not even counting the issue of statistical significance). As expected the majority of these patients were over 50 years of age. Of the 503 cases, one was discarded as an extreme statistical outlier, possibly reflecting an erroneous transcription. The dependent variables were, (I) the incidence (occurrence or nonoccurrence) of metastatic progression regardless of site, within five years following operation; (2) the latency between operation date and the time of any such M + diagnosis within a 5 to lO-year span. The difference in follow-up times reflects decisions made at different stages in 60~ 0 g

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