Proceedings of the Tenth Canadian Cancer Research 9781487579494

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PROCEEDINGS OF THE TENTH CANADIAN CANCER RESEARCH CONFERENCE

19 7 3

EDITORIAL ADVISORY BOARD

A.A. AXELRAD

Department of Anatomy, University of Toronto, Toronto, Ontario J.M.M. DARTE

Dr Charles A. Janeway Child Health Centre, St. John's, Newfoundland R. SIM A RD

Department of Cell Biology, University of Sherbrooke, Sherbrooke, Quebec L. SIMINOVITCH

Department of Medical Genetics, University of Toronto, Toronto, Ontario H . F. STICH

Cancer Research Unit, University of British Columbia, Vancouver, British Columbia R . M. TAYLOR

National Cancer Institute of Canada, 25 Adelaide St. E., Toronto, Ontario J.E. TILL

Division of Biological Research, Ontario Cancer Institute, Toronto, Ontario

PROCEEDINGS OF THE TENTH CANADIAN CANCER RESEARCH CONFERENCE Honey Harbour, Ontario, 1973 Edited by P.G. Scholefield

PUBLISHED FOR THE NATIONAL CANCER INSTITUTE OF CANADA 25 Adelaide St. E., Toronto, Ontario, Canada

BY UNIVERSITY OF TORONTO PRESS

© University of Toronto Press 1974 Toronto and Buffalo Printed in Canada Reprinted in 2018

ISBN 0-8020-2086-0 ISBN 978-1-4875-8066-7 (paper) LC 55-8263

Preface The formal sessions at the Tenth Canadian Cancer Research Conference were devoted to discussions of Endogenous Viruses, Enviromental Factors, Tumor Immunology, and Chemotherapy. In selecting the speakers for these sessions, the Program Committee (Dr H .F . Stich, Chairman; Dr G.J. Goldenberg; Dr G.A. LePage; Dr J.A. Mccarter ; Dr R. Simard, and Dr J.E. Till) was guided by the desire to have at least one paper in each session concerned primarily with the disease as it occurs in man. All speakers were reminded that, since the audience at these conferences is heterogeneous with respect to discipline and degree of experience, the presentation should aim to stimulate the beginner as well as to inform the expert. Follow-up of the formal sessions was based on the experience of the previm!s Conference and took two forms. The first arose from the fact that delegates interested in specific fields were able to meet with the main conference speakers during the subsequent workshop sessions when it was possible to discuss problems of mutual interest at the desired level. The entire third day was devoted to such workshops. The following topics were discussed and the name of the individual who chaired each workshop is indicated. Wednesday 13 June 1973 Morning workshops

Alternoon workshops

RNA tumor viruses A.F. Graham

RNA tumor viruses J.K. Ball

DNA tumor viruses P. Bourgaux

DNA tumor viruses P. Cheevers

Morphology of tumors and tumor viruses A.F. Howatson

Morphology of tumors and tumor viruses G. Tremblay

Carcinogenesis D .R. McCalla

Carcinogenesis G . de Lamirande

Cell surfaces (Receptors) F . Paraskevas

Cell surfaces (Biochemistry) H . Schachter

Cell differentiation J .S. Haskill

Cell differentiation V.I. Kalnins

vi

PREFACE

Chemotherapy D.E. Bergsagel

Immunotherapy G.J. Goldenberg

Radiation sensitizers G.F. Whitmore

Hormonal control N. Bruchovsky

Epidemiology A.B. Miller

Somatic cell genetics L. Siminovitch

Antigens and human cancer J.M. Gold Regulation of gene activity R. Simard

A second type of follow-up was designed for the benefit of the younger delegates. On the Tuesday evening, at the conclusion of the formal sessions, the main conference speakers made themselves available in separate cabins for informal discussions ('rap' sessions) to which only graduate students and postdoctoral fellows were invited. No record was kept of the proceedings of the workshops or of the 'rap' sessions, but the subsequent comments have suggested that these experiments in communication have added greatly to the value of the Canadian Cancer Research conferences to those who participated. It is unfortunate that these experiences can not be shared through the medium of the printed word, but their contributions to the stated objectives of this series of conferences, and to the reinforcement of the material presented at the formal sessions, will merit much consideration in future planning. Editor P.G . Scholefield

Editorial Advisory Board A.A. Axelrad J.M.M. Darte R. Simard

L. Siminovitch H.F. Stich

R.M. Taylor J.E. Till

Contents Preface

V

ENDOGENOUS

VIRUSES

The role of herpes viruses in human cancer Harald zur Hausen Genetics and evolution of RNA tumor viruses G.S. Martin and R.A. Weiss Occurrence, properties, and interrelationships of leukemia and sarcoma viruses of the mouse J.A . McCarter, J.K. Ball, and P.K .Y. Wong ENVIRONMENTAL

10

31

CARCINOGENESIS

Record linkage for studies of environmental carcinogenesis Howard B . Newcombe Transplacental carcinogenesis A . Koestner The use of DNA repair in the identification of carcinogens, precarcinogens, and target tissue H.F. Stich. D. Kieser, B.A. Laishes, and R.H.C. San TUMOR

3

49

65

83

IMMUNOLOGY

Mechanisms of humoral tumor immunity in malignant melanoma M.G. Lewis Immunotherapy for acute myelogenous leukemia Ray Powles Pitfalls in tumor immunology Richmond T. Prehn

113 130 136

CHEMOTHERAPY

Selection of regimens for clinical chemotherapy Thomas C. Hall The measurement of the effects of anti-cancer agents W.R. Bruce Some model systems in cancer chemotherapy G.A. LePage

149 162 171

The role of herpes viruses in human cancer HARALD

ZUR

HAUSEN

lnstitut fur klinische Virologie, Universiti:it Erlangen-Nurnberg, 852 Erlangen, Loschgestr. 7, Germany

Herpes-group and oncorna viruses represent the prime candidates as etiological agents of specific human tumors. Although several serotypes of human adenoviruses have been shown to be oncogenic in rodents, there exists no evidence that they also transform human cells. Today, the EpsteinBarr virus (EBY), as a representative member of the herpes virus group, is the prime suspect among additional candidates of the same group as well as in a comparison with the oncorna viruses. The serotype 2 of herpes simplex virus has also been shown to possess properties in human cells which are characteristic for tumor viruses. The participation of human cytomegalovirus in Kaposi's sarcoma is still inconclusive. Oncornaviruses have not yet been demonstrated consistently in any human malignancy. Their presence might be revealed, however, by the demonstration of virus-specific enzymatic activities and the existence of products which show physical properties similar to those of oncornaviruses of animal origin. The major part of this paper will discuss the presence of Epstein-Barr virus in human tumor cells and summarize results which incriminate this virus as the etiologic agent of Burkitt's lymphoma and possibly also of the anaplastic carcinoma of the nasopharynx. Attention will be focused on the demonstration of viral nucleic acids within these tumor cells. About three years ago we started to purify radioactive EB viral DNA and initiated DNA-DNA homology studies with DNA from tissue culture cells of Burkitt tumor origin. In first experiments we demonstrated significant hybridization of EB viral DNA with DNA derived from cells of the 'virusfree' Raji line, as compared to various control preparations of human KB and hamster Nil-2 cells. 1 Similarly, increased annealing of Raji cell RNA with EB viral DNA, as compared to the above controls, was observed, indicating the continued in vivo transcription of EBY-specific RNA within these cells. No cross-hybridization of DNA from herpes simplex virus-, cytomegalovirus-, or varicella virus-infected cells with EBY-DNA was detected.

H. ZUR HAUSEN

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These results showed that EBY may act like well-known DNA tumor viruses. Its DNA persists in Raji cells and at least part of this DNA is biologically active. Subsequent experiments by Ham par et al. 2 and Gerber 3 showed that Raji cells as well as several other non-virus-producing lymphoblastoid lines contain the complete, or at least almost complete, viral genome. Treatment of these cells with 5-BUdR or 5-IUdR resulted in induction of viral structural antigen and viral particle synthesis. DNA-DNA hybridizations with radioactive EB viral DNA were also applied to biopsy material derived from Burkitt lymphomas and from various other tumors of the head and neck and of the lymphatic system. 4 They revealed an increased annealing of viral DNA with DNA from Burkitt lymphomas and nasopharyngeal carcinomas as compared with a number of other preparations of different origin. These included DNA from Hodgkin biopsies, various lymphomas, and carcinomas. There existed a considerable variation in hybridized counts among the Burkitt lymphoma and nasopharyngeal carcinoma biopsies tested. DNA from multiple tumors of the same donor, however, annealed with EBY-DNA in a similar range. The extremely low yields of purified EBY-DNA and the low specific radioactivities of this material were circumvented by in vitro transcription of EBY-DNA with the aid of E.coli RNA-polymerase. The resulting complementary RNA was used in further tests. Although it has not yet been clarified which parts of the viral genome are transcribed in vitro, the use of this method has been successfully applied to the detection of latent EB viral genomes by Nonoyama and Pagano5 and by our group. 6 This test permitted the detection of EB viral DNA in all those immunoglobulin-producing lymphoblastoid cell lines tested thus far which were devoid of viral structural antigens. 7 Although most of them were subsequently shown to be inducible for EBY-antigen-synthesis by BUdR or IUdR, some of them appeared to be refractory to these compounds. 8 DNA from two immunoglobulin-negative lines, both derived from acute lymphatic leukemias, failed to hybridize significantly with EBY-specific RNA. Reconstruction tests with EBY complementary RNA (cRNA) as well as reassociation kinetic experiments with labelled EBY-DNA after addition of cellular DNA of various origin 9 demonstrated that the cells of some of the lymphoblastoid lines were 'loaded' with EBY-DNA. Approximately 0.25 per cent of the total cellular DNA was virus-specific in one line. 7 The presence of viral DNA in cells of these lines is regularly paralleled b-, the appearance of apparently virus-specific complement-fixing antigens. 10 -11 They seem to represent an expression of persisting viral genomes. Recently Reedman and Klein12 visualized such antigens by anticomplementary immunofluorescence and demonstrated their intranuclear localization.

Herpes viruses in human cancer

5

Besides the capacity to transform normal lymphocytes into continuously growing cells, EBY thus shares at least two properties with known DNA tumor viruses: it persists at a subviral level within the transformed cells and exerts at the same time an apparently specific genetic activity. Annealing of DNA from tumor biopsies with EBY-cRNA basically confirmed the results obtained by DNA-DNA hybridizations. 13 DNA from all Burkitt biopsies tested thus far revealed increased hybridization as compared to various controls. In view of the rather uniform histology of Burkitt's lymphoma, it seems reasonable to assume that the annealed label detects virus-specific DNA within tumor cells. In addition, this is supported by the presence of EBY-specific complement-fixing (Klein, personal communication) as well as membrane antigens in fresh biopsy cells and the synthesis of EBY-specific structural antigens soon after explanation of these tumor cells into tissue culture. Thus, Burkitt's lymphoma represents the first example of a human malignancy consistently associated with a transforming virus. The regular demonstration of EBY-DNA in nasopharyngeal carcinomas by DNA-DNA hybridizations was carefully re-evaluated by annealing experiments with EBY-cRNA. With few exceptions 28 DNA preparations from different biopsies of nasopharyngeal carcinomas hybridized above the background of various control preparations. The hybridization data of some of these tumors were excessively high, indicating a considerable 'load' with EBV-DNA. The nasopharyngeal carcinoma reveals histologically a mixed cell population : besides epithelial tumor cells it contains varying amounts of infiltrating lymphocytes which justify its original name 'lymphoepithelioma.' Until now it has been an open question whether the epithelial tumor cells or the lymphocytes,or both, harbour EB viral genomes. Our first attempt to analyse this problem was the comparison of hybridization data with the tumor histology. It was rather surprising that predominantly epithelial tumors which contained more than 50 per cent of epithelial cells annealed most efficiently with EBY-cRNA. If exclusively lymphocytes would harbour viral DNA, more than 0.5 per cent of their total DNA had to be virus-specific. This concentration exceeds all other experimental data obtained with persisting EBY genomes. Biopsies which were negative in the hybridization tests or annealed only to a low extent represented histologically either non-tumorous tissue or predominantly lymphoid tumors. This result suggested that epithelial cells harbour the EBY genomes. A more direct approach to localize the EBY-positive cells was the application of the anticomplementary fluorescence method 11 to frozen sections of nasopharyngeal carcinoma (NPC) biopsies. Sections from two nasopharyngeal carcinomas and three other tumors were examined by using EBV-

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reactive as well as non-reactive sera. Cells of the Raji line, derived from a Burkitt lymphoma, served as positive control. The 'Molt' line derived from a patient with acute lymphatic leukemia, was included as negative control. No anticomplementary fluorescence was observed in Molt cells and sections of non-lymphoepithelial tumors. Raji cells and apparently epithelial cells of the nasopharyngeal carcinomas, however, revealed a clearly positive fluorescence. In Raji cells, this fluorescence was confined mainly to the nuclear regions, whereas in the nasopharyngeal cells it appeared to be more granular, and was also observed in the cytoplasm. The brilliant staining depended on the presence of antibodies against EBY-associated antigens. No fluorescence characteristic for EBY early or structural antigens was detected within those sections. To further substantiate these results, sections of two lymphoepitheliomas and three control tissues ( one adenoma and two salivary glands) were denatured by NaOH treatment and subjected to in situ hybridizations with EBY-specific cRNA. We showed previously that this procedure permits the localization of viral DNA in EB virus-producing as well as in viral genomecarrying tissue culture cells. 6 After hybridization the tumor sections were treated with RNase, exposed for three weeks to autoradiography, developed, and stained. Whereas the control sections did not reveal any clustering of grains or accumulation of label over nuclei, this was clearly the case in nasopharyngeal carcinomas. Sections of one tumor which annealed with about 1900 c.p.m. in filter hybridizations were heavily labelled. This tumor consisted mainly of epithelial cells. The heavy labelling of the majority of nuclei, however, made their definite identification difficult, especially in view of the deteriorated morphology after NaOH denaturation. In the second tumor, which annealed with about 850 c.p.m. in membrane hybridizations, the situation was much better. Numerous foci of clustered nuclei were clearly labelled within those sections, whereas nuclei of the surrounding tissue were not. The arrangement of labelled clusters of these nuclei corresponded to similar foci of epithelial cells in non-denatured sections of the same tumor. Nuclei of lymphocytes and other cellular components of this tumor seemed to be unlabelled. These results permit the conclusion that the epithelial cells of nasopharyngeal carcinomas harbour those viral genomes which are demonstrated by nucleic-acid hybridizations. Thus, this tumor represents the first example of an infection of human non-lymphatic cells with EBY. The role of the infiltrating lymphocytes remains to be clarified. They might be involved in the immune response against antigens specified by the viral DNA within epithelial cells. The presence of EBY within the epithelial tumor cells of

Herpes viruses in human cancer

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nasopharyngeal carcinomas substantially supports the circumstantial evidence for a casual role for EBV in this malignancy. Little is known about the state of EB viral DNA within human tumor cells. Nonoyama and Pagano claimed to demonstrate non-integration of EBV-DNA with the host cell chromosomes14 in Raji cells. This was based on the sedimentation pattern of DNA hybridizable to EBV-cRN A in alkaline glycerol gradients. Their approach, however, might be questioned in view of alkali-labile spots within herpes virus DNA as reported by Bachenheimer et al. 15 The constant concentration of viral DNA in Raji cells observed over a period of two years of continuous cultivation12 favours covalent linkage of viral and host-cell DNA as the most reasonable explanation. Further experiments are needed to substantiate these results. Recently another member of the herpes virus group, herpes simplex virus (HSV) type 2, was suspected of playing a role in the induction of human cervical carcinoma. This was suggested by seroepidemiological studies 16 •17 and also by the presence of virus-specific antigens within some carcinoma cells. 18 Duff and Rapp19 showed the transforming capacity of partially UV-inactivated HSV 2 for hamster cells. Darai and Munk reported transformation of human lung cells after long incubation at 42° C shortly after infection. 20 It was expected that nuclei acid hybridizations would be of considerable help in elucidating the relationship of HSV 2 to cervical carcinoma cells. Dr Schulte-Holthausen in my department tested DNA from thirteen biopsies derived from cervical carcinomas in nucleic-acid homology tests with HSV 2-cRNA (H. Schulte-Holthausen, unpublished results). He was unable to observe increased hybridizations in any of the preparations tested. Although the failure to demonstrate HSV 2-DNA in these biopsies did not exclude a possible etiologic role for this agent in human cervical cancer, it showed clearly that we are not dealing with a relationship similar to EBV and Burkitt's lymphoma and nasopharyngeal carcinoma. The sensitivity of most of the HSV 2-experiments would have permitted the detection of more than one genome equivalent per tumor cell. Thus, a small content of virusspecific molecules could escape their discovery by this method. By using a more sensitive assay, Frenkel et al. 21 reported HSV 2-specific RNA in a virus-free cervical tumor by analysing DNA-RNA hybrids by hydroxylapatite column chromatography. The viral RNA transcripts were complementary to 5 per cent of HSV 2-DNA and corresponded to both early and late transcripts. Their analysis of this cervical DNA for the presence of HSV 2-DNA sequences revealed that only a fragment, representing approximately 40 per cent of the total HSV 2-DNA was present. In addition, the concentration of this fragment corresponded to about 1 mole per mole of

H. ZUR HAUSEN

8

cellular DNA. Finally, they showed that at least parts of this fragment are covalently linked to host DNA. It remains to be seen whether fragments of HSY 2-DNA are found consistently in human cervical carcinoma cells and whether they caused the transformation of these cells. The available data reveal already the enormous versatility of herpes virus-host cell interactions. It might be worthwhile to re-evaluate the oncogenic potential of any number of this group of viruses. REFERENCES 1 zur Hausen, H. and Schulte-Holthausen, H . Presence of EB virus nucleic acid homology in a 'virus-free' line of Burkitt tumour cells. Nature, 227 :245-248, 1970 2 Hampar, B., Derge, J.G., Martos, L.M., and Walker, J.L. Synthesis of Epstein-Barr virus after activation of the viral genome in virus-negative human Iymphoblastoid cells (Raji) made resistant to 5-bromodeoxyuridine. Proc. Natl. Acad. Sci. u .s., 69 :78-82, 1972 3 Gerber, P. Activation of Epstein-Barr virus by 5-bromodeoxyuridine in virus-free human cells. Proc. Natl. Acad. Sci. u.s., 69:83-85, 1972 4 zur Hausen, H ., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P., and Santesson, L. EBV-DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature, 228 : 1056-1058, 1970 5 Nonoyama, M. and Pagano, J.S. Detection of Epstein-Barr viral genome in nonproductive cells. Nature 233 : 103-105, 1971 6 zur Hausen, H . and Schulte-Holthausen, H . Detection of Epstein-Barr virus genomes in human tumor cells by nucleic acid hybridization. In Oncogenesis and Herpes viruses (P.M. Biggs, G. de The, and L.N. Payne, eds.), pp. 321-325. Lyon: IARC, 1972 7 zur Hausen, H ., Diehl, V., Wolf, H., Schulte-Holthausen, H ., and Schneider, U. Occurrence of EB viral genomes in human Iymphoblastoid cell lines. Nature, 237, 189-190, 1972 8 Klein, G . and Dornbos, L. Relationship between the sensitivity of EBY-carrying Iymphoblastoid lines to superinfection and the inducibility of the resident viral genome. Intern. J . Cancer, 11 :327-337, 1973 9 Nonoyama, M . and Pagano, J.S . Homology between Epstein-Barr virus DNA and viral DNA from Burkitt's lymphoma and nasopharyngeal carcinoma determined by DNA-DNA reassociation kinetics. Nature, 242:44-47 , 1973 10 Pope, J.H., Horne, M.K., and Wetters, E.J. Significance of a complement-fixing antigen associated with herpes-like virus and detected in the Raji cell line. Nature, 222:186-187, 1969 1 I Vonka, V., Benyesh-Melnick, M. , and McCombs, R.M. Antibodies in human sera to soluble and viral antigens found in Burkitt lymphoma and other lymphoblastoid cell lines. J. Natl. Cancer Inst., 44 :865-873, 1970 12 Reedman, B.M. and Klein, G. Cellular localization of an Epstein-Barr virus (EBV)-associated complement fixing antigen in producer and nonproducer Iymphoblastoid cell lines. Intern. J . Cancer, 11 , 499-520, 1973 13 zur Hausen, H. Epstein-Barr virus in human tumor cells. In Intern. Rev. Exptl. Pathology, Academic Press, 11:233-258, 1972

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14 Nonoyama, M. and Pagano, J.S. Separation of Epstein-Barr virus DNA in nonvirusproducing cells. Nature, 238 : 169-171, 1972 15 Bachenheimer, S.L., Kieff, E.D., Leer, L., and Roizman, B. Comparative studies of DNA's of Marek's disease and herpes simplex viruses. In Oncogenesis and Herpesviruses (P.M. Biggs, G. de The, and L.N. Payne, eds.), pp. 74-81. Lyon: IARC, 1972 16 Nahmias, A.J., Josey, W.E., Naib, Z.M., Luce, C., and Guest, B. Antibodies to herpes virus hominis types 1 and 2 in humans. Women with cervical cancer. Am. J. Epidemiol., 91:547-552, 1970 17 Rawls, W.E., Tomkins, W., and Melnick, J .L. The association of herpesvirus type 2 and carcinoma of the cervix. Am. J. Epidemiol., 89, 547-554, 1969 18 Royston, J. and Aurelian, L. Immunofluorescent detection of herpesvirus antigens in exfoliated cells from human cervical carcinoma. Proc. Natl. Acad. Sci. u .s., 67 :204-212, 1970 19 Duff, R. and Rapp, F. Oncogenic transformation of hamster cells after exposure to herpes simplex virus type 2. Nature, 233 :48-50, 1971 20 Darai, G. and Munk, K. Human embryonic lung cells abortively infected with herpes virus hominis type 2 show some properties of cell transformation. Nature, 241 : 268-269, 1973 21 Frenkel, N., Roizman, B., Cassai, E., and Nahmias, A. DNA fragment of herpes simplex 2 and its transcription in human cervical cancer tissue. Proc. Natl. Acad . Sci. u.s. 69 :3784-3789, 1972.

Genetics and evolution of RNA tumor viruses G.S. MARTIN

AND

R.A. WEISS

Imperial Cancer Research Fund Laboratories, P.O. Box 123, Lincoln's Inn Fields, London, WC2A 3PX. U.K.

RNA tumor viruses represent a homogeneous group, with similar physical and chemical properties and modes of replication. A detailed account of the biology and molecular biology of RNA tumor viruses is provided in a recent book on tumor viruses edited by Tooze. 68 Here we shall briefly discuss some aspects of the biology of these viruses in order to discuss some ideas about their origin and genetic structure, placing emphasis on the avian tumor viruses as the accompanying contribution by Mccarter reviews the murine viruses. The RNA tumor virus particle contains a 60-70S RNA genome comprised of 3-4 subunits. Associated with this genome are several internal proteins designated group-specific (gs) antigens because they share common antigen specificities within each group of viruses, e.g. the avian leukosis group or the murine leukemia group. The virus particle is enclosed in a lipoprotein envelope ( derived from the cell membrane) which bears virus-coded envelope antigens that elicit neutralizing antibodies; these antigens are glycoproteins14 which are required for the virus to penetrate the cell via receptor sites at the cell surface. The avian tumor viruses have been classified into six subgroups, A to F, on the basis of their envelope properties; members of the same subgroup interfere with each other, show antigenic cross-reactivity, and have similar host-range properties. The replication of RNA tumor viruses (Fig. 1) shows an early sensitivity to inhibitors of DNA synthesis, and a continuous sensitivity to inhibitors of DNA-dependent RNA synthesis. These observations led Temin 61 to postulate that these viruses replicate via a DNA intermediate, the provirus. This idea is supported by the discovery that the virion contains an enzyme, 'reverse transcriptase,' which is capable of using viral RNA as a template for DNA synthesis. 65 Virus mutants which lack functional reverse transcriptase are unable to replicate or to transform cells. However, a reverse transcriptase mutant of Rous sarcoma virus is complemented by phenotypic mixing with

Genetics and evolution of RNA tumor viruses

Figure I

11

Replicative cycle of an RNA tumor virus.

'helper' leukosis viruses, 25 indicating that the enzyme is required only for the initiation of infection. Furthermore, temperature sensitive reverse transcriptase mutants 27 are able to replicate if allowed an initial period at the permissive temperature, confirming that the enzyme is required only for an early step in virus growth. In vitro experiments show that the enzyme is capable of copying all the sequences in the viral RNA, 12 but the DNA is synthesized as short segments 19 and it is still obscure how complete provirus synthesis occurs in vivo. It appears that a DNA-RNA hybrid is formed and then a doublestranded DNA provirus which probably integrates into one of the host-cell chromosomes. Even after provirus formation, cellular DNA synthesis and probably a subsequent mitosis are required for the formation of progeny virus and for cell transformation. 02 , 74 To form the genomes of progeny virus, RNA strands are transcribed from the DNA provirus. Since negative strand RNA is not found in infected cells, it is assumed that the viral messenger RNA is identical with genome RNA, which may itself act as messenger. Viral replication is completed by budding from the cell membrane, which is modified by the insertion of the viral glycoproteins. The budding and release of virus particles does not kill the cell, so that it is possible for the cell to become converted into a malignant cell and at the same time produce viral progeny. RNA tumor viruses are classified into three groups, the sarcoma, leukemia, and mammary tumor viruses, according to the type of cell that they transform. Leukemia viruses can be classified further into lymphoid, myeloid, and erythroid leukemia viruses. Sarcoma and leukemia viruses of a given host species share common gs antigens, but the murine mammary tumor viruses differ from murine leukemia viruses antigenically and morphologically. Lymphoid leukemia viruses (LLV) and murine mammary tumor viruses (MMTV) occur commonly in nature. The neoplasms associated

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12

with LLV and MMTV have long latent periods and the viruses are frequently transmitted 'vertically,' from mother to offspring. For instance, viremic hens produce large quantities of avian LLV in the ovary and oviduct9 which infect the cells of the early embryo. Although the embryo rapidly becomes viremic, it usually grows normally and does not develop leukemia before maturity. Thus the bird may live to pass on the virus to the next generation as well as by horizontal infection. The sarcoma viruses and non-lymphoid leukemia viruses cause rapidly developing neoplasms; there may be strong natural selection against these viruses, therefore, and vertical transmission is not known. However, under artificial laboratory conditions these viruses are maintained easily and they have been used extensively in experimental studies. Many sarcoma viruses and non-lymphoid leukemia viruses are defective for replication and infectious stocks are maintained through complementation by 'helper' LLVs; the significance of defectiveness will be discussed below. Sarcoma viruses transform fibroblasts or cell lines in vitro. The transformed cells show altered growth properties, such as the ability to grow at high cell density or in agar suspension (reviewed by Macpherson39 ). They also show a variety of cell surface changes, including alterations in cell morphology, membrane glycoproteins and glycolipids, increased transport of small molecules, and increased agglutinability by plant lectins. The infection of a cell in culture by a single infectious sarcoma virus particle leads to the production, by cell division or successive cycles of re-infection or both, of a group of transformed cells: this is the basis of the focus assay for sarcoma viruses. 66 The leukemia viruses, on the other hand, although capable of replication in fibroblasts, do not usually transform them and other assays are therefore required. An assay for avian myeloblastosis virus, based on the transformation of yolk-sac myeloblasts in vitro, has been developed by Moscovici and Zanetti,4 5 but transformation of hemopoietic cells in vitro by strains of lymphoid leukemia viruses has not yet been achieved. Some leukemia viruses will produce plaques in cultures of certain cell types.54 , 32 , 21 Other methods for detecting and assaying leukemia viruses include interference tests, where pre-infection of cells with leukemia virus confers resistance to superinfection with sarcoma virus, and virus particle production by assaying gs antigens or reverse transcriptase. Infection with murine mammary tumor viruses cannot yet be assayed in vitro. In addition to proviruses which may be acquired by infection, all chickens and mice also appear to carry endogenous or 'germinal' proviruses which are transmitted genetically in the chromosomes of the germ cells from one generation to the next. Since endogenous viral genomes are not transmitted as virus particles, the genetic transmission by-passes host-range restrictions (see below) as well as requirements for the synthesis of viral RNA and

Genetics and evolution of RNA tumor viruses

13

proteins. They are inherited just like other segments of host chromosomal DNA and do not depend on independent replication. Compelling evidence for the existence of inherited viral genomes 68 has accrued from the discovery of viral antigens and of DNA homologous to viral RNA in embryonic tissues, the spontaneous release of infectious virus from certain host cells, and perhaps most convincing, experiments in which normal, 'uninfected' cells are induced chemically to release virus in culture. With mouse cells the most effective inducing agents are iodo- and bromo-deoxyuridine, 38 while with chick embryo cells treatment with methylcholanthrene or nitroquinoline oxide activates virus. 78 It is not yet clear whether all genetically transmitted viruses are actually oncogenic. However, the genetic transmission of LLV and MMTV is now well established, though the lymphoid and mammary neoplasms typically appear only after the viral genomes become activated to replicate as viruses. Once activated they can also be infectiously transmitted to susceptible hosts and cause further tumors in those hosts. INFLUENCE OF HOST GENES ON INFECTION BY RNA

TUMOR

VIRUSES

The susceptibility of the host cell to infection by an RNA tumor virus is controlled by a variety of genetic factors. Some of these factors affect the ability of the virus to penetrate into the host cell, while others affect subsequent intracellular events in the replication cycle of the virus. The host genotype also controls whether the cell will respond to infection by becoming transformed. In chickens, four loci, the 'tumor virus' or tv loci (tv-a, tv-b, tv-c, and tv-e), have been described, each affecting susceptibility to a specific virus subgroup ( the subgroups A, B, C, and E). In each case the allele for susceptibility is dominant, suggesting that these genes govern the synthesis of a product required for virus growth. It is most likely that this product is a receptor required for virus penetration, 73 since the resistance can be overcome either by phenotypic mixing with a virus of another subgroup (i.e. with different envelope glycoproteins) or by using Sendai virus to promote entry of the virus into the cell. Virus does adsorb to genetically resistant cells, so that the receptors must be required for a later step in penetration. Another locus which affects susceptibility to subgroup E viruses has been identified by Payne et al. 49 At this locus, the /• locus, resistance is dominant, but it can be overcome by phenotypic mixing with virus of another subgroup, indicating that the block is again at the level of penetration. Resistance appears to result from saturation of the surface receptors with E subgroup envelope antigen produced by the endogenous virus which belongs to subgroup E, because it is correlated with the presence of E subgroup antigen at the cell

14

G.S. MARTIN AND R.A. WEISS

surface and with other indications of the expression of endogenous viral genes. 76 In the mouse, loci governing the synthesis of surface receptors have not been found. Murine leukemia viruses can be classified according to their host range into three types : N-tropic, B-tropic, and NB-tropic viruses. N-tropic viruses plate 100-1000 fold more efficiently on NIH-Swiss mouse embryo cells than on Balb/ c cells. B-tropic viruses plate more efficiently on Balb/ c than on NIH-Swiss cells; and NB-tropic viruses plate with equal efficiency on both types of cells. All mouse strains resemble either NIH-Swiss or Balb/ c in showing a greater sensitivity to either N-tropic or B-tropic viruses. Crosses between these strains show that a single locus is responsible for these differences in sensitivity. Resistance to each class of virus is dominant : thus the heterozygote is resistant to both N-tropic and B-tropic viruses. The locus is denoted Fv-1, since it has been found to be identical with a previously identified locus governing sensitivity to Friend leukemia virus. 36 The resistance appears to be produced at a later step than penetration, because the tropism of a virus strain is not correlated with the serotype (envelope antigens) and cannot be overcome by phenotypic mixing. Host genes which appear to affect the ability of the infected cell to become transformed have also been identified. One example is the Fv-2 locus in the mouse: the recessive allele produces an absolute resistance to spleen focus formation by Friend leukemia virus but probably does not affect the replication of the virus in fibroblasts in vitro or in the spleen in vivo.35 The mutation thus appears to affect the ability of the erythroid target cell to become transformed by the virus. In chickens it has been found that lines with the same susceptibility to infection by lymphoid leukemia virus strain RPL-12 differ in their incidence of leukemia following infection, 47 suggesting a genetic effect on the ability of the target cell to become transformed. However, it may be argued that the transformed cells are more readily rejected in the resistant lines. Clearly in vitro systems for studying transformation by leukemia viruses are needed to determine the mechanism by which resistance is produced. GENETIC CONTROL OF ENDOGENOUS VIRUS EXPRESSION

As described above, normal cells which are not producing virus can become induced to release genetically transmitted, endogenous viruses. The frequency of induction of endogenous virus, both in vivo and in vitro, is also subject to genetic control. Cells of the high leukemic mouse strain AKR can be readily induced to produce an N-tropic virus in vitro; since the cells are sensitive to N-tropic viruses, that is, they are Fv-1° the induced virus grows to high titre. Crosses of AKR mice to low-virus strains which are also Fv-Jnn indicate that inducibility, both in vivo and in vitro, is dominant to non-inducibility 0,

Genetics and evolution of RNA tumor viruses

15

and is determined by two independently segregating genes, V 1 and V 2, either of which can give a positive phenotype. 51 When AKR mice are mated to Fv-Jbb strains carrying B-tropic viruses that appear in later life, no induction of the B-tropic virus occurs in the F 1 or in back-crosses to the Fv-Jbb parent, indicating that the induction loci in the AKR strain are specific to the virus carried by that strain. 52 Indeed, these virus-inducing loci may represent viral rather than host genes. Essentially similar results have been obtained using the Balb/c line. 59 Inducibility loci have also been demonstrated in chickens of lines 7 and 100: some of the chickens of these strains produce spontaneously the endogenous E subgroup virus of chickens, and crosses between inducible and non-inducible chickens demonstrate that, as with the induction of mouse leukemia virus, inducibility is dominant. 8 Even when the inducibility alleles are present, virus is spontaneously activated in a small proportion of cells only. Mice and chickens become viremic relatively early in life if they carry both the inducibility allele and the appropriate susceptibility alleles to allow horizontal spread of the virus from cell to cell once it is activated. In Line 100 chickens, where the virus appears fairly early in embryonic development and persists throughout life, the birds become immunologically tolerant. In AKR mice the virus appears too late in embryonic development 53 to induce classical immunological tolerance, but the antibody response elicited is unable to cope adequately with the infection.46 It has not yet been reported whether endogenous viruses induced in vitro are tumorigenic in vivo, although the results of such experiments should soon be known. However, it is likely that at least some genetically transmitted viruses are oncogenic. Meier et al. 43 have shown that in crosses between the AKR and a low cancer strain, those mice showing infectious virus early in life ( that is those which receive one of the two virus-inducing genes from the AKR strain) show a much higher incidence of leukemias and other tumors later in life than those which do not show early signs of virus production. Similarly, the incidence of mammary cancer in mice expressing the genetically transmitted virus is 100 per cent. 2 However, in Line 100 chickens in which spontaneous production of the endogenous virus of chickens occurs, viremic birds do not appear to have a raised incidence of leukemia, suggesting that the virus is not leukemogenic, at least in this strain. 8 Genes similar to those controlling the induction of leukemia viruses have also been identified in studies on mouse mammary tumor virus (MMTV) . Some strains of this virus, including the classical Bittner strain, or MMTV-S, are transmitted through the milk, but when this is prevented genetic transmission can be demonstrated. This has been most clearly shown in the GR strain of mice, which carries the Muhlbock or 'plaque-inducing' strain of mammary tumor virus, MMTV-P. Crosses between GR mice and low mammary cancer strains indicate that a single autosomal dominant gene is re-

G.S. MARTIN AND R.A. WEISS

16

sponsible for MMTV-P induction. 2 Bentvelzen and Daams3 used the appearance of an MMTV-specific antigen in the serum of young mice to study the expression of MMTV in crosses between high and low mammary cancer strains. Their results were consistent with the interpretation that a single locus controlled the appearance of MMTV-specific antigen. If this is, in fact, a viral gene, these results imply that the viral genome is integrated at the same site in different strains of mice. In some heterozygotes the antigenpositive allele was dominant, whereas in others the antigen-negative allele was dominant. On the basis of these and other results Bentvelzen 2 has proposed that control of MMTV expression involves a repressor similar to those involved in controlling the expression of the genes of lysogenic bacteriophage; dominant antigen-positive alleles can be interpreted as operator mutants, while recessive alleles can be interpreted as repressor mutants. Bishop et al. 6 have measured the amount of MMTV-specific RNA in tissues of a variety of different strains of mice. In general, the amount of RNA correlates with virus production, but in some cases large quantities of MMTV RNA were found in the absence of any detectable MMTV or viral antigen production, suggesting that control may occur at the level of translation. Partial expression of the endogenous viral genome can also occur and is again under genetic control. Cells which are not producing complete virus may synthesize some gene products specified by the endogenus virus, e.g. gs antigens. 48 •60 In chickens a dominant allele at an autosomal locus determines gs antigen expression48 and in most strains of chicken viral envelope antigen synthesis appears concomitantly with gs antigen and has been detected by the ability to complement the envelope defect of Bryan strain RSV when introduced into cells of that genotype, i.e. the endogenous viral gene products perform the function of a helper virus. 75 Measurements of viral RNA by nucleic acid hybridization suggest that gs negative cells do not produce viral RNA, whereas gs positive cells do. 26 •6 Thus it appears that the gs locus in chickens regulates the partial transcription of the viral genome. Like the inducibility loci, it may represent a viral locus rather than a host locus, and it would be interesting to know if the gs locus is allelic with the inducibility or V locus. In both cases, viral gene expression is dominant, and in chickens which are homozygous for the recessive alleles, exogenous viruses replicate normally. This suggests that the endogenous viral genome is not controlled by a repressor, unless one invokes a widespread occurrence of operator mutants, as Bentvelzen2 has suggested for mouse mammary tumor virus. In some mouse strains, gs antigen expression appears to be recessive. 00 MUTANTS OF AVIAN SARCOMA VIRUSES

Genetic analysis of the avian sarcoma viruses has shown that the genome of avian sarcoma viruses contains genes necessary for the maintenance of

Genetics and evolution of RNA tumor viruses

17

the transformed state of the cell. This first became evident when 'transformation-defective' mutants of sarcoma viruses were isolated from virus stocks treated with UV-light 20 , H9 or hydroxylamine. 2 3 These mutants replicate in, but can no longer transform fibroblasts, although some of them induce leukemias in vitro, 5 and we use the term 'transformation-defective' to mean non-transforming for fibroblasts: in this sense lymphoid leukemia viruses are transformation-defective. It was found later that similar transformationdefective mutants segregate spontaneously from clones of several strains of non-defective sarcoma viruses.70 , 41 These mutants have smaller genomes than the non-defective sarcoma viruses from which they arise13 and it appears that they arise by deletion of genetic information necessary for transformation of fibroblasts but not necessary for viral replication. The term 'oncogenes' has been proposed for such information by Huebner and Todaro 2 7 and we shall use this term in discussing genes necessary for neoplastic transformation, irrespective of whether this information is contained in cells or viruses. Transformation-defective mutants have the converse properties to 'replication-defective' viruses, such as Bryan strain Rous sarcoma virus, which transform cells but require helper viruses for replication. Thus transformation is not necessary for the production of infectious virus, and virus production is not necessary for cell transformation. Further evidence for the existence of viral 'oncogenes' as distinct from 'virogenes' (the genes necessary for viral replication) has come from the isolation of temperature-sensitive mutants of sarcoma viruses which are capable of viral replication but not cell transformation at the non-permissive temperature.40 Many temperature-sensitive mutants for transformation have now been isolated68 and all so far behave in a similar way: a cell infected and transformed by one of these mutants at the permissive temperature reverts to a phenotypically normal state when shifted to the non-permissive temperature, and will retransform when shifted down to the permissive temperature. Alterations in cellular growth properties and membrane organization change concomitantly with changes in cell morphology.3 1 , 42 Wyke 8 1 has shown recently that temperature-sensitive mutants of transformation isolated from the Prague strain of Rous sarcoma virus fall into at least four complementation groups, but it is not yet known if these represent distinct genes. In addition to mutants which affect the maintenance of the transformed state, mutants which are temperature-dependent for viral replication or both replication and transformation have been isolated. For example, Friis and Hunter 18 have described a mutant of Rous sarcoma virus which is temperature-sensitive for a late step in replication but does not affect cell transformation; as would be expected, this mutant is complemented by leukemia viruses. On the other hand, two reverse transcriptase mutants 37 show an early, transient defect in both replication and transformation, presumably because the provirus is not formed.

18

G.S. MARTIN AND R.A. WEISS

Thus the temperature-sensitive mutants can be classified into three types according to their behaviour at the non-permissive temperature: transformation mutants, defective in transformation but not replication; replication mutants, able to produce stable cell transformation but unable to produce infectious virus; and co-ordinate mutants, unable either to replicate or to transform cells at the non-permissive temperature. THE STRUCTURE OF THE VIRAL GENOME

AND THE

MECHANISM OF RECOMBINATION

The RNA extracted from the virions of a non-defective strain has a molecular weight of about 10 x 106, but dissociates on heating or treatment with dimethyl sulphoxide into 3 or 4 subunits of molecular weight about 3 x 106 •11 The association of subunits into the 'native' molecule occurs extracellularly shortly after the virus has matured. 15 The RNA subunits can be resolved by gel electrophoresis in two size classes, a and b. 13 Duesberg et al. 15 prepared stocks of sarcoma viruses from clones of singly infected cells, in order to eliminate any heterogeneity due to the accumulation of transformationdefective mutants or other genetic variants during successive cycles of infection, and found that the RNA of sarcoma viruses grown in this way contained only the higher molecular weight class of subunit, the a subunit. Stocks of leukemia virus, or transformation-defective mutants of a sarcoma virus, contain only the lower molecular weight class of subunit, the b class. (Stocks of sarcoma virus prepared by successive cycles of infection contain both a and b subunits; it is not clear if the a and b subunits are present in distinct particles, or if there are some virions containing both a and b subunits.) Oligonucleotide fingerprinting and hybridization studies 15 indicate that the a subunits contain the sequences present in the b subunits, as would be expected from the observation that transformation-defective mutants can arise from sarcoma viruses. The concept that the viral genome is composed of independently replicating subunits is supported by studies on viral recombination. It was found by Vogt 71 and by Kawai and Hanafusa33 that cultures doubly infected with a sarcoma virus and a leukemia virus of another subgroup produced, at high frequency, recombinant viruses containing the host-range (antigenic) marker of the leukemia virus and the transformation marker of the sarcoma virus. Weiss et al., 79 in a study of recombination between exogenous and endogenous viruses, found that many recombinants were heterozygous for the hostrange marker, that is, they produced progeny of both host-range types. This indicated that the genome is at least partially polyploid, though the progeny rapidly segregated into one or the other host-range type. Furthermore, recombinants were only produced in cells in which the endogenous viral

Genetics and evolution of RNA tumor viruses

19

genome was transcribed into RNA suggesting that the recombination occurred at the RNA level rather than by exchange between DNA molecules. Two models of the viral genome are consistent with these observations.72 In the first model each subunit is genetically distinct, and since each gene is represented only once, the viral genome is haploid. Thus, if the genes within a subunit are represented by numbers enclosed by parentheses, the a subunits of the sarcoma virus genome could be represented as ( 1 2 3 4 5) ( 6 7 8 9 10) ( 1112 13 14 15), while the b subunits of a leukosis virus, or of a transformation-defective mutant of a sarcoma virus, would be ( 3 4 5) ( 8 9 10) ( 13 14 15). On this model, simultaneous or successive deletions in each subunit would be necessary to generate a transformation-defective virus. On the second model the subunits of the viral RNA are genetically identical and since each gene is represented several times, the genome is polyploid. Thus, on this model the genome of a sarcoma virus released from a clone of singly infected cells would be represented as (1 2 3 4 5) (1 2 3 4 5) (1 2 3 4 5), while the subunits of a transformation-defective mutant would be ( 3 4 5) ( 3 4 5) ( 3 4 5) . A variant of this second model would be to assume that only one subunit is successful in initiating infection, so that the virus would be functionally haploid: deletion in one subunit could then result in the production of a transformation-defective variant. Recombination might involve subunit reassortment or the formation of a heterozygote at virus maturation, followed possibly by molecular recombination: this latter step could occur on provirus formation during the next replicative cycle, either by copy-choice transcription of DNA off different RNA subunits or by breakage-reunion between DNA proviruses. The distinction between the haploid and polyploid models may perhaps be elucidated by further recombination studies, which may indicate if there are linkage groups in the genome. It may also be possible to distinguish the models by inactivation studies on sarcoma viruses released by singly infected cells: if the inactivation is multi-hit, then the genome would appear to be polyploid. It should be added that if the genome is polyploid, then the total genome size is only about 3 X 106, and it is difficult to see how all the proteins, believed to be coded by the viral genome on the basis of genetic and biochemical work, can be accommodated in a genome of this size. RECOMBINATION-DEFECTIVE VIRUSES

Many transforming viruses are defective for replication. Infectious stocks of these viruses always contain 'helper' transformation-defective viruses which complement the defects in replication of the transforming viruses, often by phenotypic mixing, i.e., the helper virus provides structural components for the transforming virus. The classical example of this phenomenon is the

G.S. MARTIN AND R.A. WEISS

20

Bryan strain Rous sarcoma virus (BH-RSV), which apparently does not code for envelope antigens. 56 When a cell is singly infected with BH-RSV, the cell transforms, but the progeny virus particles it releases are not infectious. If, however, the cell is superinfected with a leukemia 'helper' virus, progeny BH-RSV is now released and carries the envelope specificity of the helper virus; it is called a pseudotype. When BH-RSV is re-cloned, non-infectious virus is produced once more. This indicates that it is only carried by phenotypic mixing and does not recombine with the helper virus with respect to the envelope antigen. 33 , 79 Most isolates of murine sarcoma virus are also defective 1 though McCarter will describe a non-defective form in this volume. Most isolates of myeloid and erythroid leukemia virus are also defective, for example, avian myeloblastosis virus, 45 avian myelocytoma 29 virus, 22 avian erythroblastosis strain R, 28 and Friend erythroleukemia virus of mice. 16 •58 Many other transforming viruses, for example, strains of feline sarcoma virus and avian sarcoma virus, may prove to be defective because it is known that infectious stocks contain excess transformation-defective viruses which can act as helper viruses. Thus the helper-independent strains of avian sarcoma viruses, which have been used extensively for genetic studies, may prove to be exceptions among transforming viruses. Since the defective character of helper-dependent viruses are stable on long-term passage with helper viruses, they are defective for both replication and recombination. However, it is possible that defective viruses can recombine with their helper viruses for some gene loci but not others. For example, there is suggestive evidence 25 that BH-RSV recombines with helper viruses in respect to the reverse transcriptase gene but not the envelope antigen gene. Until we know more about the organization of the viral genome and the mechanism of recombination, we cannot say which stage of recombination does not function in defective viruses. If the genome is essentially haploid and recombination takes place by reassortment of unique segments, there must be a block to reassortment in defective viruses. Since the defective genome is transmitted in virions coded by the helper virus, there is no apparent restriction on the assembly of the genome segments with the helper virus proteins. Therefore one must assume that the genome segments of the two parents cannot be packaged together. Lack of space seems an unlikely reason for this in view of the genetic redundancy implicit in heterozygotes; the genome segment bearing transformatoin genes should be able to displace one of the duplicated segments to yield a haploid, non-defective virion. If the viral genome is polyploid, the block to recombination might occur either for reassortment of genetic elements during virion assembly or for recombination of proviral elements in the next replicative cycle. Defective proviral recombination could be a result of the non-homologous nature of the

Genetics and evolution of RNA tumor viruses

21

genomic segments. If this were the case, defective viruses could transmit the helper virus genome without forming a stable recombinant. The transmitted helper virus genome would be lost as quickly as heterozygotes segregate to homozygotes or haploids 79 but one should be able to select for non-defective reassortments, at least for a few generations. McCarter's competent murine sarcoma virus ( this volume) may be an example of this phenomenon. ORIGIN' OF TRANSFORMING VIRUSES

'rhe apparent ubiquity of :RNA tumor viruses in mice and chickens has led to two types of models for cancer, both of which stress the importance of RNA tumor viruses. The first type of model, which includes those proposed by Gross, 24 Lieberman and Kaplan, 34 Bentvelzen et al.,4 and Huebner and Todaro, 27 is based on the activation of latent, vertically transmitted RNA tumor viruses. The most specific model is that of Huebner and Todaro, who have proposed a universal mechanism of oncogenesis in that all cancer is due to the activation, by carcinogens or age, of genetically transmitted RNA tumor viruses. These viruses are presumed on the model to contain genetic determinants of transformation functions, called 'oncogenes,' in addition to those of viral replication functions, called 'virogenes'27 or 'germinal provirus.'2 The central feature of the viral oncogene model is that the genes necessary for transformation are present in the germ-line and need only be expressed for transformation to occur. Thus the model is phylogenetic, in that the oncogenic information is believed to have been preserved in evolution, and it is also epigenetic in that the oncogenic information is not generated during somatic development but is simply 'switched on' at some time during the life of the organism. Temin, 63 , 64 on the other hand, has argued that oncogenic information would be eliminated by natural selection since it is not of advantage to the host. Although sarcoma viruses do have functions necessary for the transformation of fibroblasts, it is not yet known whether the endogenous, genetically transmitted viruses have analogous functions involved in leukemogenesis and mammary carcinogenesis. Temin64 points out that transformation by lymphoid leukemia viruses and mammary tumor viruses is inefficient; there is usually a long time-lag between the onset of virus production and the development of tumors. He therefore considers that those viruses, rather than carrying specific oncogenes, 'behave more like non-viral carcinogens in their mechanisms of neoplastic transformation; that is, they increase the probability of a misevolution of cellular genetic elements, the protoviruses.' Temin63 argued originally that cancer results from somatic mutation of protoviruses, which are involved in growth control and which replicate in

G.S. MARTIN AND R .A. WEISS

22

a way similar to that of RNA tumor viruses, i.e. by information transfers from DNA to RNA and back to DNA, because the protoviruses can specify reverse transcriptase. This allows not only for mutation but for changes in location of genetic elements. The RNA tumor viruses, on Temin's model, have evolved from protoviruses and therefore have homology with them. However, reverse transcriptases with properties similar to the viral enzyme have not been detected in normal somatic cells80 •80 and the evolution of virogenes and oncogenes from protoviruses remains highly speculative. The protovirus and oncogene models are not wholly mutually exclusive, and Todaro and Huebner 67 have adopted some of the precepts of the protovirus hypothesis, such as the independent control and location of virogenes and oncogenes. It may well be that neither hypothesis is correct, and that most natural cancers are caused neither by RNA tumor viruses nor by elements replicating in the same way as these viruses. Nevertheless, it seems plausible to generalize from studies with transforming viruses and postulate that malignant growth depends on the expression of specific cellular genes, whether these are present in the germ-line or not, although these genes need have no association with viruses. What, then, is the origin of the oncogenes borne by RNA tumor viruses? As mentioned in the introduction, RNA viruses which cause rapidly developing tumors, such as the sarcoma viruses and myeloid or erythroid leukemia viruses, do not occur commonly in nature. One would certainly expect strong natural selection against the perpetuation of such highly oncogenic viruses, or at least against their expression, particularly when the oncogenes of these viruses are not required for viral replication. In Figures 2 and 3 we present a model which is a collage of the protovirus and oncogene concepts. We propose that oncogenes evolve not from protoviruses, but from normal genes involved in growth control. These genes may mutate in somatic cells to cause neoplastic growth. Viruses which are not transforming for any type of tissue - virogenes without oncogenes - presumably originate from cellular elements replicating via reverse transcriptase, that is, protoviruses. The oncogenes are not homologous to virogenes and would not commonly be transmitted or encapsulated as RNA tumor viruses. However, we propose that on rare occasions this may happen, giving rise to infectious, transforming agents. Such a virus would arise by a rare recombination event in which the oncogenes would become linked to the unrelated virogenes. We further propose that transforming viruses can arise from completely non-oncogenic viruses or from transformation-defective viruses by recombination with oncogenes (Fig. 3). It is significant that the murine sarcoma viruses that have appeared de novo in the laboratory have arisen from animals or cells infected with lymphoid leukemia viruses (LLVs). 68 Pre-

Genetics and evolution of RNA tumor viruses Normal genetic elements

Gcnctk clements maintained in some species or cells hy natural or artificial selection •

23 Genetic elements introduced into cells hy infection

PROTOVIIWS-------+VIROGENES --------,-----NON-TRANSFORMING VIRUS Capahle of replication and trnnslocation via reverse 1ranscriptase

KNA transcript can become

padagcd into virus partides

,:omposcd of its own gene prodU\.' lS

REPLICATION HELPER DEFECTIVE-INDEPENDENT TRANSFORMING TRANSFORMING VIRUS VIRUS Requires helper for transmission

Transmit oncogenes causing

transformation with varying Jegrees target cell specificity

or

GENES involved ----+ONCOGENES in growth con1rol: Genetic information for neoplastic tissue specific, not transformation; not normally rcplic;lle

2 -i

60

0

z

40

Cl 20

2

4

6

B

10

12

14

HOUFlS

Figure 2

DNA repair synthesis in cultured human fibroblasts following short term exposure to 4NQO and measured as unscheduled incorporation of 3HTdR (autoradiography, V) or as a shift in the sedimentation rate in an alkaline sucrose gradient ( ■). By about 12 hours, post-treatment DNA repair synthesis ceases and the sedimentation profile approaches the original pattern.

Bouyant density cesium chloride gradients were repeatedly used to separate newly synthesized DNA from parental DNA and to measure the incorporation of BrdU into non-replicating, yet repairing DNA strands. 6 This is quite an elegant procedure of distinguishing DNA replication from DNA repair, but is hardly applicable as a rapid analytical method to screen hundreds of carcinogenic agents. Incorporation of BrdU into DNA also renders the position of incorporation labile to the effect of long-wave ultraviolet radiation. 54 This photosensitizing procedure has been successfully applied to estimate the extent of DNA repair. 49 Cultured mammalian cells were UV irradiated or treated with a chemical carcinogen; thereafter, they were allowed to undergo repair replication in the presence of BrdU and exposed to a pulse of 313 nm light. Repair-replicated regions containing BrdU are rendered alkaline-labile by the irradiation. The extent of DNA breakage and repair is deduced by the degree of shift in the DNA peak following centrifugation in an alkaline sucrose gradient. Only recently introduced, this approach is not yet popular. The technique of choice appears to be the autoradiographic detection of radioactive precursors applied during the period of DNA repair synthesis. By exposing non-proliferating cells to exogenous physical6 or chemical8 •58 ,62 , 63 agents and then allowing them to engage in DNA repair synthesis in the presence of 3HTdR, a measure of unscheduled repair synthesis can be readily obtained.58 Furthermore, the autoradiographic preparations provide

Use of DNA repair in identification of carcinogens

87

a record of the cytological location of the incorporated thymidine, the identification of the cell type engaged in DNA repair synthesis, the proportion of cells involved in DNA repair, and the variation of DNA repair levels within a cell population.58 Since hundreds of preparations can be handled simultaneously, and the evaluation of the autoradiographic preparations can be semi-mechanized, this procedure can be applied in large-scale screening tests. 1 DNA REPAIR AND IDENTIFICATION OF CARCINOGENS -

BIOASSAYS

Several new factors contribute to what may be considered a critical period in screening programs for carcinogens. The yearly input of new compounds into man's environment occurs at an unprecedented rate. The present drugoriented society tries to solve its organic and psychic ills by consuming vast quantities of compounds in all imaginable permutations and combinations. The needs of the new megalopolis demand the long-term preservation of virtually all food products, which in turn requires the large-scale use of effective preservatives. The rapidly increasing trade between nations leads to a worldwide spread of new compounds which are either man-made or appear as naturally occurring contaminants in the imported products. The standard 'rodent painting and feeding tests' for carcinogenicity can hardly cope with this newly evolved situation. The cost4 5 of these experiments is prohibitive ( up to $30,000 for testing a single compound on 200 mice), and the completion of these tests requires a relatively long time ( up to 2 years). These facts are not readily acceptable to the highly competitive business community. Moreover, the logistics of handling and screening millions of mice or rats would be staggering. Thus there is an urgent demand for an economic, rapid, and relevant screening procedure, or at least for a bioassay, which could be used in a prescreening program. In this paper we should like to discuss the pros and cons of using DNA repair synthesis of mammalian cells as an indicator for a carcinogenic and / or mutagenic activity of a compound. The idea is based on the assumption that chemical carcinogens interact with DNA and that the ensuing DNA changes will result in a DNA repair synthesis. This working hypothesis was examined by comparing the extent of DNA repair in hamster and human cells after their exposure: ( 1) to several key agents belonging to different groups of carcinogens (Table I); (2) to highly, weakly, and non-oncogenic compounds61 (Fig. 3); and ( 3) to precarcinogens and their active proximate metabolites (Fig. 4) .60 •62 A short exposure of cultured mammalian cells to several key carcinogens results in a DNA repair synthesis whereas the application of non-oncogenic agents (e.g., 3NQO, 5NQO), precarcinogens

H .F. STICH

et al.

88

TABLE I DNA repair synthesis as a bioassay for chemical carcinogens Unscheduled 3 HTdR (uptake detected)

Unscheduled 3 HTdR uptake (not detected)

4NQO (& 16 carcinogenic derivatives)

DMBA BP MCA, MCA-DIOL DBA, DBA-DIOL BA, BA-DIOL

3-Me-4NPO 3-Me-4HAPO 2,3-diMe-4NPO 2,3-diMe-4HAPO 2-Me-4NPO 3-Et-4NPO 2,3-diEt-4NPO Rugulosin MNNG MMS EMS NMU HN 2 t-Butyl-hydroperoxide ICR-191 N-OH-AAF N-Ac-AAF

N-OH-AAS N-Ac-AAS N-OH-AAP N-Ac-AAP N-Ac-AABP N-Ac-3-AAF N-Myristoyloxy-AAF BA-epoxide MCA-epoxide

DMN n-amyl-nitrite Cyclophosphamide Aflatoxin B 1 Daunomycin Acriflavine neutral Ethidium bromide

AAF AS AAS AAP AABP ABP Cycadin 3-0H-xanthine guanine-n-oxide 3-0H-guanine

(e.g., AAF, AAS) and non-oncogenic metabolic products (e.g., diols of benz(a)anthracene; 4AQ02 5 a metabolite of the carcinogen 4HAQO) did not elicit a detectable level of unscheduled 3 HTdR incorporation. A correlation between the oncogenicity of a compound and the level of DNA repair synthesis is particularly evident within a group of related carcinogens. For example, cells respond to the highly and weakly oncogenic derivatives of 4NQ0 26 with a high and low level of unscheduled DNA synthesis respectively. The non-oncogenic 4NQO derivatives lack the capacity to evoke a detectable DNA repair synthesis. The K-region epoxide of benz(a)anthracene, which is a potent transforming18 and mutagenic 24 compound and probably represents the proximate carcinogen, 20 is a highly active inducer of DNA repair, as compared to the parental benz(a)anthracene or the BA-cis-5,6-dihydrodiol which is a further metabolic product. 60 The application of N-hydroxy and N-acetoxy derivatives 38 •39 of AAF, AS,

89

Use of DNA repair in identification of carcinogens

0

20

GRAINS/ NUCLEUS 40 60 80

100

I 4NQO 4HAQO 2-methyl-4NQO 5-methyl-4NQO 6-methyl-4NQO 7-methyl-4NQO 6-n-butyl-4NQO 6-chloro-4NQO JI 3-methyl-4NQO 8-methyl-4NQO 3-Huoro-4NQO 6-carboxy-4NQO 2-methyl-4NPO 3-methyl-4NPO 3-methyl-4HAPO 3-ethyl-4NPO 2,3-dimethyl-4NPO 2,3-dimethyl-4HAPO 2,5-dimethyl-4NPO 3,5-dimethyl-4NPO

Ill 3NQO 5NQO 6NQO 7NQO SNQO 4-NHo·QO 4-0H:-Qo 4-phenylsulfonyl-QO QO

Figure 3 DNA repair synthesis in cultured human fibroblasts exposed to highly (group I), weakly (group II) and non-oncogenic (group III) derivatives of 4NQO and 4NPO. GRAINS

0

10

/

NUCLEUS

20

30

40

BA BA-EP0XI0E BA-0I0L

MCA MCA-EP0XIDE MCA-0I0L

Figure 4 DNA repair synthesis in cultured human fibroblasts exposed for 5 hours to benz( a) anthracene (BA), BA-5 ,6-epoxide, BA-cis,5,6-dihydrodiol; methylcholanthrene (MCA); and the epoxide and dihydrodiol of MCA. Autoradiography.

H.F . STICH

et al.

90

AAS results in cells responding with a DNA repair synthesis, whereas the non-activated AAF, AS, AAS at equimolar concentrations lack the capacity to elicit a detectable level of DNA repair in cultured human or hamster fibroblasts. However, there are two groups among the compounds listed in Table I which deserve a closer examination. Several potent mutagens including acridines, daunomycin, and ethidium bromide lacked the capacity to evoke a detectable level of DNA repair synthesis although they can affect DNA molecules. Although these agents seem to intercalate between the nucleotides of DNA and lead to frameshift mutations, they do not fall into the category of carcinogens. The postulated relationship between oncogenicity of a compound and its capacity to elicit DNA lesions which result in a DNA repair synthesis seems to apply to the intercalating type of agents. In this connection it is of interest to note that quinacrine-mustard ( ICRI91), which has an alkylating activity apart from its intercalating capacity, induces a low but definite level of DNA repair synthesis. As can be seen from Table I there are several so called 'false negatives.' These compounds are definitely carcinogenic, but they do not lead to a DNA repair which is detectable by an unscheduled incorporation of 3 HTdR. Several factors may be responsible for this exceptional behaviour. Low solubility in water, DMSO, or ethanol is one of the simplest reasons for a 'false negative.' For example, lipophilic derivatives of 4NQO (e.g., benzylsulfonylquinoline I-oxide) are good representatives of this type of compound. A second set of carcinogenic agents may affect cells without acting directly on DNA or they may produce DNA alterations, which are not recognizable and repairable by the 'cut and patch' repair mechanism. For example, the oncogenic human adenovirus type 12 and 18 and the Simian adenovirus SA7 neither induced a measurable DNA repair synthesis nor inhibited a DNA repair synthesis elicited by single UV dose in Syrian hamster cells in which the viruses do not replicate but induce neoplastic transformation (Table II). Finally the cultured fibroblast may not represent the proper target cells for those carcinogens which need activation by an organ-specific enzyme system. This topic is discussed in the following section. DNA REPAIR AND IDENTIFICATION OF PRECARCINOGENS

Many of the proposed in vitro test systems which rely on bacteria, yeast, fungi, or cultured mammalian cells appear to be highly sensitive indicators for activated carcinogens and mutagens, but they seem to miss precarcinogens and premutagens that require activation to potent metabolites. Unfortunately, many noxious compounds in man's environment belong in this latter category. The ubiquitous nitrosamines, the widely spread polycyclic

Use of DNA repair in identification of carcinogens

91

TABLE II DNA repair synthesis in XPE and control cells infected with inactivated human adenovirus type 12 and exposed to UV-radiation Grain per nucleus

UV' (900 erg/mm 2 ) AD12 50 PFU 2 AD1210PFU AD 12 50 PFU plus UV AD12 10 PFU plus UV 1

Normal

XP

62 0 0 57 56

13

0 0 11 12

The cited samples were taken 12 hours postinfection (3 hours absorption time). Comparable results were obtained when the infected fibroblast cultures were sampled at 4, 24, and 36 hours postinfection with the UV inactivated AD12.

aromatic hydrocarbons, and the naturally occurring hepatocarcinogens of fungal origin ( e.g., aflatoxins, sterigmatocystin) typify this category of compounds. Thus, a successful utilization of any in vitro system - including DNA repair synthesis of human fibroblasts - will depend to a large extent on the introduction of effective and reliable activation procedures for a wide array of precarcinogens. For one example - dimethylnitrosamine (DMN) - we should like to illustrate the combined application of DNA repair synthesis and in vitro activation as a procedure that may be suitable in the massive screening of exogenous precarcinogens. The activation preparation30 •36 consists of a postmitochondrial supernatant fraction resulting from a 9000xg centrifugation (10 minutes) of a mouse liver homogenate. The microsome fraction (200300 mg liver-equivalents per cell culture contained in a Leighton tube) is then fortified with 8.5 ftmoles NADPH, 25 ftmoles magnesium chloride, and 20 ftmoles glucose-6-phosphate to allow for possible regeneration of NADPH via the phosphogluconate pathway during the course of the incubation.13 DMN was mixed into the fortified microsome preparation and the mixture was then added to non-proliferating cells. The cultures were flushed with 0 2 and left without further agitation at 37 ° for the required incubation period. For analysis of DNA repair synthesis, the cell cultures were processed in the usual way to assess the level of unscheduled 3H-TdR uptake by the autoradiographic analysis. For an analysis of the changes in molecular weight of DNA, a modified version of the McGrath and Williams technique was employed.9 The results, which are given in Figure 5 show a relatively high level of unscheduled 3H-TdR incorporation in cultured human fibroblasts exposed to DMN plus the complete activation mixture, whereas DMN alone induces just a trace of DNA repair synthesis. Similarly, the alkaline

H.F. STICH et

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TABLE III Toxicity of 13-2'-deoxythioguanosine (13-TGdR) and its modification by arabinosylcytosine (Ara-C)* Treatment

No. deaths

Max. wt. loss (%)

13-TGdR, 13.6 mg/kg, daily, X9 Ara-C, 10 mg/kg followed in 10 min by 13-TGdR, 13.6 mg/kg, daily, X9 Ara-C, 10 mg/kg followed in 16 hr by 13-TGdR, 13.6 mg/kg, daily, X9

10

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* Groups of 10 C3H female mice 12-14 weeks old, initially weighing 20 gm each, were used. Surviving mice had regained initial weight by day 20. Those dying did so in the period 14-16 days from the start of the treatment.

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Mecca lymphosarcoma recovers rapidly and greatly exceeds control values during the period 8-24 hours. It should be vulnerable to /3-TGdR during this overshoot period. The L1210 is very sensitive to Ara-C and does not recover appreciably before 16 hours. However, when redosed at 24 hours, a second recovery was much more rapid. Such redosed Ll210 cell populations do not take up and phosphorylate any less Ara-C. Their behaviour is not as yet explained. The data of Figures 6 and 8 show that there are great variations between bone marrow and tumors in rates of recovery of DNA synthesis from Ara-C. These differences could probably be exploited for therapeutic pur-

Model systems in cancer chemotherapy

177

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poses. It is also necessary to know how long ,8-TGdR and the resulting nucleotides would be present in the target cells in appreciable amounts in order to design therapy schedules. Figure 9 presents data on the amounts of mononucleotide in mouse bone marrow at intervals after a dose of ,8-TGdR at 8.5 mg/kg. The content in DNA is also given, though this is less significant since it is a composite of continued incorporation and cell death. The mononucleotide had a half-life of approximately 3 hours and had declined to a relatively low level by 6 hours. Figure 10 gives the data for a similar experiment on L1210 cells, again indicating a half-life of 3 hours. Thus a model system has emerged from these studies where we have a drug (,8-TGdR) that is toxic only to the bone marrow of the host, is toxic

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only during S-phase, has a relatively short residence time in cells, and does not interfere with the cycling of cells. We appear to have a second agent (Ara-C) that permits us to protect the bone marrow from toxicity by reversibly inhibiting DNA synthesis. The DNA synthesis of experimental tumors can be manipulated with Ara-C in a manner such as to discriminate between bone marrow and tumor. Further studies of the model system with

Model systems in cancer chemotherapy

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