Advances in Cancer Research Volume 39

ADVANCES IN CANCER RESEARCH VOLUME 39

Contributors to This Volume Michael Baum

Beverly E. Griffin

David A. Berstock

A. Marchok

Thierry Boon

Dorothy A. Miller

Stephen M. Dilworth

Orlando J. Miller

Paula J. Enrietto

I? Nettesheim

E. Gorelik

John A. Wyke

ADVANCES IN CANCERRESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm. Sweden

SIDNEY WEINHOUSE Fels Research Institute Temple University School of Medicine Philadelphia, Pennsylvania

Volume 39- 1983

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ISBN 0-1 2-006639-4 PRINTED IN THE UNITED STATES OF AMERICA

83 84 85 86

9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBUTORS TO V O L U M E

39 . . .

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ix

Neoplastic Development in Airway Epithelium NETTESHEIM AND

A. MARCHOK

1. Introduction . . . . . . . . . . . . . . . . . . . . . . Experimental Approaclies and Methodologies. . . . . . . . 111. Morphology of Acute Phase Responses-Early Iiiduction Pliasc I\'. Chronic. Phase Hespoiises . . . . . . . . . . . . . . . . v. Crll Popiilatioii Studies during Neoplastic Development . . . VI. klodifiers of Neoplastic Developiiient in Airway Epithelium . VII. Conclusions . . . . , . . . . . . . . . . . . . . . . . 11.

Kcfcrciicc,s .

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1

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lh 24 44

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59 62

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63

Concomitant Tumor Immunity and the Resistance to a Second Tumor Challenge E.

GORELlK

Introductiorr , . . , . . . . . . . . . . . , . . . . . . . . . Tuinor Excision a d Iiesistancc to a Second Tumor Challenge: Sinecoinitant Immunity . . . . . . . . . . . , . , . . . . . . 111. Resistancc, of the Tumor-Bearing Host to a Second Tuiiior Cliiillengt: Coiicomitant Tumor Immunity . . , . . . , , . . , . . , . . . I\< Metastases as a Second Tiunor Grat't: Antiilletastatic Concomitant Immunity . , . . . . . . . . . . , . v. Is tlic- Status of Concomitant Immunity L1iiiqiic for n Tiuiior-Host Relationship?. . , . , , , . . . . . . . . . , VI . Mrclimisms of Resistancc of tho Tumor-Rearing Host to a Second Tumor Graft. . . . . . . , , . . . . . . . . . . . VII. h4echanisms of thc Inhibition of M b y tlic Pririitiry Tumor. . . . . . , . , , . . . . . . . . . . . VIII. Concliision and Suiniiiary . . . . , . , , , , . . . . . . . . , R e l ' e r ~ ~ c.e s. . . . . . . . . . . . , . . . . . . . . . , . . 1.

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Antigenic Tumor Cell Variants Obtained with Mutagens THlERRY

1 I1

BOON

Ill troduction . . . . . . . . . . . . . . . . . . . , . . . . . . . . Evidence for the Production of' Tiiiiior Cclls with Incrcascd Iiniiiiiiiogciiicity b y Slutagcn Treatment . . . . . , . . . . . . . . , . . . . . . . .

121 12.3

vi

CONTENTS 111.

Iniinunogenic Variants Obtained in Vitro with Nitrosogiianidine Derivative MNNG . . . . . . . . . . . . . I V. Immunogenic Tumor Cell Populations Ohtaiiicd i t ] Vico with Triazenylirnidazole Derivative DTIC . . . . . . . . . . . . . V. Discnssion . . . . . . . . . . . . . . . . . . . . . . . . . . . Refkrencrs . . . . . . . . . . . . . . . . . . . . . . . . . . .

125

136 111 117

Chromosomes and Cancer in the Mouse: Studies in Tumors. Established Cell Lines. and Cell Hybrids DOROTHY A . MILLERA N D ORLANDO J . MII.I.ER

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . T-CeIl 1. eukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 . 13-Cell Leukemias . . . . . . . . . . . . . . . . . . . . . . . . . . . I V. Plasnracy tomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1'. Evidence from Cell Lines For the Hole of' Cliromosomr 15 . . . . . . . . . v1. Virwcs a i i d C~cncsRclated to Tiiniorigciicsis . . . . . . . . . . . . . . . \'I1 . 110 All Tiiinors I-lave Specific Chromosome Changes? . . . . . . . . . . . V I I I . Gene Aniplification: I lomogcnconsly Stiiiiiing Hegions and h u b l c M i n u t c (:hromosoincs . . . . . . . . . . . . . . . . . . . IX . Cenernl C:hal.actcristics of kforise Coll Lines . . . . . . . . . . . . . . . X . Suppression of Tumorigeuicity in Hyl)rid Cells . . . . . . . . . . . . . . XI . Chroinosornc Loss in Tumorigenic I-lybritls . . . . . . . . . . . . . . . . XI1 . Chromosome 4 i n Vitro Growth and 7rumorigenicity . . . . . . . . . . . XI11 . Conil)l(~iii"iitatioirAnalysis iciitl the Nuin1)c.r of C c n c ~Involvcd ill Tumorigenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . XI\! Trisomv 15 in Twnorigenic Flyliritls . . . . . . . . . . . . . . . . . . . XV. Suppression of Tiiiiiorigc,nicity DNA Fragnients . . . . . . . . . . . . XVI . SV1O-liiducecl T~iiisfi)riiiationand Tiii)ii)rig[.iii(!it. iii k1yl)rid C;eIls . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVI I . (:ommon MLlrcliaiiisnis of' Tunror Suppression i l l Interspecific I1yl)rids . . . . . . . . . . . . . . . . . . . . . . . . XVIII . ( h e t i c Basis (if Traiisforniation and Tiimorigcnicitv . . . . . . . . . . . . XIX . Altcrcd 1ic.xosc Transport in Mnlignnnt . 60-

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PRIMARY ANTISERUM DILUTION FIG. 288. FIG.28. Evidence for cross-reactingtumor antigens in rat tracheal carcinomas and neoplastic tracheal cell lines. (A) Transplantation resistance in Fisher 344 rats repeatedly immunized with syngeneic tracheal carcinoma cell lines. Rats in each group were immunized against one tumor line and were challenged with 5 X TD,, of the other tumor lines as well as the same line. Horizontally shaded squares indicate the existence of cross-protection among the carcinoma lines, and diagonally shaded squares indicate additional cross-protectionbetween DMBA, and sarcoma and between DMBA, and fetal antigens. ND, Not done; ?, cross-reactivity uncertain. (From Jamasbi and Nettesheim, 1977.) (B) Antibody binding activity of 2-10-1 immune rat serum for neoplastic rat tracheal epithelial cells (2-10-1,8- 10-2, 1000M,and 165-4-0), murine line 1 alveolar carcinoma cells, and murine A3 1 fibroblasts. Nonspecific binding was determined for each cell line by substituting normal F-344 rat serum for immune serum and was subtracted from the experimental results. (From Braslawsky ef a/., 1982.)

60

P. NETTESHEIM AND A. MARCHOK

cigarette smoking and exposure to asbestos results in a markedly increased risk to develop lung cancer (e.g., Hammond et al., 1979). On the other hand, several studies have provided suggestiveevidence that dietary factors, particularly carotenoids and retinoids, might reduce the risk to develop lung cancer even though this notion seems far from being proved (Peto et al., 1981). It is not possible to draw any conclusions from epidemiologic data regarding the nature of such interactions. Experimental models, however, can serve as powerful tools to elucidate pathogenetic mechanisms underlying multifactor etiologies suggested by repidemiologic data, even though extrapolation of such results to human disease always has to be done with caution because of possible species differences. Since the general topic of host and environmental factors in respiratory tract carcinogenesis has recently been reviewed (Nettesheim et al., 1981a), we can limit this discussion to an examination of existing evidence suggesting modulation of the cellular evolution of neoplastic disease in the airways. In other words, what evidence is there to indicate that the cells which have been recruited into the neoplastic process by exposure to carcinogen can be influenced during the “postinitiation phase” in their progression to the malignant state? Is there evidence for tumor promotion in respiratory tract epithelium analogous to two-stage skin carcinogenesis or for inhibition of progression or promotion analogous to that described for mammary as well as skin carcinogenesis (Moon et al., 1976; Sporn et al., 1976; Verma et al., 1979; for review Nettesheim, 1980; Sporn and Newton, 1979)? This question seems to be of particular importance in view of the issues raised by the in vivo-in vitro studies on the cellular evolution of airway neoplasia (Terzaghi and Nettesheim, 1979, 1982). These studies suggest that without further stimulation only a fraction of the carcinogenaltered cells present in carcinogen-exposed target tissues progress to the neoplastic state, seemingly driven by an internal force, while others, possibly the majority, do not fully develop or express their neoplastic potential unless triggered by some “external” factor or condition (such as tissue dissociation and multiple rounds of cell proliferation). Several lines of evidence suggest that respiratory epithelia of different origins are indeed responsive to tumor-promoting stimuli. Thus, it has been shown that the alveolar and/or bronchiolar epithelium of mice initiated with urethan can be promoted with phorbol (Armuth and Berenblum, 1972) and butylated hydroxytoluene (Witschi et al., 1977). Furthermore, it was demonstrated that rat tracheal epithelium exposed in vivo to DMBA (Table VII) or in vitro to MNNG can be promoted by the phorbol ester TPA (Steele et al., 1980; Topping and Nettesheim, 1980b). In the in vitro two-stage carcinogenesis studies, promotion resulted in an enhanced transformation frequency as well as a shortening of the time required for neoplastic transformation to occur.

61

NEOPLASTIC DEVELOPMENT IN AIRWAY EPITHELIUM

TABLE VII RAT TRACHEAL TRANSPLANTS USINGDMBA AS THE INITIATOR AND TPA AS THE PROMOTOR

TUMORPROMOTION IN

Number of tracheas at risk

TPA DMBA old Experiment 1” 188

CWpeW

loo

188 Ob

Experiment F 35 35 35 a

22 20

ob

I00

20

1 OOb

56 38 26

O* -d

,

Number of tracheas with carcinomas 16

4 0 19

2 1

Percentage 73 20 0 34 5 4

Modified from Topping and Nettesheim (1980). Blank pellets devoid of DMBA and TPA. A. J. P. Klein-Szanto and P. Nettesheim (unpublished). No pellet inserted.

Promotion-like effects have also been reported with subtumorigenic doses of asbestos (Topping and Nettesheim, 1980a). A number of dietary factors have been shown to be effective modifiers of carcinogenesis (Clayson, 1975; Fink and Kritchevsky, 1981; Rogers and Newberne, 1975; Wattenberg, 1975). Among them are vitamin A-related compounds, the so-called retinoids. In recent years, a number of studies have demonstrated the effectiveness of natural and synthetic retinoids as inhibitors of tumor promotion in skin carcinogenesis as well as mammary and bladder carcinogenesis during the postinitiation phase (Moon et al., 1976; Newberne and Rogers, 1981; Sporn and Newton, 1979; Sporn et al., 1976; Verma et al., 1979). Similar efforts to inhibit carcinogenesis in the respiratory tract have met with mixed success. Some studies seem to indicate inhibition by retinoids while others fail to do so (for review see Nettesheim, 1980). The reason for this ambiguity is not certain but it is conceivable that promoting factors in most in vivo lung tumor models used for testing anticarcinogenic effects of retinoids are of less importance than they are in, e.g., skin, mammary, or bladder carcinogenesis models. However, it was recently shown (Marchok and Huang, unpublished observations) that inhibition of neoplastic transformation by retinoids can be readily accomplished in vitro. Tracheal organ cultures exposed to MNNG in the manner described in Section V,A,1 and subsequently cultured in

62

P. NETTESHEIM A N D A. MARCHOK

medium containing retinyl acetate form healthy epithelial outgrowths. These, however, cannot be maintained for extended periods as primary cultures suggestingthat the cells are programmed to terminally differentiate due to the retinoid exposure. As described in Sections V,A, 1 and 2, without the retinoid treatment such outgrowths become stable primary cultures which can be subcultured and become neoplastically transformed at a high frequency. However, the in vivo studies have provided strong evidence that vitamin A depletion increases the susceptibility of pulmonary epithelia to carcinogenesis and enhances the progression of neoplastic development (for review see Nettesheim, 1980). The mechanism of this vitamin A deficiency effect is not clear. It may be related to the increased cell replication which is known to occur in airway mucosa of vitamin A-deficient animals (Boren et al., 1974)and/or to the disturbance of normal differentiation ofthe mucociliary epithelium resulting in widespread epidermoid metaplasias (Clark et al., 1980; Harris et al., 1972; Marchok et al., 1975). VII. Conclusions

Much has been learned over the last two decades about the development of metaplastic -dysplastic lesions in the conducting airways of individuals at high risk to develop bronchogenic carcinoma. Their histological and cytological features resemble in many ways presumed preneoplastic lesions described at other organ sites, notably the cervix uteri. However, the key issue5 regarding the exact nature of such lesions and their place in bronchogenic carcinoma development are as yet unresolved. Are these lesions obligatory steps in the pathogenesis of bronchogenic carcinoma? Does their regression, which has been reported to occur in humans as well as in experimental animals following cessation of the carcinogenic insult, indicate reversion of the neoplastic process? These and other questions still remain to be answered. The fact that the same types of discrete dysplastic lesions which can be observed in humans (e.g., in heavy smokers or uranium miners) have also been induced in experimental models with well-defined chemical as well as physical carcinogens lends considerable support to the hypothesis that these epithelial abnormalities are preneoplastic in nature but does not prove such a relationship. Recent advances in experimentation with heterotopic transplantation of airways with xenografts of animal and human tracheas and bronchi and with tissue and cell cultures of airway epithelium of animal and human origin have opened up new avenues to study the pathogenesis of bronchogenic carcinoma. With the methodologies now existing, fruitful studies should be possible to examine, for example, the dependency of the rate of progression (or reversion) of dysplastic lesions on the type or the dose of

NEOPLASTIC DEVELOPMENT IN AIRWAY EPITHELIUM

63

carcinogen or on the effect of promoters (or antipromoters).With the recent introduction of in vivo-in vitro approaches for the study of neoplastic development and the advances in culture systems which can be used for investigating transformation of airway epithelial cells, the cellular analysis of the biologic potential of dysplastic airway lesions and the progression of initiated cells is now feasible. Such studies not only will enlarge our understanding of the development of bronchogenic carcinoma but also will elucidate many important facets of carcinogenesis and the evolution of neoplastic cell populations, in general. Future investigationswill no doubt lead to the discovery of new markers of neoplastic transformation and to a more complete understanding of the multiphasic process of neoplastic development as it occurs in the epithelial lining of the airways. This, we expect, will increase our fundamental knowledge of carcinogenesis and will at the same time provide new and useful tools with clinical application.

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CONCOMITANT TUMOR IMMUNITY AND THE RESISTANCE TO A SECOND TUMOR CHALLENGE E. Gorelik* Surgery Branch. National Cancer Institute, Division of Cancer Treatment National Institutes of Health, Bethesda, Maryland

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ce to a Second Tumor Challenge:

Sinecomitant Immunity . . ..... .. ......... ... . .. .. . ... .. . ... ... ... . .. . .. . .. . . . . . .. Ill. Resistance of the Tumor-Bearing Host to a Second Tumor Challenge: Concomitant Tumor Immunity . .... .. ... ... . .. . . . . . .. . ... ... ... . .. . .. . . . . . . . A. Resistance to a Second Tumor Challenge: Dependence on Size of Growing Tumor and Size of Second Tumor Inoculum . .. . .. . .. . ... . . . .. . .. ... . .. . .... . B. Immunogenicity of Tumor Cells and Concomitant Tumor Immunity.. . .. . .. C. Antigenic Specificity of Concomitant Tumor Immunity.. .... . .. . .. .. . . . . . . . . D. Immunologic Reactivity of the Tumor-Bearing Host and Concomitant Tumor Immunity.. ................. ... ........ ........ ... . ,.. ... . . . ... .. .. . ...... ...... E. Concomitant versus Sinecomitant Immunity IV. Metastases as a Second Tumor Graft: Antimetastatic Concomitant Immunity.. . A. Resistance of the Tumor-Bearing Host to an Intravenous Challenge of Tumor Cells ... . .. . ... .,. ... ... ......... ...... .. . .. . ... .. . ... ....... ....... B. Resistance to Local versus Systemic Intravenous Tumor Challenge.. . . .. . . ... C. Inhibition of Spontaneous Metastatic Growth in the Tumor-Bearing Host . .. V. Is the Status of Concomitant Immunity Unique for a Tumor-Host Relationship?. . .... ...... ..... . .. ... .. . ... ... ... .. ... . ... ..... VI. Mechanisms of Resistance of Tumor-Bearing Host to a Second Tumor Graft.. .. A. Immunologic Mechanisms. . .. . .. . .. . ... ... .. . ... ............................. B. Nonimmunologic Mechanisms of the Inhibition of a Second Tumor Graft Growth in the Tumor-Bearing Animals.. . .. .. . .. ... ......... ............ ..... VII. Mechanisms of the Inhibition of Metastases Development by the Primary Tumor .. . ... .. . .. . ... .. . .. . .. . ., . . . ... . .. .. . . . . .. . .. . .. . .. . . .. . .. VIII. Conclusion and Summary.. . .. . ... ...... ... ... . . . . . . .. . ... ... ... .. , .. .... ........ References. ............ . ............. . .. . .. . . . . . . . ... .........................

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I. Introduction

At the beginning of the twentieth century the mechanisms of tumor growth were intensively studied. In parallel with the success of these investigations, great expectations arose concerning the potential ability of the immune system to prevent or treat malignant diseases. It was found that Present address: Biological Therapeutics Branch, Division of Cancer Treatment, National Cancer Institute, Frederick, Maryland 2 1701. 71 ADVANCES IN CANCER RESEARCH. VOL. 39

Copyright 0 I983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN-0-12-0066394

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animals could be immunized and become resistant to the growth of transplantable tumor cells. Similar resistance to the reimplantation of tumor cells was observed in mice which “spontaneously” rejected the first tumor graft (Ehrlich, 1906; Bashford et al., 1908; Russel, 1908; Woglom, 1929). In 1906, Ehrlich found that the growth of a second tumor cell inoculant may be suppressed in mice bearing a progressively growing tumor. Bashford et al. (1908) confirmed this finding and conducted further study bf this phenomenon. They came to the conclusion that resistance of a tumor-bearing host to a second tumor challenge is mediated by immunologic mechanisms. This phenomenon was termed concomitant tumor immunity. However, a short period of naive enthusiasm in tumor immunology was followed by a long period of great disappointment. During the genesis of experimental oncology, all studies were performed using outbred animals and transplantable nonsyngeneic tumors. Selection of homozygotic inbred strains of mict with high and low cancer risk had only just begun. Later using inbred strains of mice, genetic factors were found to play an important role in the transplantation of normal and malignant tissue (Little and Strong, 1924; Strong et al., 1938; Snell, 1948, 1953). Successful transplantation of malignant and normal cells requires that the histocompatibility genes (H genes) of the recipient and the donor be identical. Incompatibility in the H genes causes the rejection of transplanted cells (Snell, 1948, 1953). It was shown that immunologic reactions are responsible for this rejection (Gorer, 1942, 1956). It became clear that most previous observations of resistance to tumor growth in tumor immunology studies would have to be reassessed in light of the evidence that this resistance results from reactions against histocompatibility antigens rather than specific antitumor reactions. This resulted in severe skepticism as to the existence of specific host-mediated antitumor responses. The renaissance of tumor immunology started in 1957- 1960. This was made possible by the use of inbred strains of mice, syngeneic tumors, and new methodologic approaches. Prehn and Main (1957) succeeded in showing that following excision of growing methylcholanthrene-induced tumors, mice became resistant to the second tumor challenge. This resistance was tumor antigen specific and was not mediated by the histocompatibility antigens. Tests for resistance of tumor-excised mice to a second tumor challenge became a favorite experimental model for the study of antitumor immune response, immunogenicity, and antigenic specificity of tumor cells. At the same time it became possible to assess the immunologic reactions against tumors by using immune lymphocytes in the neutralization test (Winn, 1959; E. Klein and Sjogren, 1960; Klein et al., 1960) or humoral

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antibodies as cytotoxic agents in vitro or in vivo (Gorer and OGorman, 1956;Gorer, 1956). With these or other immunologic techniques developed later it was shown that an immune system may recognize tumor cells and respond by the production of immune lymphocytes or humoral antibodies (Old and Boyse, 1964;Klein, 1966, 1976;Law, 1969;Baldwin, 1973;Hellstrom and Hellstrom, 1974;Herberman, 1974;Witz, 1977;Old, 1981).The immunologic mechanisms involved in the response against histocompatibility antigens and tumor-associated transplantation antigens were similar. These data stimulated Thomas (1959)and Burnet (1964,1970) to assess the biologic meaning of the homograft reaction and the general role of the immune system in the multicellular organism. It was suggested that the biologic value of the immune system was the performance of immune surveillance and the maintenance of genetic homeostasis of somatic cells in the multicellular organism by elimination of invading microorganisms, parasites, or mutant somatic cells including potentially malignant cells (Burnet, 1964, 1970). The immune surveillance theory stimulated numerous investigations of malignant processes in different immunologic situations. Based on these studies, several objections have been made to the theory (Prehn, 1971, 1976; Stutman, 1975,1981 ;Baldwin, 1977;Mollerand Moller, 1979).Most ofthe apparent contradictions to the immune surveillance theory have actually been related to the postulated central role of T cells in host defenses against tumors (Burnett,-1970). However, the data obtained do not reject the existence of antitumor immune surveillance mechanisms in the organism and may only indicate that tumor development is a multicomplex process and that different defense mechanisms on different levels are probably involved.

1. Antitumor defense starts at the cellular level and includes all mechanisms that prevent malignant transformation of somatic cells. Xeroderma pigmentosum is a good example of this assumption. Cells of patients with this disease have a defect in the mechanism for repair of UV-induced DNA damage; this results in the accumulation of malignant cells and the development of multiple tumors (Cleaver, 1969;Trosko and Chu, 1975). 2. Somatic cells with malignant potency can be eliminated or their proliferation can be inhibited by immunologic or nonimmunologic mechanisms responsible for control of cell proliferation, regeneration, and tissue growth. In evaluating the antitumor defense role of immune mechanisms it is necessary to consider that in some situations immune reactions may stimulate malignant growth (immunostimulatory hypothesis; Prehn, 1971, 1976).

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Unfortunately, the immune system is not as efficient as is desirable and does not always recognize “self” and “nonself,” and, hence, it does not always eliminate “nonself.” This is true for malignant cells as well as for some microorganisms and parasites that may cause lethal infections in man and animals. However, this does not mean that an immune surveillance system does not exist. There are numerous “holes” in the immune system that provide escape of tumor cells from immune destruction (Hellstrom and Hellstrom, 1974; Klein and Klein, 1977; Naor, 1979; Klein, 1980). It is possible to assume that the antitumor surveillance mechanisms do not function with the optimum efficiency in the complete prevention of malignant disease, but are, instead, mostly “aimed” at postponing the development of malignant diseases in the postreproductive period. Numerous attempts to prove or reject the immune surveillance theory were recently summarized by G . Klein (1980): “In conclusion, IS (immune surveillance) cannot be discussed in all-or-none terms, either in relation to tumors in general or in relation to one specific type of effector.” Abundant evidence indicates that the immune system is especially efficient in the elimination of malignant cells induced by different oncogenic viruses. For some oncogenic viruses “tumor development is a biologic accident of heavy, polyvalently acting immunosuppression” (Klein, 1980). Strong immune responses are evoked by some chemically induced tumors, while spontaneous tumors are unable to elicit detectable rejection reactions (Foley, 1953; Prehn and Main, 1957; Baldwin, 1966; Prehn, 1976; Hewitt et al., 1976). It is possible that spontaneous tumor cells are produced during long periods of tumor progression and that these cells have lost “lethal” antigenicity and have become independent from immune destruction (Klein and Klein, 1977; Klein, 1980). Although spontaneous tumor cells do not evoke resistance to the second challenge and do not stimulate a detectable level of cytotoxic immune lymphocytes and antibodies, involvement of the immune system in the protection against spontaneous tumor is supported by the fact that the incidence of malignancy under immunosuppressive conditions among transplant recipients or patients with immunodeficiency increased by 100- to 1000-fold. These types of malignancy that arise are mostly non-Hodgkin’s lymphomas, carcinomas, or Kaposi’s sarcoma (Penn, 1978; Spector et al., 1978; Mitchison and Kinlen, 1980; Macek, 1982). This fact indicates that the previous assumption (Ehrlich, 1906; Burnet, 1970; Stutman, 1975) that neoplastic cells constantly arise in all normal tissues and organs might not be correct. On the basis of this assumption, substantial increases in tumors of different histologic types would be expected in the immunosuppressed organism. However, the rate of malignant transformation of somatic cells is under genetic control (Heston, 1948;

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Trosko and Chu, 1975; Knudson, 1977). Investigations of genetic cancer have shown that there is no general genetic predisposition to cancer but that this predisposition is always tissue or organ specific (Heston, 1948, 1976; Heston and Dunn, 1951; Knudson, 1973, 1977). Low incidence of tumors in some strains of mice is not a result of effective function of the antitumor immune surveillance but rather a result of the low rate of malignant transformation of somatic cells in certain organs or tissues of these mice. In the absence of the transformed malignant cells, immunosuppression does not result in an increase of tumor incidence. Thus, immunosuppressive procedures do not increase, for example, the incidence of lung or mammary tumors in C57BL/6 mice to the level observed in A/J or C3H mice. Therefore, the genetic background of nude or beige mice should be taken into consideration when the influence of the immune status on tumor development is studied. Experiments performed by Kripke (1974, 1981) with UV-induced skin tumors led to a new view in the understanding of the relation between tumors and the host immune system. These experiments clearly demonstrated that it is not general immunosuppression but rather selective T-cellmediated suppression of immune reactions against highly antigenic UVinduced tumor cells that may be responsible for permitting their growth. These investigations demonstrate that even though the cells of UVinduced tumors are antigenic, they are not able to elicit an immune response in the autochthonous host. Is this situation unique for UV-induced tumors, or is it an example of the processes that occur during development of different spontaneous or induced tumors in man and animals? Absence of a detectable immune response against spontaneous tumors does not mean that antigenicity is absent from these tumor cells. The ability to produce monoclonal antibodies against different human or animal tumor cells may indicate that these cells are antigenic (Herlyn et al., 1979; Levy et al., 1979; Olsson and Kaplan, 1980; Old, 1981; Sikora and Wright, 198 1). Interest in the immune surveillance theory was renewed by the discovery of a subset of normal nonsensitized lymphocytes that can destroy tumor cells in vitro and in vivo. These cells have been termed natural killer (NK) cells. T-cell-depleted nude mice have a high level of NK reactivity, which may be partially responsible for antitumor defense in these mice. Based on these data the concept of natural cell-mediated immunity was proposed. It has been suggested that natural cell-mediated immunity may be an alternative and additional mechanism that participates in the immune surveillance and elimination of invading microorganisms or potential malignant cells (Herberman and Holden, 1978; Kiessling and Haller, 1978). Natural cellmediated immunity occurs through the action of different effector cells including natural killer cells, natural cytotoxic cells, macrophages, and

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granulocytes (Hibbs et al., 1978; Adams and Snyderman, 1979; Fisher and Seffer, 1978; Herberman and Holden, 1978; Kiesling and Wigzell, 1979; Stutman, 198I ; Keller, 1980; Korec, 1980). The main characteristics of these cells are the ability to react immediately against tumor cells without apparent antigenic stimulation or to be activated by various agents or lymphokines (e.g., interferon, MAF, IL-2) and to destroy malignant cells in a nonspecific manner. The identity of the cell surface determinants recognized by these cells and the mechanism by which they distinguish malignant and nonmalignant cells are, for the most part, unknown. Natural cell-mediated immunity is considered as a first line of defense in antitumor immune surveillance; it operates before the specific immune response can be evoked. In addition, this immune mechanism may recognize and destroy tumor cells that are nonimmunogenic for T-cell-mediated immunity (Herberman, 1982; Keller, 1980;Kiessling and Wigzell, 1979). The possible involvement of NK cells and macrophages in antitumor defense is supported by numerous findings that these effector cells can be very efficient in the elimination of tumor cells in vivo at the local site of their growth and in the control of metastatic spread and growth (Fidler, 1974; Alexander, 1976; Hibbs et al., 1978; Haller et al., 1980; Adams and Snyderman, 1979; Karre et al., 1980; Hanna and Fidler, 1980, 1981; Gorelik and Herberman, 1981; Gorelik et a/., 1982a). The contribution of the specific and nonspecific immunologic mechanisms in the antitumor or antimetastatic defense may depend on the properties of tumor cells and the stage of malignant disease. It is obvious that in some cases both the specific and nonspecific mechanisms of antitumor immune surveillance fail to prevent the clinical appearance of malignant tumors, their spread, and growth. In spite ofthis apparent failure there is still great hope that immune mechanisms could be exploited in the immunotherapy of malignant disease. “The nonrejectability of spontaneous tumors does not necessarily justify a pessimistic view about the possibilities of utilizing immunologic approaches in tumor therapy” (Klein and Klein, 1977).Great efforts are under way to develop efficient methods for the immunotherapy of cancer (Terry and Rosenberg, 1977; Goodnight and Morton, 1980; Wright and Bernstein, 1980; Spitler, 1980). Further investigations of the mechanisms that help tumor cells prevent immune reactions and escape from immunologic destruction might result in the development of immunologic methods that can be effective in the treatment of cancer patients. Numerous reviews have summarized the investigations of antitumor immune mechanisms (Old et al., 1962; Old and

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Boyse, 1964; Klein, 1966, 1976; Law, 1969; Balwin, 1976; Hellstrom and Hellstrom, 1974; Herberman, 1974, 1980). The present article concentrates on the studies of the mechanism of concomitant tumor immunity, which has not attracted much attention and in many respects remains unclear. A comparison of the resistance to a second tumor challenge in the tumorexcised and the tumor-bearing host might bring some clarification in the understanding of the mechanisms involved in concomitant tumor immunity. Since metastases can be considered as a second tumor graft that has occurred spontaneously, the mechanisms of antimetastatic concomitant immunity are also discussed. The results obtained in the investigation of antitumor immunity are extraordinarily contradictory. Some of these data can be explained by the differences in the tumor systems used by different investigators or by differences in the experimental procedures. Critical analysis of these results seems impossible without including some details concerning factors such as dosage of tumor inoculation or challenge, time period of tumor growth, and strain of animals. Although it may be considered as unnecessary for this kind of article, we have found that such details may be useful in explaining the contradictions in the results of tumor immunology studies. II. Tumor Excision and Resistance to a Second Tumor Challenge: Sinecomitant Immunity

Host resistance to a second tumor challenge after surgical excision of a growing tumor was discovered by Uhlenhuth, Haendel, and Steffenhagen in 1910 (Woglom, 1929). It is remarkable that this antitumor resistance to a second challenge in surgically treated rats had not been observed when tumor removal was incomplete and recurrent tumor appeared. This resistance was termed “operation immunity” and seemed very obscure. The phenomena remained forgotten for 40 years. Lewis and Aptekman (1952) were probably the first to reintroduce the idea of testing for antitumor resistance after removal of the primary tumor. This idea was based on the observation that animals with tumors that had undergone spontaneous regression became resistant to a second tumor challenge. Since spontaneous regression of a syngeneic tumor in inbred strains of mice or rats is an exclusively rare event, Lewis and Aptekman decided to induce artificial regression by strangulation of a growing tumor. Allen (1 940) had demonstrated that occlusion of the blood circulation in the tumor by a rubber tourniquet caused atrophy of the growing tumor. Using this technique, Lewis and Aptekman (1952) found that ligation of a growing

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syngeneic sarcoma or carcinoma in inbred rats for 1 day resulted in complete tumor regression. Rats with regressed tumors were able to inhibit growth of the reinoculated identical tumor cells. However, Fardon and Prince (1952), using the same technique, failed to repeat these results. The experiments were performed using inbred DBA/ 1 mice and transplantable spontaneous mammary carcinomas. Foley ( 1953) confirmed the observation that regression of ligated, transplanted, spontaneous mammary carcinomas in inbred mice did not make these mice resistant to the challenge with the same tumor cells. In contrast, regression of ligated sarcomas induced by methylcholanthrene (MC) in C3H/He mice increased resistance to challengewith the same MC-induced tumor cells, but did not increase resistance to challenge with mammary carcinoma cells. Prehn and Main (1957) tested 14 fibrosarcomas induced by MC and found that temporary growth of these tumors in inbred strains of mice or their F, hybrids made these mice resistant to the second tumor challenge. Resistance was tumor specific, although partial cross-reactions were observed among the tumors. However, antitumor immunity was not produced by any of seven histologically similar spontaneous sarcomas. These results supported the hypothesis that immunity to MC-induced tumors was not caused by heterogeneity among the mice. In addition, immune mice did not reject normal skin graft from the donors of MC-induced tumors. Inoculation of normal cells did not make the mice resistant to the growth of MC-induced tumors and did not cause rejection of skin grafts from syngeneic mice. Furthermore, Prehn and Main (1957) performed ligation or surgical excision of the growing tumors and found that both these procedures left mice equally immune to reimplantation of the identical tumor cells. Since publication of their study, this method of surgical removal of a growing tumor has been used in numerous investigations of the immunogenicity and antigenic specificity of tumor cells. In 1970, Fisher et al. proposed to term the resistance to the second tumor challenge in the host after excision of the local tumor as “sinecomitant immunity,” in contrast to “concomitant immunity,” which denotes antitumor resistance in the presence of a growing tumor. Although both sinecomitant and concomitant tumor immunity are measured by the ability of the recipient to prevent growth of reinoculated tumor cells, they have many striking differences. The main characteristics of sinecomitant immunity are as follows: 1. The resistance to a second tumor challenge is usually observedfollowing excision of relatively small tumors: 5 - 10 mm in diameter. Removal of larger tumors usually did not allow mice to suppress the growth of reinocu-

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lated tumor cells (Prehn and Main, 1957; Prehn, 1960; Old et al., 1962; Sjogren, 1965; Baldwin, 1966). 2. Antitumor resistance of tumor-excised mice is rather weak and can protect the growth of relatively small tumor cells inoculum (I03-l O5 cells). Increased doses of reinoculated cells may overcome this resistance (Old et al., 1962; Klein et al., 1960; Old and Boyse, 1964; Sjogren, 1965; Baldwin, 1966). 3. Resistance to a second tumor challenge appears only after a lag period following tumor excision. In early experiments performed by Foley (1953) no resistance was observed if the tumor challenge was performed 2 - 6 days before or on the day of a tumor ligation. Mice were immune only if the second tumor reinoculation was performed 2 -6 days after tumor ligation. When MC-induced tumors were surgically removed, identical results were obtained. No resistance was found in the host when antigenic tumor cells were reinoculated immediately or 1 day after radical resection of the primary tumor. Strong resistance to the same tumor cells did, however, appear when the interval between excision and inoculation was at least 7 days (Riggins and Pilch, 1964;Stjernsward, 1968;Kaibara et al., 1970). This interval is probably required for rehabilitation of the immune system reactivity, which was suppressed by growing tumor (Mikulska et al., 1966). 4. Only immunogenic tumors stimulate protection against a second tumor challenge in tumor-excised mice. Animals have a high level of resistance after removal of tumors induced by carcinogenic polycyclic hydrocarbons or some oncogenic viruses. The immunogenicity of these tumor cells is confirmed by the fact that these heavily irradiated tumor cells can immunize mice against challenge with live cells (Klein et al. 1960, 1962; Old et al., 1962; Old and Boyse, 1964; Sjogren, 1965). After tumor excision the spleen or lymph node cells of mice can be specifically cytotoxic in vitro or in vivo against tumor cells (Klein et al., 1960; Old and Boyse, 1964; Sjogren, 1965; Bard el al., 1969; Deckers et al., 1971; Baldwin, 1976; North and Kirstein, 1977). Tumors induced by different chemical carcinogens vary in their ability to stimulate antitumor immunity, as shown by studies of tumor-excised mice. However, numerous attempts have failed to show resistance of tumor-excised mice against spontaneous tumors (Foley, 1953; Prehn and Main, 1957; Baldwin, 1966; Hewitt et al., 1976). 5 . Resistance of tumor-excised mice is antigen specific.Antigenic specificity of tumor cells was demonstrated by testing the ability of tumorexcised mice to inhibit the growth of reinoculated cells that were identical and nonidentical to those from the removed primary tumor. Using this approach it was found that tumors induced by chemical carcinogens with 3-methylcholanthrene, 3,4-benzpyrene, 3,4,9,10-dibenzpyrene, and

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1,2,5,6-benzanthracene have unique antigenic specificity (Prehn and Main, 1957; Prehn, 1960; Old et al., 1962; Globerson and Feldman, 1964; Baldwin, 1976). Partial cross-reactions between certain chemically induced tumors have been observed (Prehn and Main, 1957). Basombrio (1970) tested 25 syngeneic carcinogen-induced tumors. All tumors had individual antigenic specificity. Although weak cross-reactivity was found, it was not easily reproduced. Using tumor excision and challenge, Globerson and Feldman ( 1964)have found that two sarcomas produced by 3,4-benzpyrene in a single mouse had different antigenic specificities. The high specificity of antitumor resistance observed in tumor-excised mike permitted analysis of antigenic differences between tumor cells derived from the primary MC-induced tumor and its pulmonary metastases (Sugarbaker and Cohen, 1972). Antigenic heterogeneity of a tumor cell population was established by challenging tumorexcised mice with tumor cells obtained from the different poles of the same tumor or with tumor cells obtained from the same malignant transformed clone of 3T3 cells (Prehn, 1970; Basombrio and Prehn, 1972). Using the tumor excision and challenge technique, Pimm and Baldwin (1977) have found that recurrent tumors in rats had different antigenic specificities in comparison to excised primary tumors. The tumor excision and challenge method was used for the investigation of the immunogenic and antigenic properties of the virus-induced tumors. Tumors induced by the same virus have been shown to share the same tumor-associated transplantation antigens, although some unique antigens in virus-induced tumors could be found (Sjogren, 1965; Klein, 1966; Morton et al., 1969; Miller and Heppner, 1979; Olsson and Ebbesen, 1979). In general, the method of tumor excision and challenge has been found to be a fruitful assay for the investigation of the development antitumor immune response, immunogenicity, and antigenic specificity of the investigated experimental tumors. 111. Resistance of the Tumor-Bearing Host to a Second Tumor Challenge: Concomitant Tumor Immunity

Although the resistance of the tumor-bearing host to a second tumor graft was described by Ehrlich (1906) and Bashford et al. (1908), new attention was brought to this phenomemon by Gershon 60 years later (Gershon et al., 1967, 1968; Gershon and Kondo, 1971). Using spontaneous lymphoblastic lymphoma, Gershon et al. (1967) have shown that in hamsters bearing this lymphoma > lo5 times more tumor cells were rejected than is required to produce tumors in normal control animals. Lausch and Rapp (1969) made

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a similar observation in hamsters bearing dimethylbenzanthracene (DMBA)-induced tumor. The phenomenon of concomitant immunity was demonstrated by Bard et al. ( 1969) and by Fisher et al. ( 1970) using inbred strains of mice and rats. Resistance of cancer patients to reinoculation of autologous tumor cells was found by Southam ( 1964) and Brunschwig et al. (1 965). In general, investigation of the mechanism of concomitant tumor immunity attracted less attention than the study of immunity in tumor-excised mice. It is remarkable that in experiments in which resistance to a second tumor challenge was tested in tumor-excised mice this challenge was not performed in parallel in mice bearing progressively growing tumors (Foley, 1953; Prehn and Main, 1957; Prehn, 1960; Klein et al., 1960; Globerson and Feldman, 1964; Old and Boyse, 1964; Sjogren, 1965). There are several possible explanations for this fact. In experiments performed by Lewis and Aptekman (1 95 I), Foley ( 1 953), and Prehn and Main (1957), the main notion was that the resistance to a second challenge developed only in mice in which the primary tumor had regressed (after ligation) or had been surgically removed. This hypothesis received strong experimental support. Many attempts to show that resistance had developed during progressive tumor growth failed if the challenge was performed in the presence of the first tumor. However, this resistance appeared after excision of the primary tumor (Riggins and Pilch, 1964; Mikulska et al., 1966; Jonsson and Sjogren, 1966; Stjernsward, 1968; Kaibara et al., 1970; Vaage, 1971). In addition, lymphocytes of the tumor-bearing host did not reveal any antitumor activity in the neutralization assay, and the inhibitory effect of spleen cells or peritoneal exudate cells appeared 1 - 3 weeks after surgical excision of the growing tumors (Old et al., 1962; Mikulska et al., 1966; Alexander el al., 1969; Barski and Young, 1969). Transplanted spleen cells of tumor-bearing mice can transfer the antitumor resistance to normal mice. However, this resistance appeared only if the challenge with tumor cells was performed 1-2 weeks after spleen cell transfer (Deckers et al., 197 I; Vaage, 197 1). These data were interpreted to be a result of immunologic exhaustion in the presence of growing tumor (Mikulska et al., 1966). The analysis of the resistance of tumor-bearing mice to a second tumor graft encounters some methodologic difficulties. The growth of the second tumor graft is measured in the presence of the growing first tumor. When the inoculum size of the second tumor is small, its latent period is relatively long, in which case mice may die from the first tumor before the second develops to a visible tumor. On the other hand, high cell doses of the inoculated second tumor may overcome the effect of concomitant immunity and may grow in the same way as in control mice. It is necessary to find

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some balance between the primary tumor volume and the size of the second tumor inoculum. These factors should be determined for the individual tumor, since the growth characteristics of tumors may vary to a great extent. Resistance of tumor-bearing mice to a second tumor graft has several characteristics that may indicate differences in the mechanisms of antitumor resistance observed in tumor-bearing and tumor-excised mice. A. RESISTANCE TO A SECOND TUMORCHALLENGE: DEPENDENCE ON SIZEOF GROWING TUMOR AND SIZEOF SECOND TUMORINOCULUM In experiments performed by Gershon et a!. (1 967), different numbers of lymphoma cells ( lo6- lo8) were reinoculated into hamsters 7, 14, and 21 days after the first transplantation of lo7lymphoma cells. Seven days after lymphoma transplantation growth of reinoculated 1 O6 lymphoma cells was suppressed in six of eight hamsters tested. However, increased doses of the reinoculated cells ( lo7- lo8) grew uninhibited in these hamsters. With progression of the first tumor during 2 - 3 weeks, hamsters became resistant to the reinoculation of increased doses ( lo7- lo8) of lymphoma cells. In hamsters bearing DMBA-induced tumor for 7 - 16 days, Lausch and Rapp (1969) found that tumor growth was prevented when lo3- lo5but not lo6 tumor cells were reinoculated. Antitumor resistance was higher in hamsters bearing the first tumor graft for 16 days then for 7 days. Strong concomitant immunity was observed in mice bearing highly immunogenic TU-5 tumor induced by SV40 virus (Zarling and Tevethia, 1973). Mice were inoculated sc with 2 X lo5 TU-5 tumor cells. These mice were completely resistant to reinoculation 7 days later with lo5, lo6, or 5 X lo6 identical tumor cells. Resistance to a second tumor graft did not decline with progression of the primary tumor and was strongly expressed even 2 1 days later in the presence of the large tumor mass. These data may indicate that expression of concomitant tumor immunity increaseswith the time of tumor growth and probably depends on the size of the growing tumor, although, in the studies mentioned above, the precise size of the first tumor at the moment of the challenge was not presented. Kearney and Nelson ( 1973) transplanted l O7 MC-induced sarcoma cells into CBA/J mice. No resistance to the second challenge of lo6 cells was observed 6 days later when the primary tumors were about 1 cm in diameter. When these tumors reached 1.5-2 cm in diameter 14 days later, the mice exhibited substantial resistance to the second tumor inoculum. However, the inhibition of a second tumor graft depends not only on the size of the primary tumor but also on the size of the second tumor inoculum. In the rats bearing MC-induced tumor the inhibitory effect was more profound against 2 X lo5than against 1 X lo6 reinoculated tumor cells (Fisher et al.,

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1970). Antitumor resistance of tumor-bearing mice to a second tumor challenge as a function of both tumor size and number of reinoculated tumor cells was further investigated by North and Kirstein (1977) and Gorelik et al. (198 lb). AB6F, mice were inoculated in a foot pad with lo5 SA1 tumor cells; 3 days later no visible tumors were evident, and mice reinoculated at that time with lo6 SAl cells did not show any resistance to the second challenge. When the primary tumor started to grow, 6-9 days later, the growth of the reinoculated cells was partially inhibited. At day 12 after primary inoculation the growth of lo5reinoculated cells in AB6F, mice was completely prevented. Inhibition of the second tumor growth was partial when the number ofthe reinoculated cells increased to lo6- lo7 (North and Kirstein, 1977). Complete resistance to the growth of 2.5 X lo5reinoculated 3LL cells was observed in mice bearing 3LL tumor > 1.5 cm in diameter, although the growth of the second 3LL tumor was not completely inhibited when increased numbers of 3LL cells (5 X lo5- 1 X lo6) were reinoculated (Gorelik et al., 1981b). In the presence of larger primary 3LL tumors (>2 and > 3 cm in diameter) growth of the second tumor grafl was prevented even when 1 X lo6 or 5 X lo6 3LL cells were reinoculated. In control mice, visible tumors appeared 2 days after inoculation of 5 X lo6cells into the food pad (Gorelik et al., 1981b). These data demonstrate that tumor-bearing mice can show a high level of resistance to a second tumor challenge while the first tumor continues to grow progressively until the mice die. The antitumor resistance of tumorbearing mice is a function of both the primary tumor size and the dosage of the challenge inocula. The level of resistance to a particular number of reinoculated tumor cells increases with the size of the primary tumor. In mice with a constant tumor size, there is more efficient suppression of relatively low doses of reinoculated cells, although increased doses can overcome this antitumor resistance. In these experiments, antitumor resistance increased in parallel with the increase of the primary tumor size. In some tumor systems, however, a different pattern of resistance to a second challenge was observed. In a study by Chandrasadasa ( 1973) using immunogenic MC-induced tumor MC6 in BALB/c mice, the resistance to a second challenge with 105- 7 X 1O6 tumor cells increased during 7 - 14 days of tumor growth and declined at 2 1 - 35 days. Decrease of the antitumor resistance at the later stage of tumor growth was detected mostly in response to higher challenge doses, whereas resistance to a challenge with lo5cells persisted at almost all stages of the primary tumor growth. In contrast, C57BL/6 mice bearing weakly immunogenic MC23 tumor did not show such an abrupt loss of resistance to a second

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challenge in the later period of tumor growth (Chandrasadasa, 1973). In a study by Berendt et al. (1978) mice challenged into the foot pad with 1 X lo6 Meth A tumor cells 2 days after intradermal inoculation of primary Meth A tumor cells showed significant antitumor resistance. This resistance peaked when the challenge was performed on day 4,and then underwent progressive decay. Decrease in the antitumor resistance in tumor-bearing mice began on day 6 when the primary intradermal tumors were less than 100 mg. By day 16 mice bearing intradermal tumors greater than 500 mg did not show any resistance to a second challenge. Similar data were obtained by Howell et al. ( 1975). Mice bearing transplanted mKSA tumor induced by SV40 virus showed some resistance to the reinoculation of 1 X lo6 mKSA cells in the first 1-2 weeks of tumor growth but this resistance disappeared in the following 3-5 weeks. In parallel the spleen cells of tumor-bearing mice inhibited the growth of tumor cells in the neutralization assay only when they were obtained from donors bearing tumor during 1-2 weeks. Spleen cells of mice bearing this tumor lost their antitumor activity after 3-5 weeks (Howell et al., 1975). Belehradek et al. (1972) observed resistance to a second challenge in mice bearing MC-induced sarcoma TBL CL2 for 10 days. This resistance disappeared with progression of the initial tumor (30 days of tumor growth). However, it was possible to restore resistance to a second challenge if the primary tumor was excised at day 23 of tumor growth and if mice were challenged 7 days after tumor excision. In all of these experiments the challenge was performed during a certain period after the primary inoculation of tumor cells or when the primary tumor reached a specific volume. In the development of concomitant tumor immunity, the relative importance of the presence of established tumor is still unclear, as is the time interval between the first tumor graft and the challenge. In some experiments the challenge was performed within 6 - 7 days after the first tumor graft. By this time, the primary tumor had become established and antitumor resistance was detected (Gershon et al., 1967; Lausch and Rapp, 1969; North and Kirstein, 1977). In other studies, a period of 6 - 7 days between the first and the second transplantation was too short for a particular type of tumor cell or certain initial doses to develop visible tumors and resistance to a second challenge (Fisher et al., 1970; North and Kirstein, 1977). To further distinguish the importance of the tumor size on the period between transplantation and challenge in the expression of concomitant tumor immunity, additional experiments were necessary. Specifically,there was a need to evaluate the effect of a challenge at a specific time following initial transplantation of varying numbers of tumor cells (constant time period and variable tumor size) and to assess the results of reinoculation in

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mice bearing tumors of identical size but at different periods of their growth (constant tumor size and variable time period). Such experiments were performed by North and Kirstein (1977). AB6F, mice were inoculated in the foot pad with lo4, lo5, or lo6, SAl sarcoma cells. Every 3 days after transplantation groups of mice were challenged by injection in the second foot pad with the standard dose of 1 X 1 O6 SA 1 cells. In mice challenged 6 days after primary inoculation with a high dose of tumor cells (1 X lo6), the growth of the second inoculum was inhibited whereas no resistance was found at that time in mice whose primary inoculum was lo5 or lo4 tumor cells. Resistance in these mice appeared when the challenge was performed 9 and 18 days after primary inoculation with 1 O5 or 1 O4 SA 1 cells, respectively. In all groups of mice, the level of antitumor resistance further increased in parallel with an increase of the primary tumor size. Analysis of these data indicates that irrespective of the time period between the primary and secondary tumor inoculations resistance to a second challenge appeared when the primary tumor reached a critical size, which was similar in all three groups of mice investigated. Resistance to reinoculation with lo6 tumor cells was found with a primary tumor that was about 2.0 mm in diameter at the foot pad. This tumor size appeared in mice 6 , 9 , or 18 days after transplantation of lo6, 105,or lo4SA 1 cells, respectively (North and Kirstein, 1977). The model of concomitant tumor immunity is very dynamic, with varying parameters in two growing tumor cell populations, which may influence both the host and each other. As was discussed above, the number of cells reinoculated has been shown to be extremely important for the detection of antitumor resistance in tumor-bearing mice. High doses of the challenge inocula can be suppressed by an established, large primary tumor present at the moment of challenge. On the contrary, by decreasing the number of reinoculated cells it is quite possible to minimize the size of the primary tumor cell population required for expression of eoncomitant immunity. Therefore, it is expected that inhibition of the second tumor graft growth can be demonstrated when small numbers of tumor cells are reinoculated even at the latent period of the first tumor growth. Data obtained by Deckers et al. (197 1 ) and Zarling and Tevethia ( 1973) experimentally support this hypothesis. In the investigations performed by Deckers et al. (1971) mice were challenged with 5 X lo3 MC-induced MCA-I0 sarcoma cells at 1 -28 days after initial transplantation of lo5 MCA-10 cells. Mice reinoculated 1 day after tumor transplantation did not show any resistance, whereas mice challenged 7 -28 days after primary tumor inoculation showed equal and strong resistance to the second tumor reimplantation. Mice challenged on day 7 had no visible primary tumors; however, primary tumors developed later in all of these mice, whereas the

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second tumors grew only in 8% of mice. Tumors developed in 92% of control mice inoculated with challenge doses of tumor cells (5 X lo3). Similar results were obtained using SV40-induced TU-5 tumor cells (Zarling and Tevethia, 1973). TU-5 tumor cells were transplanted in BALB/c mice and challenge with lo5 TU-5 cells was performed 1-6 days later. The second tumor growth was completely prevented if the mice were challenged even in the absence of established primary tumor (4 - 6 days after inoculation with primary tumor cells). When the interval between inoculation and challenge was only 1 day, development of the second tumor was significantly inhibited. Whereas normal recipients developed palpable tumors by day 7, mice that had received a second reinoculation 1 day after the first TU-5 cell transplantation did not develop secondary tumors until day 15. Growth of the first tumor was not changed. These data indicate that in some tumor systems the presence of an established tumor at the moment of the challenge is not obligatory for the detection of concomitant tumor immunity. Furthermore, it is quite possible that the time interval between the primary tumor transplantation and the challenge is not absolutely required. In other experiments performed by Zarling and Tevethia ( 1 973), the same number of TU-5 tumor cells (lo5)were simultaneously inoculated into BALB/c mice at two distant sites. Tumors at both sites developed at the same rate, as did tumors in mice given only one dose of tumor cells. However, simultaneous inoculation of different numbers of tumor cells may give different results. Simultaneous transplantation of the small and large tumor inocula into mice may result in the development of a visible tumor from the large dose, which may prevent the growth of the small tumor inoculum. Indeed, Lausch and Rapp (1969) simultaneously inoculated lo1,lo2, lo3,and lo4DMBAinduced tumor cells into four different sites on a single hamster. Local tumors developed at the sites of inoculation of lo3 and lo4 cells. In the presence of these tumors, growth of 10' and lo2cell inocula was completely prevented. Tumors developed in 25 and 75%of control hamsters inoculated separately with 10' or lo2cells, respectively. These data demonstrate the importance of the volume of the first tumor mass but not the time interval between initial tumor transplantation and challenge. The size of the challenge dose is critical for assessment of the expression of concomitant tumor immunity. OF TUMOR CELLSAND CONCOMITANT B. IMMUNOGENICITY TUMOR IMMUNITY

Several of the experimental tumors used for investigation of concomitant tumor immunity were strongly immunogenic.

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Gershon et al. (1967) and Gershon and Kondo (1971) transplanted a lymphoblastic lymphoma, that had arisen spontaneously in a random-bred hamster, into an inbred line of MHA Syrian golden hamsters. This allogeneic lymphoma induced a strong immune response but grew progressively. Peritoneal exudate cells (PEC) or spleen cells of tumor-bearing hamsters suppressed tumor growth in the neutralization test even at a ratio between tumor and PEC of 1 : 1. In addition, following surgical excision of this lymphoma growing locally, hamsters were resistant to a second challenge with high doses of lymphoma cells ( 106- lo7). AB6F, mice bearing SA1 sarcoma for 10 days were resistant to a second tumor challenge. Lymphocytes from lymph nodes of these mice were specifically immune, as demonstrated by the neutralization assay (North and Kirstein, 1977). Several chemically induced tumors that elicit concomitant immunity in mice and rats are immunogenic. This is evidenced by their ability to induce both concomitant and sinecomitant tumor immunity (Bard et al. 1969; Fisher et al., 1970). Immunogenicity of EL4 and Meth A tumors has been demonstrated by numerous experiments (Old and Boyse, 1964; Berendt et al., 1978; Rogers and Law, 1981). These tumors are efficient in the induction of concomitant tumor immunity (Berendt et al., 1978; Gorelik et al., 1981b; Gorelik, 1983). MC-induced S- 10 sarcoma showed strong concomitant tumor immunity, and lymphocytes from tumor-bearing mice were efficient in the adoptive transfer of antitumor immunity to the normal recipients (Deckers et al., 1973). Spontaneous tumors such as the Lewis lung carcinoma (3LL), B16 melanoma, and Madison lung carcinoma (M 109) are nonimmunogenic or weakly immunogenic. Lymphocytes or serum of mice bearing these tumors had no detectable level of cytotoxic activity in vitro or in vivo. After surgical removal of these tumors, resistance to a second tumor challenge was not observed (E. Gorelik, unpublished observation). Nevertheless, mice bearing these weak immunogenic tumors (3LL, B 16, or M 109) were resistant to a second tumor challenge, even when reinoculated doses were as high as 1 X 106-5 X lo6 tumor cells (Gorelik et al., 1981b; Gorelik, 1983). Similarly, mice bearing the weakly immunogenic tumor MC23 were found to be resistant to a second challenge. However, lymphocytes of mice bearing this tumor did not show any antitumor protection in the neutralization assay (Chandrasadasa, 1973). Resistance to spontaneous autologous tumor cells was demonstrated in cancer patients (Southam and Brunschwig, 1961; Southam, 1964; Brunschwig et al., 1965).Tumor cells were obtained from patients with advanced cancer of the ovary or uterus. Autologous tumor cells (104-108) were inoculated sc at marked sites on the anterior region of the thigh. After inoculation of lo8cells, tumors appeared in all patients, whereas injection of lo4 cells did not result in visible tumors in any case. When 1O6 autologous

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tumor cells were used for reinoculation, great differences in the growth of reinoculated cells were found. Resistance to autotransplantation of lo6 cells was more profound in patients with localized cancer than in those with regional or distant metastases. Patients with less aggressive cancer disease showed higher antitumor resistance, as measured by their ability to suppress growth of 1 X lo6 autotransplanted tumor cells. In parallel, patients whose ability to reject allograft was impaired were less resistant to transplantation of autologous tumor cells (Brunschwig et al., 1965). All these data demonstrated that resistance of the tumor-bearing host to a second tumor challenge can be induced by both immunogenic and nonimmunogenic tumor cells.

c. ANTIGENICSPECIFICITY OF CONCOMITANT TUMORIMMUNITY Contradictory results were obtained when the antigenic specificity of the resistance of tumor-bearing mice to the second tumor graft was analyzed. Using six different MC-induced sarcomas, Kearney and Nelson (1 973) studied the specificity of antitumor resistance in CBA/J mice. They found that CBA mice bearing C-6 sarcoma exhibited two phases of resistance to challenge tumors: 14 days after transplantation of a primary C-6 tumor (diameter, 1.5 - 2 cm) mice were resistant to C-6, partly resistant to C-4, and not resistant to C-8, C-9, or C- 10 sarcomas. Twenty-one days after a primary C-6 tumor growth (diameter 2 3 cm) mice were resistant to all tested MC-induced sarcomas. North and Kirstein (1977) found that mice bearing sarcoma SA1 had nonspecific tumor resitance to the challenge with syngeneic benzpyreneinduced fibrosarcoma BP3 or MC-induced fibrosarcoma MC5. However, mice bearing SA 1 sarcoma for 10 days were nonspecifically resistant if the challenge dose of BP3 or MC5 was 5 X l O5 cells. Mice bearing SA 1 sarcoma that were reinoculated with a higher dose of tumor cells (2 X lo6) showed resistance against SAl cells only. In the neutralization test, T lymphocytes from regional lymph nodes draining the site of the primary SAl tumor specifically suppressed the growth of SA 1 but not BP3 tumor cells. Nonspecific resistance to the second inoculum was observed in C57BL/6 mice bearing 3LL tumor > 2 cm in diameter. The growth of reinoculated 3LL, B16, and EL4 tumor cells was inhibited in these mice. Similarly, in mice bearing B 16, EL4, or T- 10 tumors, the growth of reinoculated 3LL tumor cells was arrested (Gorelik et al., 1981b). In some experiments, specific resistance to a second challenge was found. Mice bearing S-10 sarcoma were resistant to a second inoculum with lo3 S- 10 cells, whereas an S- 1 1 tumor grew unrestricted (Deckers et al., 1973). In mice bearing Dunn osteosarcoma growth of reinoculated 2 X lo5 cells of

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Dunn osteosarcoma was inhibited but growth of 2 X 1O6 C 1.18 tumor cells was not inhibited (Gatenby and Basten, 1980). Concomitant immunity induced by strongly immunogenic TU-5 tumor cells transformed by SV40 virus appeared to be specific for virus-induced, tumor-associated transplantation antigens. Mice bearing TU-5 tumor were resistant to the challenge with another type of SV40-induced tumor cell (SV40-VLM), whereas no resistance was detected when the challenge was performed with spontaneously transformed TUT3 No. 2 tumor cells (Zarling and Tevethia, 1973). Analysis of the specificity of the resistance of tumor-bearing mice to a second challenge may be accomplished by the difference in the growth characteristics of the tumors investigated. Challenge of mice bearing a tumor of a certain size with one dose of tumor cells may give inappropriate results concerning the specificity of the antitumor resistance. D. IMMUNOLOGIC REACTIVITY OF THE TUMOR-BEARING HOST AND CONCOMITANT TUMORIMMUNITY Nonspecific antitumor resistance of tumor-bearing mice was demonstrated in several experiments, indicating that nonspecific immunity might be mediated by cells other than T cells. Alternatively, nonimmunologic mechanisms also could be responsible for this phenomenon. Therefore, investigations of concomitant tumor immunity in the immunosuppressed or immunodeficient host make a particular contribution toward the understanding of the mechanisms of this phenomena. In thymectomized, lethally irradiated (900 R), and bone marrow reconstituted mice (B mice) that bear intradermal Meth A or SA 1 primary tumors resistance to a second challenge was substantially decreased. However, some antitumor resistance remained; hence, the growth of the second graft in B mice was significantly inhibited (North and Kirstein, 1977; Berendt et al., 1978). In some experiments, mice were irradiated with 900 R, protected with 2 X lo6 bone marrow cells, and inoculated id with Meth A tumor cells 4 - 6 weeks after irradiation. In these mice, as well as in nonirradiated, control, tumor-bearing mice, the growth of reinoculated 1 X lo6 Meth A cells was completely prevented (Berendt et al., 1978). B mice or ICR nude mice bearing 3LL tumor were resistant to the second challenge of 2 X 105-5 X lo6 3LL cells. BALB/c nude or conventional BALB/c mice bearing M 109 tumor were equally efficient in the suppression of the growth of reinoculated 5 X lo5 M109 cells (Gorelik et a/., 1981b). These data may indicate that with some experimental tumors resistance of tumor-bearing mice to a second tumor challenge is not mediated via a

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T-cell-dependent immune reaction. North and Kirstein ( 1977) suggested that macrophages may be primarily responsible for this antitumor resistance. This suggestion was based on the findings that expression of concomitant tumor immunity was in parallel with an increase of resistance of tumor-bearing mice to iv inoculation of lo5 Listeria monocytogenes (North and Kirstein, 1977; Berendt et al., 1978). NK cells can also be considered as cells that may participate in the nonspecific suppression of the second tumor growth in tumor-bearing mice. This assumption was studied using mice with suppressed macrophage and NK cell functions (Gorelik, 1983). In C57BL/6 mice, these functions were suppressed by silica treatment. Silicatreated C57BL/6 mice and NK deficient beige and normal C57BL/6 mice bearing 3LL tumor were challenged by inoculation in the foot pad with 1 X lo6 3LL cells. Growth of the second graft was strongly inhibited in all tumor-bearing mice regardless of the activity of NK cells or macrophages. These data indicate that inhibition of a second graft in mice bearing 3LL tumor is not mediated by NK cells or macrophages.

E. CONCOMITANT VERSUS SINECOMITANT IMMUNITY The main characteristics of the concomitant and sinecomitant immunity are summarized in Table I. Concomitant tumor immunity can be rather strong and can prevent growth of the high doses of the reinoculated cells. However, it could be that this antitumor resistance is not tumor specific and can be observed even in the immunosuppressed or immunodeficient mice, Furthermore, mice bearing immunogenic or nonimmunogenic tumors may demonstrate strong resistance to a second tumor challenge. Tumor-excised animals are resistant to a second tumor challenge with the TABLE I RESISTANCEOF TUMOR-EXCISED AND TUMOR-BEARING MICETO A SECONDTUMORCHALLENGE

CHARACTERISTICS OF THE

Characteristic Ability to inhibit the growth of a second tumor graft Immunogenicity of reinoculated tumor cells Antigen specificity of tumor cells Host

Tumor-excised mice

Tumor-bearing mice

Weak

Strong

Only immunogenic

Immunogenic and nonimmunogenic Can be nonspecific

Specific Immunocompetent

Can be immunosuppressed or immunodeficient

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same immunizing tumor. This strong antigen-specific response is exerted only against immunogenic tumors. The discrepancy in the main characteristics of sinecomitant and concomitant immunity could result from differences in the mechanisms responsible for these phenomenon. Most of the investigations of the mechanisms of sinecomitant and concomitant immunity were performed separately with different tumor systems. However, several attempts to compare the antitumor resistance of the animals in the presence or absence of growing tumor were performed under the same experimental conditions and rather contradictory results were obtained. Resistance to a second tumor challenge can be observed (1) only after excision of the primary tumor, (2) equally in the presence or absence of the growing tumor, or (3) only in the presence of the progressing tumor. 1. In mice with immunogenic tumors the developing immune response may be neutralized or suppressed by growing tumor mass. Therefore, no resistance to a second challenge was found in the tumor-bearing host. There is a required time period following tumor excision before antitumor resistance becomes detectable (Foley, 1953; Riggins and Pilch, 1964; Mikulska et af., 1966; Stjensward, 1966). Recovery of the antitumor reactivity after tumor excision can be abrogated by the reinoculation of high doses of tumor cells or antigenic material (Vaage, 1971). Cytotoxic activity of spleen or peritoneal cells became apparent only if the period between tumor removal and testing exceeded 7 days (Mikulska et af., 1966; Stjernward, 1966; Alexander et af.,1969; Deckers et af.,1971). 2. In some experimental tumor systems resistance to a second challenge was equally expressed in the presence of growing tumor or after its removal. Growth inhibition of reinoculated tumor cells was equally efficient in C3H/HeN mice while they bore MC-induced tumor or after its excision. Immunogenicity of the tested tumor was supported by the ability of the lymph node cells of tumor-bearing mice to transfer antitumor immunity to normal recipients adoptively (Bard et al., 1969). In Lewis rat, MC-induced tumor equally elicited strong concomitant and sinecomitant immunity (Fisher et af., 1970). Hamsters bearing allogeneic lymphoma or after its removal showed a high level of resistance to a second challenge (Greene and Harvey, 1960; Gershon and Kondo, 1971). 3. Using the same lymphoblastic lymphoma, Gershon and Kondo (197 1) found that early removal of the growing tumor may impair antitumor resistance. Thus, growth inhibition of reinoculated lymphoma cells was less efficient in tumor-excised hamsters than in tumor-bearing hamsters. PEC of these tumor-excised hamsters were also less efficient in the neutralization

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test than PEC of tumor-bearing hamsters. As mentioned above, the same assay performed 2 weeks after tumor growth showed equal resistance to a second challenge in tumor-excised and tumor-bearing hamsters. After further study of immunity in the tumor-excised and tumor-bearing hamsters the investigators found that the antitumor resistance of these animals depends not only on the time of tumor excision but also on the initial “immunizing” tumor dose; for example, hamsters were inoculated with different doses of lymphoma cells ( lo5, lo6, or lo7) and the resulting tumors were removed 7 - 28 days after transplantation. Antitumor resistance of tumor-bearing and tumor-excised hamsters were tested by reinoculation of lo6 lymphoma cells. The prevention of growth of the second graft was less efficient in all tumor-excised hamsters than in tumor-bearing hamsters. However, this was true only if the tumor excision was performed 7 days after inoculation of 106- 1 O7 cells or 14 days after transplantation of lo5 cells. Antitumor resistance was fully expressed if tumor excision was performed later: 14 or 2 1 days after transplantation of lo6- lo7 or lo5 tumor cells, respectively. Similar data were obtained when the PEC of tumorexcised and tumor-bearing hamsters were tested in the neutralization test. Unfortunately, these experiments give no indication of the size of the excised tumor. It would be predicted that 7 - 14 days after transplantation of lo5- lo7 tumor cells the developing tumors would have different volumes. Thus, the time periods between transplantation and excision and tumor bulk are both critical for the stimulation of antitumor immune reactions. Based on these experiments, Gershon and Kondo ( 197 1) concluded that antitumor immunity can be divided into two phases: (1) a developmental or inductive phase and (2) an established phase. Only the first phase is significantly affected by tumor removal. Tumor excision at the inductive phase removes the antigenic stimuli and prevents further development of the antitumor response. At this phase, antitumor reactions can be boosted by reinoculation of 1 O7 lymphoma cells into tumor-excised hamsters. This stimulation was tumor specific, since only the lymphoma cells that were identical with removed tumor stimulated PEC antitumor activity of tumorexcised hamsters (Gerson and Kondo, 1971). Later, it was suggested that tumor excision in hamsters was accompanied by an increase in the activity of the suppressor cells, which are responsible for the decay in antitumor resistance (Gershon, 1974). These experiments were performed with an allogeneic lymphoblastic lymphoma that was apparently immunogenic. At the period when immune reaction was established antitumor resistance was expressed equally in animals bearing the immunogenic tumor or after its removal. When weakly immunogenic tumors are used, the results of testing antitumor resistance in tumor-excised and tumor-bearing mice may give

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patterns that are different from those described above. Kearney and Nelson ( 1973) found resistance to a second challenge with MC-induced C-6 tumor

cells only in the presence of the growing primary tumor. Following tumor excision this resistance disappeared. Resistance of mice bearing C-6 tumor was tumor nonspecific. In mice bearing large tumors growth of various types of reinoculated tumor cells was inhibited (Kearney and Nelson, 1973). When local 3LL tumor was removed, mice did not demonstrate any resistance to a second tumor challenge. In contrast, inhibition of the second implanted tumor was observed in the presence of the growing 3LL tumor (DeWys, 1972). This inhibition of the second tumor growth may be partly reversible after removal of the primary tumor. Mice bearing 3LL tumor were challenged with an injection in the second leg. Seven or fourteen days later, in mice bearing two tumors the primary tumors were removed. Mice in which the initial tumor was amputated on day 7 showed only slight reduction growth of the second tumor. More profound inhibition was found if the primary tumor was removed later - 14 days after challenge. Maximum inhibition of the second tumor growth was observed in mice in which the initial tumor remained in situ (DeWys, 1972). Similar results were obtained when 3LL and T- 10 tumor cells were used to challenge the mice in which the 3LL primary tumor was removed or allowed to grow (Gorelik, 1983). Growth of reinoculated 3LL cells was similar in the tumor-excised and control mice, whereas in mice bearing the first 3LL tumor, the growth of the second 3LL tumor was substantially suppressed. This inhibition was tumor nonspecific because growth of reinoculated MC-induced T- 10 tumor cells was also inhibited in the presence of growing 3LL tumor. In contrast, there was no inhibition of T- 10 tumor growth in mice that had had excision of the 3LL tumor (Gorelik, 1983). These data show that for some weakly immunogenic tumors, the mechanisms involved in the resistance to a second challenge can be different in the tumor-bearing or the tumor-excised host. Highly immunogenic tumors can be equally efficient in the induction of concomitant and sinecomitant immunity. IV. Metastases as a Second Tumor Graft: Antimetastatic Concomitant Immunity

Growth of tumor cells reinoculated into animals bearing a primary tumor mimics the situation that is observed during metastases formation. Metastases can be considered as a second tumor graft developed spontaneously during the primary tumor growth. Therefore, understanding the mechanisms controlling growth of the second tumor graft may be extremely important in understanding the mechanisms of metastatic growth and

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antimetastatic immunity. The investigations using experimental model primary tumors and artificial or spontaneous metastases may also provide additional information in understanding the mechanisms of concomitant tumor immunity. A. RESISTANCE OF THE TUMOR-BEARING HOSTTO AN INTRAVENOUS CELLS CHALLENGE OF TUMOR In the experiments discussed above, the resistance developed during tumor growth was tested by reinoculation of tumor cells sc, im, or in the foot pad. Antitumor resistance can be assessed by iv inoculation of tumor cells into the tumor-bearing host, and the number of artificial metastatic foci developed in the lungs of tumor-bearing versus control animals serves as an indication of the antitumor resistance to a second challenge. Induction of artificial metastases in the lungs or other organs following iv inoculation of tumor cells into healthy animals is widely used for investigation of the mechanisms of metastatic growth (Fidler et af., 1978). However, this experimental model does not encounter the immunologic and nonimmunologic changes in the tumor-bearing organism that may interfere with surviving, extravasation and growth of tumor cells. Involvement of concomitant tumor immunity in the antimetastatic defense was investigated in several studies using the models of artificial and spontaneous metastases. The number of artificial lung metastases that develop after iv inoculation of tumor cells into tumor-bearing mice can be classified as equal, higher, or lower than in normal non-tumor-bearing mice. These controversial results may be due to differences in several properties of the tumor cells used for investigation and to various complex changes in the tumor-bearing host, which can be qualitatively different at different stages of tumor growth. In experiments performed by Miles et al. (1974), the development of artificial metastases in the lungs of C3HF/Bu mice bearing MC-induced FSA fibrosarcoma was substantially suppressed. This inhibition of metastases formation was a function of the size of the initial tumors (or time of tumor growth). Primary tumors started to grow after sc transplantation of 4 X lo5FSA cells. Mice were challenged with lo5 FSA cells iv 3 hours to 14 days later. At the time of challenge (3 hours to 7 days after primary tumor growth) some mice had no visible tumor and others had tumors with diameters up to 5.3 mm. Local tumors had developed in all mice at 15 days after iv inoculation, but the number of metastatic colonies in the lungs was suppressed threefold in comparison to normal mice inoculated iv with FSA tumor cells. Mice that were challenged 14 days after local tumor growth (9.9 mm in diameter) had eight times fewer metastases in the lungs than

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control mice. Spleen or lymph node cells were capable of adoptively transfering immunity to iv inoculated tumor (Miles et al., 1974). Using injections of weakly immunogenic line 1 carcinoma, Yuhas et al. (1975) achieved similar results. BALB/c mice bearing sc growing line 1 carcinoma that were challenged iv at different periods of tumor growth exerted strong resistance to the development of metastatic foci in the lungs. In mice bearing sc tumor for 14 days, the growth of pulmonary metastases was almost completely prevented. Tumor size, but not interval between sc transplantation of tumor cells and iv challenge, was largely responsible for the antimetastatic resistance in tumor-bearing mice. BALB/c mice were inoculated sc with 5 X lo4, 5 X lo5, 5 X lo6, or 5 X lo7 line 1 carcinoma cells; 2 hours later, all mice received iv 2 X lo4 tumor cells. Twenty-one days after sc transplantation of tumor cells, the weight of developed local tumors was a function of inoculated sc cell dose. The level of inhibition of iv inoculated tumor cells was directly proportional to the weight of developed local tumors. Mice bearing the larger sc tumors (5.0 g after inoculation of 5 X lo7 cells) were able to inhibit, almost totally, the development of lung tumors after the iv injection of tumor cells. In these experiments, Yuhas et al. (1975) found that pulmonary tumors developed after iv inoculation of line 1 carcinoma cells into mice bearing relatively small sc tumors exerted an inhibitory effect on the growth of these local tumors. Similar iv injections of tumor cells at later stages of local tumor growth had little or no effect on the primary tumor growth. Mice that received 500 R 2 hours before sc transplant did not show any resistance to iv reinoculated cells at any stage of local tumor growth. However, the numerous lung tumor colonies that developed in irradiated recipients inhibited the growth of the sc tumors. In some experiments, the resistance of tumor-bearing mice was tested by assessing their ability to eliminate [ 12SI]IUdR-labeledtumor cells (Fidler et al., 1977; Proctor et al., 1979). In C57BL/6 mice bearing sc B16 melanoma during 10 or 21 days, the elimination of the radiolabeled tumor cells from the lungs was more efficient than in normal mice. In parallel, the number of artificial metastases in the lungs of these tumor-bearing mice was one-half that in control mice (Fidler et al., 1977). In the experiments performed by Proctor et al. (1979) radiolabeled mammary carcinoma 1699 cells were inoculated iv at different time periods after initiation of the primary tumors with 5 X 105-5 X lo4 tumor cells. Between 5 and 10 days after tumor implantation, depending on the numbers of cells used for tumor induction, the rate of elimination of radiolabeled cells from the lungs of the tumor-bearing mice was significantly higher than in the control mice. In parallel, at the same time points, iv

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inoculation of nonradiolabeled cells into tumor-bearing mice was accompanied by a dramatic decrease in the number of developed lung tumors (Proctor et al., 1979). The rate of elimination of tested tumor cells returned to normal 10- 20 days after im inoculation of 5 X lo5- 5 X l O4 tumor cells, and later, the ability of tumor-bearing mice to eliminate radiolabeled cells was substantially depressed. No inhibition of the growth of artificial pulmonary metastases was observed in mice bearing large tumors (Proctor et al., 1979). On the contrary, in the experimental system used by Ando et al. ( 1979) local tumor growth resulted in high sensitivity to iv challenge with the same tumor cells. C3HJ/Bu mice were inoculated in the hind legs with 5 X lo5spontaneously arisen fibrosarcoma NSFSA cells. Mice were challenged iv with 1 X lo5cells 3 , 7 , and 14 days later; tumor size on those days was 0,5.4, and 11.8 mm, respectively. The number of pulmonary metastases that developed after iv inoculation of tumor cells was similar in the control mice and the mice challenged 3 and 7 days after the first transplantation of tumor cells. However, iv challenge of mice bearing 1 1.8-mm tumors resulted in a threefold increase in the number of developed metastatic foci in the lung (Ando et al., 1979). The accelerated growth of the iv inoculated tumor cells in tumor-bearing mice did not depend on the challenge doses. Enhancement in mice bearing the tumor for 2 weeks was shown not to be due to metastases seeded from the primary tumor. The specificity of lung colony enhancement was investigated by comparing the yield of lung colonies in mice that, for 14 days, had borne antigenic non-cross-reactive spontaneous NFSA fibrosarcoma or MC-induced fibrosarcoma. Mice bearing NFSA or FSA fibrosarcoma were challenged iv with NFSA tumor cells. A substantial increase was observed in the number of metastatic foci in the lungs of all tumor-bearing mice, regardless of the origin of the primary growing tumor. Depression of the resistance to iv inoculation of tumor cells disappeared after tumor excision (Ando et al., 1979). Irradiation of mice before or 12 days after inoculation with primary tumor cells resulted in a dramatic increase in the number of metastatic foci in the lungs following iv inoculation of tumor cells. This acceleration of metastases formation was equal in control and tumor-bearing mice. Suppression of antimewstatic defense was also found in thymectomized, irradiated, and bone marrow reconstituted mice (B mice). In these mice, the presence of a growing tumor did not exert any additional influence on the growth of metastatic lung tumor (Ando et al., 1979). These data demonstrate that, depending on the tumor used for investigation, tumor-bearing mice can be resistant, or, on the contrary, more sensitive to iv reinoculation of tumor cells. In addition to these patterns of rejection or enhancement, iv challenge of tumor cells in

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tumor-bearing mice may reveal changes from resistance to high sensitivity depending on the size (period) of the developed local tumor (Janik et al., 1981). BALB/c mice were challenged iv on days 9, 16, and 28 after sc transplantation of 5 X lo4 cells of the spontaneous Ll sarcoma. After reinoculation of 2 X lo5 L1 cells into mice bearing small palpable local tumor (9 days of tumor growth) the number of tumor nodules that developed in the lungs of these mice was similar to that observed in normal mice. This number decreased if the challenge was performed in the presence of a 0.5 cm3tumor (16 days of growth). In mice bearing large tumors (3-4 cm3; 28 days of growth) the number of artificial metastases in the lungs was higher than in normal control mice. Resistance or susceptibility to the iv challenge can be adoptively transferred into normal mice by spleen cells of tumorbearing mice. The higher sensitivity of mice bearing large tumors was attributed to the presence of suppressor cells (Janik et al., 198 1). B. RESISTANCE TO LOCALVERSUS SYSTEMIC INTRAVENOUS TUMORCHALLENGE The efficiency with which different immunization protocols produce antitumor resistance was assessed by the ability of mice to destroy tumor cells inoculated locally or iv. Experimental data demonstrate that results of the testing of antitumor resistance can be different if the tumor challenge is performed sc, im, or iv. Wexler et al. (197 1) performed excision of MC-induced tumor and the second challenge of the identical tumor cells was done im or iv. Growth of im reinoculated tumor cells was completely inhibited in tumor-excised mice. Identical cell suspensions inoculated iv, however, produced tumors in the lungs of all immunized mice (Wexler et al., 197 1). Using mammary carcinoma cells Vaage (1977) came to the opposite conclusion:the immune resistance to iv inoculated tumor cells appears to be significantly more effective than resistance to sc transplanted tumor cells. When the number of tumor cells that produces 100%growth sc in normal mice was injected into immunized (tumor-excised) mice, tumor growth occurred in about 50% of the mice. The dose of tumor cells that results in 100% growth afier iv inoculation in the lungs of normal mice induced pulmonary tumors in only 10%of immunized mice. The resistance to iv challenge was higher than to sc challenge in mice with progressively growing mammary carcinoma (Vaage, 1977). Ando et al. (1979) found that in mice bearing spontaneous fibrosarcoma ( 1 1.8 mm in diameter) in the hind leg for 14 days the growth of im reinoculated tumor cells was completely prevented. However, similar iv reinoculation of tumor cells revealed that in tumor-bearing mice, antimeta-

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static resistance was suppressed and the number of tumors developed in the lungs was higher than in the control mice. Local or systemic iv transplantation of tumor cells into normal nonsensitized mice also reveals great differences in the tumor development. Local tumors developed in 100% C57BL/6 mice after ifp inoculation of 1 X lo4 3LL tumor cells. These number of tumor cells failed to establish tumors after iv inoculation. In order to receive tumor growth in the lungs it is necessary to increase the number of iv inoculated 3LL cells at least up to 2.5 X lo5.In contrast, B16 melanoma cells were more efficient in the initiation of tumor growth after iv than after sc transplantation (E. Gorelik, unpublished observation). These data may indicate that different antitumor mechanisms operate at the site of inoculation and in the bloodstream. Numerous investigations have shown high rates of elimination of tumor cells inoculated into the bloodstream of normal nonimmunized mice; 90 - 99% of iv inoculated cells can be eliminated from the recipients within 3 - 24 hours (Hofer et af.,1969; Fidler et al., 1978; Liotta et al., 1978). Some insight into this phenomenon was provided when Riccardi et af. (1980) showed that the levels of retained [lZSI]IUdR-labeledtumor cells in the lungs, at 4 hours following iv inoculation, correlated well with the levels of NK activity in the recipients. Strains of mice with high levels of in vitro splenic NK activity also showed high levels of elimination of iv inoculated tumor cells from the lungs. Various treatments that stimulated or inhibited the activity of NK cells also increased or decreased the elimination of tumor cells from the lungs following iv injection (Riccardi et al., 1980). The number of surviving tumor cells in the lungs determined the number of developed metastatic foci. Stimulation with interferon or interferon inducers paralleled the decrease in the number of developed artificial metastatic tumors in the mice; inhibition of NK reactivity in mice with cyclophosphamide or antiasialo GMI serum was associated with an increase in metastatic growth (Hanna and Fidler, 1980, 1981; Gorelik et af.,1982a). Tumor cells that were resistant to the cytotoxic action of NK cells in vitro following iv inoculation were eliminated rather efficiently (Riccardi et af., 1980). However, radiolabeled NK-resistant tumor cells inoculated into the foot pad persist for long periods of time and developed local tumors (Gorelik and Herberman, 1981). C57BL/6 mice bearing sc large 3LL tumor (>2 cm) showed strong resistance to reinoculation of 3LL cells ifp. However, after iv reinoculation of radiolabeled 3LL cells elimination of tumor cells from the bloodstream in these tumor-bearing mice was less efficient than in normal mice. In addition, spleen cells of these mice lost their NK activity when they were tested against YAC lymphoma cells in a 4 hour V r release assay (E. Gorelik, unpublished observation). Using different tumors it was found that the NK

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cell activity of tumor-bearing mice declined with progression of local tumor growth (Becker and Klein, 1976; Gershon, 1980; Gorelik, 1983). Therefore, the antitumor resistance of tumor-bearing or tumor-excised mice assessed by sc or iv challenge could be determined by the contribution of specific immune cells and nonspecific NK cells. During local tumor growth NK cell activity might decrease below normal level. Since NK cells are highly efficient in the elimination of tumor cells from the bloodstream, it may result in the development of more metastatic foci in the lungs of tumor-bearing mice than in normal control mice inoculated iv with tumor cells. To assess properly the resistance of mice to local or systemic (iv) challenge, involvement of the specific and nonspecific immune reactions in the destruction of renoculated tumor cells should be taken into consideration. In addition, the results of iv challenge in tumor-bearing and tumorexcised mice may be due to changes in the platelets and coagulation system of tumor-bearing mice. These changes may influence the yield of tumor nodules in the lungs after iv inoculation of tumor cells (Gasic et al., 1973; Ambrus et al, 1978; Warren, 1981). OF SPONTANEOUS METASTATIC GROWTH IN THE C. INHIBITION TUMOR-BEARING HOST

Since growth of reinoculated tumor cells can be suppressed in tumorbearing mice, it appears that the development of spontaneous metastases may be inhibited in the presence of the primary tumor. Numerous experimental data have demonstrated that removal of the primary tumor, when tumor cells have metastasized to different anatomic locations, may lead to accelerated growth. Accelerated growth of spontaneous metastases following excision of the primary tumor was described 70 years ago (Tyzzer, 1913). Although the surgical removal of the primary tumor in mice prolonged their survival, the size of developed metastatic nodules was larger than in mice bearing the primary tumor. These observations were confirmed by Tadenuma and Okonogi ( 1924). Schatten (1958a) reexamined this observation using an inbred strain of mice (DBA) and syngeneic sarcoma 49 and S91 Cloudman melanoma. Tumors were removed when they reached 1.5 cm in diameter. Twenty-one days later there was a significant increase in the number and especially in the size of metastatic foci in the lungs. Surgical trauma (amputation of a non-tumor-bearing leg) had no influence on the metastatic growth. In the next series of experiments, Schatten (1 958b) studied the number and size of metastases in the lungs 2 1 and 28 days after excision of S9 1 Cloudman melanoma. Striking differencesin the size but not in the number of pulmonary metastases were found in tumor-excised and tumor-bearing mice.

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Metastases in tumor-bearing mice remained static in size while a rapid, progressive, growth was noted in tumor-excised mice from 21 to 28 days after tumor excision. Intensive study of the postoperative development of metastases was performed by Ketcham et af.( I96 1). They transplanted five spontaneously metastasizing mouse tumors and measured metastatic development at different time intervals following removal of the primary tumor. It was found, however, that removal of the local tumor increased the size of the pulmonary metastases but not the number. In fact, tumor-excised mice showed a reduced number of postoperative lung metastases. In these experiments a special technique was applied (Wexler, 1966) that made it possible to detect very small metastases (Ketcham et al., 1961). The number of developed metastases in tumor-excised mice depended also on the size of the removed local tumor. Following removal of small 3LL tumors (4-6 mm in diameter), the number of lung metastases in C57BL/6 mice was lower than in tumor-bearing mice. However, the number of metastases increased above the number in control mice when larger tumors (8- 10 mm in diameter) were excised (Gorelik et af.,1978). Inoculation of ['251]1UdRin vivo and its indorporation into proliferating cells in the lungs allow the assessment of the number of metastatic cells proliferated in the lungs of tumor-bearing and tumor-excised mice. It was found that the number and size of metastatic tumors in the lungs and the level of [1251]IUdRincorporation are postively correlated. In addition, the level of radioactivity in the lungs may characterize the total mass of proliferating metastatic cells in the lungs (Gorelik et af.,1980).Using this approach it was found that there are three periods of local growth, each characterized by a different effect on metastatic growth. Depending on the size of local tumor removed, the level of postoperative metastatic growth may be either lower than, identical to, or higher than that in nonoperated tumor-bearing mice. When relatively small local tumors were excised, the lungs were left with very few metastatic cells in comparison to the number in the lungs of tumor-bearing mice in which the local tumor continued to shed numerous cells. Their growth at this stage exceeded the growth of the fewer cells existing in the lungs ofthe operated mice. At the next stage of medium-sized tumors, an equilibrium can be reached between the effect of suppression exerted by local tumor and the shedding of cells. Although the removal of the local tumor stopped the continuous shedding of metastatic cells, it also removed the suppressive effect of the local tumor on its metastases. The continuous supply of metastatic cells in the tumor-bearing mice resulted in a metastatic mass similar to that reached by the fewer metastatic cells in the lungs of tumor-excised mice. At these two stages, tumor-amputated mice may show fewer metastatic

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foci in the lungs, but each focus is of a larger mass. At the third stage, when the local tumor reached a larger size, more metastatic cells settled in the lungs, and they were subject to a greater suppressive effect by the local tumor. Hence, tumor excision resulted in a higher level of proliferated cells and larger metastatic masses than those in tumor-bearing mice (Gorelik et al., 1980). Suppression of metastatic growth in the tumor-bearing host and its acceleration after local tumor removal were not restricted by mouse tumor models. Similar results were obtained in hamsters bearing lymphoblastic lymphoma (Greene and Harvey, 1960). This lymphoma growing locally produced a low incidence of pulmonary metastases (10%of the animals), although tumor cells were already present in the circulation and in different organs of all mice 7 days after tumor transplantation. Surgical excision of the local tumor caused dramatic acceleration of metastatic growth, which was a function of the size of the tumor removed or the time of its growth. Metastases were not found when excision was performed prior to day 6 of tumor transplantation. Metastases were found in 49.1% of hamsters when their local tumors were excised following 8-9 days of tumor growth. The proportion of hamsters that developed metastases increased to 70.3- 100% when the amputation was performed after 10- 13 days of tumor growth. Metastases of nonoperated, tumor-bearing hamsters were small and limited to a single organ (lungs). In tumor-excised hamsters the metastases were large and widespread. It is remarkable that hamsters bearing tumor for 7 - 15 days were completely resistant to autologous transfer of blood containing circulated tumor cells. The same inoculation into normal hamsters gave tumor growth at the place of inoculation. In many experiments, control series were included; they demonstrated that accelerated growth of metastases following tumor excision was not due to stress, surgical trauma, or anesthesia (Schatten, 1958b; Ketcham et al., 1961; Greene and Harvey, 1960; Gorelik et al., 1980). These data may indicate that growth of distant metastases can be inhibited in the presence of growing tumor. Metastatid growth can be accelerated following removal of a local tumor of a critical size. This conclusion is also supported by experiments in which accelerated metastatic growth can be abrogated by retransplantation of the removed tumor (Gorelik et al., 1978). Removal of the local 3LL tumor is accompanied by an increase in the number of visible pulmonary metastases, their size, and the total weight of lungs with metastases. By reinoculation of 1 X lo6-3 X lo6 3LL cells into the second leg of tumor-amputated mice, the expected acceleration of metastatic growth was abrogated. Smaller doses of the reinoculated 3LL cells ( 1 X lo5- 3 X los) had no influence on the growth of metastases in the

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tumor-amputated mice. The latent period for tumors that developed after inoculation of small doses ( 1 X 105- 3 X 105) was too long to influence the growth of metastatic cells that settled in the lungs. After reinoculation of 1 X 106- 3 X lo6 cells, local tumors reached the size at which the inhibitory effect exerted or supported by tumor was fully expressed (Gorelik et al., 1978). Mechanisms responsible for the inhibition of the growth of metastatic cells in the presence of the local tumor could be similar to those that suppressed the growth of the reinoculated tumor cells in the tumor-bearing host. V. Is the Status of Concomitant Immunity Unique for a Tumor- Host Relationship?

Although microorganisms and parasites are antigenically different from the host and can elicit strong immunologic reactions, the immune system is unfortunately unable to control many lethal infections in humans and animals. In some respects it resembles the situation observed in animals bearing highly immunogenic tumors. Additional parallels can be found (Nelson, 1974). Organisms infected with parasites or bacteria can be resistant to a second challenge (reinfection) with the same agent. Even at the latent period of parasite or bacterial infection, the organism can display a certain degree of resistance to reinfection. This kind of bacteria - host relationship was demonstrated, for example, for Salmonella typhimurium or Salmonella enteritidis (MacKaness et al., 1966: Blanden et al., 1966). The term premunition has been used to describe the resistance of an organism in a state of latent infection against superinfection by a parasite or bacteria of the same species (Sergent, 1963; MacKaness et al., 1966; Blanden et al., 1966). Another kind of host -bacteria balance may develop at a certain stage in the progression of tuberculosis and pertussis infections. The number of bacteria inoculated into mice can drop rapidly and then reach a constant amount, which is maintained during a long period. Attempts to change this steady state were made by reinfection or by chemotherapy. Reinoculated microorganisms were quickly eliminated in the infected host. Chemotherapy may decrease the number of microorganisms, but after it is discontinued the number of bacteria returns to the previous level. This situation has been called complaisance, the “sensitive and constant readjustment of the host immune mechanisms governing the steady state” (Cheers and Gray, 1969).In tuberculosis, bacteria persisted for at least 1 year and the mice never recovered. The steady state in pertussis terminated 5 or 6 weeks after infection, and then all visible bacteria disappeared (Cheers and Gray, 1969).

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Smithers and Terry (1969) found that a state similar to concomitant tumor immunity developed in animals with schistosomiasis.Adult forms of schistosomes were transferred into normal monkeys, and 14 days later they were challenged with 2 X lo3 cercariae. The challenge proved lethal to the control monkeys. They became ill and died 8 weeks later. All experimental animals survived, and none showed signs of illness. Egg production from established adult worms persisted during the destruction of the challenge infection. The mechanism of nonrejectability of the first parasite is mostly obscure. It is unknown whether a common mechanism is responsible for the resistance of an infected organism to reinfection and the resistance of a tumor-bearing host to a second tumor challenge. VI. Mechanismsof Resistance of the Tumor-Bearing Host to a Second Tumor Graft

A. IMMUNOLOGIC MECHANISMS

Ehrlich ( 1906) proposed the hypothesis of athreptic immunity to explain the poor growth of reinoculated tumor cells in tumor-bearing animals. According to this hypothesis nutrient factors rather than immunologic factors are responsible for this resistance. Depletion of the essential nutrient substances by growing tumor makes it impossible to support the growth of a second tumor graft. This hypothesis, which had no experimental support, was soon rejected by Bashford et al. ( 1908). It was suggested that immunologic mechanisms rather than nutrient mechanisms are responsible for growth inhibition of a second graft. This antitumor resistance of tumorbearing recipients was termed concomitant tumor immunity. What was the experimental basis for this hypothesis? Mostly the immunological nature of the resistance to a second inoculum was deducted from the observations that the protection was greatest in the mice or rats with small slowly growing tumors. Animals with quickly growing tumors were less resistant to a second challenge. Keeping in mind that all of these experiments were performed using outbred animals and allogeneic tumors, it is understandable that these tumors induced strong immune response. Some tumors grew in spite of a developed immune response. However, reinoculated tumor cells were destroyed. The immunologic nature of the resistance of the tumor-bearing host to a second tumor graft was further supported by several investigations. Resistance of the tumor-bearing animals to a second tumor challenge was in parallel with the immunological activity of the spleen, lymph node, or peritoneal exudate cells tested in a neutralization assay (Gershon et al., 1967; Bard et al., 1969; Gershon and Kondo, 1971; Deckers et al., 1971). Decline of antitumor resistance to a second challenge in mice bearing a large

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tumor mass was observed when the spleen cells of these mice lost their antitumor activity (Howell et al., 1975). Resistance to a second challenge can be observed in mice bearing a highly immunogenic tumor or after its removal (Gershon and Kondo, 1971; Fisher et al., 1970; Zarling and Tevethia, 1973). Therefore, highly immunogenic tumors induce an immune response, which is unable to prevent growth of the initial tumor but can be effectivein the elimination of the reinoculated tumor cells. At the established phase the immune response induced by immunogenic tumors did not require the concomitant presence of the growing tumor. Based on the experiments performed by North and Kirstein ( 1977) and Berendt et al. ( 1978), it is possible to suggest that for immunogenic tumors such as SA 1 and Meth A, T-cell-mediated immunity is mostly responsible for the resistance of tumor-bearing mice to.a second tumor challenge. In T-cell-depleted B mice bearing SA 1 or Meth A tumors, the level of antitumor resistance substantially decreased, but did not disappear. These data may also indicate that additional mechanisms could also be involved in the inhibition of the second tumor growth in T-cell-depleted mice. In addition, this notion can be supported by the observation that mice bearing an SA 1 tumor showed nonspecific resistance to a nonrelated BP3 tumor (North and Kirstein, 1977). What kind of mechanisms could be responsible for the resistance to a second tumor challenge in mice bearing nonimmunogenic or weakly immunogenic tumors? Spleen cells and lymph node cells of these mice have no detectable level of antitumor cytotoxic activity measured in vivo or in vitro. Resistance to a second tumor graft in mice bearing weakly immunogenic tumors was observed only in the presence of the growing tumors and disappeared following their excision (DeWys, 1972; Kearney and Nelson, 1973;Gorelik, 1983).In T-cell-depleted B mice or in nude mice bearing 3LL or M 109 tumors, the level of the resistance to a second tumor challenge was high and was similar to that observed in immunologically competent tumor-bearing mice (Gorelik et al., 1981b; Gorelik, 1983). In addition, the resistance of mice bearing weakly immunogenic tumors to a second tumor challenge was tumor nonspecific (Kearney and Nelson, 1973;Gorelik et al., 1981b). These data may indicate that resistance to a second challenge of mice bearing weakly immunogenic tumors is not mediated by T-cell immunity or that its contribution to this resistance is relatively low. Meanwhile, there are no data demonstrating that tumor-nonspecific, activated macrophages or NK cells are responsible for this resistance in tumor-bearing mice. It was found that at the latest stages of tumor growth, the functions of the immune system, including those of NK cells and macrophages, are suppressed (Nelson and Kearney, 1976; Otu et al., 1977; Gershon, 1980). Using NK deficient beige mice, or mice in which NK and

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macrophage functions were suppressed by silica, it was found that these mice bearing sc 3LL tumor suppressed the growth of reinoculated 3LL cells (Gorelik, 1983). Inhibition of a second tumor graft growth in the tumorbearing mice can result from the active elimination of the reinoculated tumor cells by host immune cells or their inability to proliferate. This question was analyzed using the radioisotope methods that allowed investigation of the fate of radiolabeled tumor cells at the local site of their inoculation (Gorelik and Herberman, 1981; Gorelik et al., 198la). BALB/c +/+ or BALB/c nude mice bearing sc syngeneic spontaneous Madison lung carcinoma (M 109) were reinoculated ifp with 2 X lo5 [1251]IUdR-labeled M 109 cells. Intact BALB/c +/+, BALB/c nude mice, and allogeneic C57BL/O and CBA/J mice received the same amount of radiolabeled M I09 tumor cells. The level of radioactivity in the foot pad of mice was determined at different periods after tumor cell inoculation (Fig. 1). No differences were found in

DAYS AFTER INOCULATIONOF RADIOLABELED TUMOR CELLS

FIG. I . Elimination of radiolabeled MI09 tumor cells from the foot pad of intact and tumor-bearing mice. Intact syngeneic BALB/c +/+ (El), BALB/c nude (A), allogeneic C57BL/6 (O), and CBA/J (0)mice were inoculated ifp with 2 X lo5 [12sI]IUdR-labeledMI09 tumor cells. Radiolabeled MI09 tumor cells (2 X lo5)were also reinoculated ifp in BALB/c +/+ (B) or BALB/c nude (A)mice bearing sc MI09 tumor ( 2 2 cm in diameter). At various periods after inoculation of radiolabeled cells the level of radioactivity remaining ifp was measured.

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the clearance of radioactivity in BALB/c +/+ and BALB/c nude mice bearing tumors or in non-tumor-bearing mice. The similarity in the levels of radioactivity remaining in the foot pads of these mice may indicate that T cytotoxic cells, NK cells, macrophages, or other potential cytotoxic factors were not active or that they were equally efficient in tumor-bearing and non-tumor-bearing and in immunocompetent and T-depleted nude mice. The potential efficiency of immune reaction was supported by the elimination of radiolabeled M109 cells in allogeneic C57BL/6 and CBA/6 mice (Fig. 1). Although the levels of radioactivity in the foot pads of BALB/c +/+ and BALB/c nude mice bearing and nonbearing sc tumor were similar, striking differences were found in cell proliferation. In intact BALB/c +/+ and BALB/c nude mice, ifp tumors appeared and grew progressively, but the growth of reinoculated M 109 tumor cells was strongly suppressed in BALB/c +/+ and BALB/c nude mice bearing sc M109 tumors (Fig. 2). Thus, proliferation of reinoculated cells is strongly inhibited in the presence of the growing primary tumor. Using immunogenic Meth A tumor, it was found that in tumor-bearing mice the clearance of radiolabeled Meth A cells from the foot pad was higher, and fewer tumor cells survived than in non-tumor-bearing mice. In addition, growth of reinoculated cells in the tumor-bearing host was suppressed. In mice bearing large Meth A tumors (>2-3 ) there was more efficient suppression of the growth of the second tumor graft than in mice bearing small (21 cm) Meth A tumors (Gorelik, 1983).

9.0

[

T

6

10

14

18

22

26

30

DAYS AFTER if p INOCULATION OF TUMOR CELLS

FIG. 2. Resistance of tumor-bearing mice to the second tumor challenge. Growth of radiolabeled M 109 tumor cells (2 X lo5)inoculated ifp in intact BALB/c +/+ (0)or BALB/c nude (A) or tumor-bearing BALB/c +/+ (m) or BALB/c nude (A) mice.

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Therefore, data demonstrated that resistance of tumor-bearing mice to a second tumor graft was mediated by immunologic and nonimmunologic mechanisms. Immunologic protection was greater in mice bearing highly immunogenic primary tumors. Involvement of nonimmunologic mechanisms in the suppression of the reinoculated cell growth was more likely for mice bearing weakly immunogenic or nonimmunogenic tumors. OF THE INHIBITION OF A SECOND B. NONIMMUNOLOGIC MECHANISMS IN THE TUMOR-BEARING ANIMALS TUMORGRAFTGROWTH

1. Nutrient Factors

Since Bashford et al. ( 1908) proposed that immunologic mechanisms may be responsible for resistance of the tumor-bearing host to a second tumor challenge, Ehrlich’s hypothesis of athreptic immunity has not been considered seriously. However, there is evidence indicating that Ehrlich was not completely wrong. First, a great body of data exists clearly demonstrating that nutrient restriction, even in healthy mice, may be accompanied by strong inhibition of the development of spontaneous or induced tumors, as well as the growth of transplanted tumors (Rous,, 1914; Bischoff and Long, 1938; Tannenbaum and Silverstone, 1952; Jose, 1979; Giovanella et al., 1982). Food restriction, although capable of inhibiting tumor growth, does not prolong, and, if anything, shortens the life span of the host (Giovanella et al., 1982). Nutrient deficiency paralleled weight loss and tissue atrophy, whereas the tumor mass progressively increased. Although weight loss occurred during starvation of the healthy animals or during tumor growth, it is possible that weight loss is due to different mechanisms, since the metabolic rate is depressed during starvation, whereas in cachectic tumor-bearing animals it is increased (Bondy, 1976). Additional support for the Ehrlich hypothesis of athreptic immunity can be obtained from the results of the investigation of the biochemistry of malignant cells and metabolic and hormonal changes in the different organs and tissues of tumor-bearing hosts. In contrast to local growth of benign tumors growth of malignant tumors is accompanied by various systemic effects on the functions of different organs and tissues in the tumor-bearing organism. This systemic influence of the growing tumors can be a result of (1) their production of different physiologically active substances and (2) changes in the metabolism and hormonal balance of the host caused by successful competition of the neoplasm with the normal tissues for vitally important metabolites and trophic factors (Mider, 195 1; Greenstein, 1954; Waterhouse, 1974; Gold, 1974; Shapot, 1979). There is a abundant evidence that tumor is a trap for glucose and nitrogen. An extremely high rate of glucose uptake by cancer

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cells resulted in very high consumption of glucose. Thus, the level of glucose in tumor tissue remained undetectable (Gullino et al., 1967; Shapot, 1979). This created an enormous glucose concentration gradient between the blood and the tumor (80 mg/100 ml versus zero). Such a gradient favors successful competition of the tumor with the host tissues (Shapot, 1979). Tumor growth is accompanied by losing of glycogen from the liver and muscle. Tumor cells not only consume the amino acids of the host’s tissue proteins but also enhance protein catabolism of the host tissues, and, at the Same time, hinder protein synthesis in them (Lundholm, 1975; Clark and Goodlad, 1975; Shapot, 1979). Tumor cells compete as successfully with normal cells for the precursors of nucleic acid (Ferdinandis et al., 197 1 ; Shapot, 1979). In this competition for vital substances tumor cells have the advantage, and an increase in tumor mass is observed at the expense of the host tissues. Rats bearing Flexner carcinoma for 5 days lost 3 1Yo of their weight and 39% of live protein. In contrast, tumor protein increased by 345% (Le Page et al., 1952). Tumor growth was accompanied not only by the disturbance of carbohydrate, protein, and lipid metabolism, but also by impairment of enzyme function in the host tissues and disturbance of the endocrine regulation. In the tumor-bearing organism, no tissue functions normally (Greenstein, 1954; Shapot, 1979). It appears that in severe biochemical disturbance the conditions for proliferation of reinoculated tumor cells cannot be as favorable as they are in the intact control animals. Tumor growth depends on the stroma formation and blood supply that may be crucial to competition of the reinoculated tumor cells for nutrient substances with an already established and progressively growing primary tumor. However, inhibition of the stroma formation cannot be considered as a main reason for suppression of the second tumor graft growth. Ascitic tumor cells grow without stroma. Nevertheless, growth of ascitic RL 6 1 tumor cells in the peritoneal cavity of mice bearing im M 109 tumor was dramatically suppressed (Gorelik, unpublished observation). Metabolic disturbances and development of cachexia increase with increase of the tumor mass. In several experiments, growth inhibition of reinoculated tumor cells was more efficient in mice bearing large tumors (Kearney and Nelson, 1973; North and Kirstein, 1977; Gorelik et al., 1981b). Nude mice or silica-treated mice developed cachexia very quickly in the presence of growing primary tumors and growth of reinoculated tumor cells was dramatically inhibited (Gorelik, 1983). Weight loss or cachexia may not be observed at the moment of reinoculation oftumor cells into the tumor-bearing host, but they can develop later, especially if the size

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of the second inoculum i s rather small and requires a long latent period for development of a visible tumor. The probability that inhibition of tumor growth results from a lack of nutrient substances can be supported by the data obtained during experimental and clinical attempts to correct the malnutrition of cancer patients or tumor-bearing animals. All attempts to correct the weight loss in tumorbearing organisms by supplying different nutrient substances intravenously resulted in acceleration of tumor growth. Carbohydrate calories alone produced no significant improvement in nutritional status and did not significantly stimulate tumor growth. Amino acids may improve nitrogen balance and increase the rate of tumor growth. Provision of adequate amino acids with carbohydrate calories further increases the rate of tumor growth (Cameron and Roger, 1977;Oram-Smith et al., 1977; Lowry et al., 1978; Buzby et al., 1980). 2. Antimitotic Factors The suppression of the growth of the second tumor inoculum may result from the production or induction by the tumor of a different kind of antimitotic inhibitor that may block the proliferation of the reinoculated tumor cells (DeWys, 1972; Sugarbaker et al., 1977; Gorelik et al., 1978, 1981b). The idea that the tumor induced the systemic effect by production of some kind of toxin was supported by Greenstein (1954) and especially by Nakahara and Fukuoka ( 1958) in their concept of cancer toxohormone. Although there is no direct evidence for the production of some kind of cancer toxins, tumor cells did produce different substances with various physiological activity (enzymes, ectopic hormones, prostaglandins, oncofetal or placental proteins). The substance produced during tumor growth may influence the growth of the second implanted graft. It has been suggested that the tumor produces antiproliferative factors that may suppress the multiplication of the reinoculated tumor cells (DeWys, 1972; Sugarbaker et al., 1977; Gorelik et al., 1978). Several substances that have an inhibitory effect on protein or DNA synthesis were found in ascitic fluid, serum of tumor-liearing animals, and supernatants of the cultivated tumor cells or were extracted from tumor cells (Sylven and Holmberg, 1965: Rounds, 1971;Bichel et al., 1975; Urusizaki, 1976). In addition, increased amounts of polyamines, prostaglandins, and cyclic nucleotides also may participate in the inhibition of the tumor cell proliferation (Heby et al., 1975; Harris et al., 1975; Honn et al., 1981; Pastan and Johnson, 1974; Heby, 1981; Droller, 1981). Another kind of antimitotic agent, chalones, may regulate cell proliferation (Bullough, 1977).Chalones are tissue-specific mitotic inhibitors, which

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are glycoproteins of molecular weight between 10,000 and 30,000. There is evidence supporting the existence of chalones for epidermis, melanocytes, fibroblasts, and some hemopoietic tissues. Their action appears to arrest replicating cells in either the G, or G2 phase of the cell cycle. Tumor cells contain a severely reduced concentration of chalones caused by an abnormally high rate of chalone loss into the circulation. With increased tumor mass the systemic chalone concentration should rise. This might result in a progressive inhibition of the mitotic activity of tissue-specific normal or malignant cells (Bullough, 1977). Inhibition of tumor growth can be achieved in experimental conditions using a certain histologic type of chalone (Bullough, 1977). Proliferation of 3LL cells in diffusion chambers implanted in the abdominal cavity of mice bearing im local 3LL tumor was significantly inhibited. This inhibitory effect disappeared after surgical excision of the local 3LL tumor. This effect was specific, since the proliferation of Ehrlich ascites tumor cells in the diffusion chamber was similar if these chambers were implanted in normal or 3LL-bearing mice. Tissue-specific challones are considered a factor responsible for this antiproliferative effect in tumorbearing mice (Oh1 et al., 1980). In recent years, numerous growth factors have been purified (e.g., epidermal, nervous, and T cell and B cell growth factors). The existence of different mitotic and antimitotic factors is re. quired for regulation and control of tissue and organ cellular homeostasis in a multicellular organism. The balance between cell death and renewal in different histologic types of tissue or organs should be maintained by a special control mechanism. The presence of numerous highly proliferative malignant cells may stimulate the specific feedback control mechanisms directed at the restriction of their multiplication. Susceptibility to these control mechanisms probably was not lost completely by malignant cells. Sugarbaker et al. (1977) suggested that the inhibitory effect exerted by growing tumor may have (1) a local inhibitory effect on the growth of solid, or ascites tumors with exponential slowing of the tumor growth rate as tumor size increases, (2) a systemic inhibitory effect on the growth of metastases or second tumor implant, and (3) a systemic inhibitory effect on the proliferation of normal cells with depression of their function and development of cachexia. Retardation of tumor growth with increased tumor size may have resulted from deterioration of the blood and nutrient supply required for tumor cell proliferation. However, there are several indications that this retardation of tumor growth started before nutritional deficiencies occurred (Sylven and Holmberg, 1965; Bichel, 1972; Hams et al., 1975; Gordon, 1979). According to Laird (1964) retardation of tumor

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growth appears to be an actively increasing depression of the specific growth rate, rather than a passive, preset limitation imposed by exhaustion of the available growth-supporting factors in the environment. Inhibitory effects exerted by growing tumor might be an additional mechanism involved in the suppression of the growth of reinoculated tumor cells or spontaneous metastasizing tumor cells. Another aspect of the “inhibitory hypothesis” is concerned with the influence of the antimitotic factors elicited by growing tumor on the proliferation of normal cells. Mitotic inhibitors produced or induced by tumor may block turnover of cells in normal tissues and organs. This may result in the dysfunction of these organs and tissues and in the development of numerous systemic effects and may cause the death of tumor-bearing hosts (DeWys, 1972; Urushizaki, 1976; Sugarbaker et al., 1977). There have been only a few studies on the kinetics of normal cell renewal in the tumor-bearing host. There was no suppression of the renewal of jejunal epithelium of hamsters bearing fibrosarcoma, duodenal crypt cells in mice bearing Krebs tumor, or bone marrow cells in mice bearing Ehrlich ascites carcinoma (Betts et al., 1966; Lala, 1972). However, tumors in these experiments were relatively small and dynamic studies of normal cell renewal during tumor growth were not performed. Although there is poor information concerning the inhibitory effect of tumor-derived products on normal cell proliferation and function, abundant evidence exists that numerous factors which are produced by the tumor-bearing host or released by tumor cells may interfere with the lymphoid system. Repeatedly observed data demonstrated the development of severe immunosuppression in both man and experimental animals during tumor growth. Numerous factors that are released by, or can be obtained from, tumor cells were efficient in the in vivo or in vitro suppression of different immunologic functions [T- and B-cell-mediated immune responses, macrophage functions, MLC, GVH, and proliferative response to antigens or mitogens (Kano and Friedman, 1977)]. In this review, Kano and Friedman presented a summary of 23 immunosuppressive factors isolated from tumor cells or tumor-bearing individuals (man and animals). Some of these factors strongly suppress proliferation of lymphocytes stimulated by mitogens (Kano and Friedman, 1977). Are all of these factors specific for suppression of proliferation of lymphoid cells? It is quite possible that some of the factors isolated from tumor cells or the tumor-bearing host may have an antiproliferative effect on the lymphoid and nonlymphoid cells. In this case, it seems likely that such factors may exert an inhibitory effect on the proliferation of reinoculated tumor cells in the tumor-bearing host.

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In spite of the attractiveness of these suggestions, direct experimental data are required to clarify the role of nutrient and inhibitory factors in the suppression of the growth of reinoculated tumor cells. VII. Mechanisms of the Inhibition of Metastases Development by the Primary Tumor

Mechanisms of the inhibition of metastatic growth in the presence of the primary tumor may be similar to those that suppress the growth of reinoculated tumor cells. The following findings support the concept that immunologic mechanisms stimulated by the local tumor participate in the suppression of metastatic growth. 1. Immunogenic tumors have poor metastatic potential in comparison with nonimmunogenic tumors (Kim, 1979). 3LL tumors can grow in allogeneic mice; however, development of pulmonary metastases was completely prevented in these mice. Incompatibility in the H-2Db complex and in some non-H-2-coded antigens is responsible for the prevention of the formation of metastases. In immunosuppressed mice, the metastatic growth in the lungs occurred in allogeneic mice bearing 3LL tumor (Isakov et al., 1981). 2. Different immunosuppressive procedures may result in an increase in the development of metastases in various anatomic locations, and immunostimulation may prevent the formation of metastases (Fidler et al., 1978). 3. Immune response elicited by the primary tumor participates in the immunoselection of the metastatic cell variants, which could be antigenically different from the cells of the primary tumor (Sugarbaker and Cohen, 1972; Fogel er al., 1979; Gorelik et al.. 1980, 1982b).

Specific T-cell-mediated immune reaction can be efficient in the prevention of metastatic growth only in mice bearing immunogenic tumor (Fidler et al., 1979; Fidler and Kripke, 1980). However, nonspecific immune reactions mediated by NK cells or macrophages can be the additional mechanisms in antimetastatic defense (Alexander, 1976; Fidler er al., 1978; Gorelik et al., 1979, 1982a; Hanna and Fidler, 1980, 1981). Nonimmunologic mechanisms are also involved in the suppression of metastatic growth. In immunosuppressed mice (T-cell-depleted B mice or cyclophosphamide-treated mice), the development of spontaneous pulmonary metastases from 3LL tumor dramatically increased. However, this growth of metastases was not optimal and can be further accelerated in immunosuppressed mice by excision of the primary tumor (Gorelik et al., 1980). Using immunogenic and nonimmunogenic tumors, the acceleration of metastatic growth was observed after removal of the primary tumor

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(Schetten, 1958a,b; Greene and Harvey, 1960; Ketcham et al., 196 1; Gorelik et d.,1978, 1980). It has been suggested that suppression of metastatic growth can result from the inhibitory influences exerted by the primary tumor (Schatten, 1958b; Ketcham et al., 1961; Sugarbaker et al., 1978; Gorelik et al., 1978, 1980). It is also difficult to exclude the possibility that deterioration of the nutrient supply and metabolic disturbance may cause the poor growth of metastatic cells in various organs of tumor-bearing host (see Section V). Therefore, immunologic and nonimmunologic mechanisms could be involved in the inhibition of metastatic growth in the presence of the primary tumor mass. VIII. Conclusion and Summary

Concomitant tumor immunity is considered as a synonym for the immunity of tumor-bearing mice in contrast to the immunity of immunized mice. Indeed, growth of immunogenic tumors is accompanied by the development of a specific immune response and by the production of immune lymphocytes or humoral antibodies that can be determined by different in vivo or in vitro techniques. Antitumor immune response was assessed by the level of resistance of tumor-bearing or immunized mice to a second challenge with tumor cells. Resistance of the tumor-bearing mice to a second tumor inoculum is considered to result from concomitant tumor immunity. From the immunologic point of view the presence of the antigenic material in increasing amounts is not required to maintain the immune reactions. Surgical removal of the growing tumor after development of antitumor response leaves these mice immune to the second tumor challenge, and immune cytotoxic cells in these mice can be detected. On the other hand, it is well known that the depression of antitumor reactions (eclipse phenomenon) can develop with the increase of tumor mass. After removal of the growing tumor the immune reactivity can be restored. It was found that mice bearing a nonimmunogenic huge tumor mass in the absence of a detectable amount of cytotoxic cells or humoral antibodies showed a high level of resistance to a second tumor challenge. An increasing body of evidence indicates the possible involvement of nonimmunologic mechanisms in the resistance of a tumor-bearing host to a second tumor challenge. Therefore, it is necessary to consider separately the mechanisms of this resistance in mice bearing immunogenic and nonimmunogenic tumors. The term concomitant tumor immunity is relevant for highly immunogenic tumors. This immunity can be antigen specific, can be mediated by immune lymphocytes, and can be expressed in the presence of a growing tumor or after its removal.

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Resistance to a second tumor graft in mice bearing weakly immunogenic or nonimmunogenic tumors has another characteristic. This resistance was observed only in the presence of a growing tumor and disappeared after its removal, it can be tumor nonspecific, and it can be observed in the immunologically deficient or immunosuppressed host. Therefore, the resistance of mice bearing weakly immunogenic or nonimmunogenic tumors is probably largely mediated by nonimmunologic mechanisms, although some contribution of immune mechanisms cannot be excluded. What kind of nonimmunologic mechanisms can be responsible for the prevention of the growth of reinoculated tumor cells? Substantial metabolic disturbances in the different tissues and organs occurred during tumor growth. It was also accomplished by disturbances in hormonal and enzyme production and regulation. The tumor is able to obtain all its requirements from the blood and to grow rapidly when the host is fasted and forced to maintain the blood constituents by catabolism of normal tissues. Hence, in these conditions the growth of reinoculated tumor cells cannot be optimal. In addition, there are numerous experimental data indicating that the tumor may produce or induce some substances with antimitotic activity. Several mitotic inhibitors were found in the ascites fluid, serum of tumor-bearing mice, supernatant of cultivated cells, or extracted from tumor cells. The possible antiproliferative effect of these antimitotic factors on the reinoculated tumor cells is considered. Using weakly immunogenic tumor it was shown that suppression of the second tumor graft growth in tumor-bearing mice was not associated with rejection of the reinoculated tumor cells, but was due mostly to the prevention of their proliferation. Since the resistance of tumor-bearing mice to a second tumor challenge may not be caused by an immunologic mechanism this experimental model cannot be applied for the assessment of antitumor immune response in tumor-bearing mice. The contribution of the immunologic mechanisms in this resistance should be specifically assessed. Involvement of the different mechanisms in the resistance of tumor-bearing animals to a second tumor graft may explain the obvious contradictions of the results obtained during investigations of this phenomena. Suppressivegrowth of reinoculated tumor cells in the tumor-bearing host simulates the situation in which the growth of metastatic tumor cells is sup€ essed in the presence of the growing primary tumor. Numerous experimental data demonstrated that the surgical excision of the primary tumor removes the inhibitory influence and accompanies the acceleration of metastatic growth. Similar mechanisms might be responsible for inhibition of the growth of spontaneous metastases or tumor cells reinoculated into tumor-bearing host.

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The primary tumor is a source of the metastatic cells. From the other side it may induce the immune reactions which can eliminate or suppress the growth of the metastatic cells. In addition, proliferation of metastatic cells in different organs and tissues can be impaired by metabolic and hormonal changes occurring in tumor-bearing organisms or actively suppressed by inhibitory factors which may be produced or induced by growing primary tumor. Depending on immunobiological properties of malignant cells the contribution of immunologic and nonimmunologic mechanisms in this inhibition could be different. Further investigations of the mechanisms of these phenomena can contribute to understanding the mechanism of malignant disease and developing effective methods for treatment of cancer patients.

ACKNOWLEDGMENTS I am particularly indebted and most grateful to Dr. Maury Rosenstein, Dr. Steven Keller, and Mrs. A. McCarthy for extensive editorial comments and corrections. The assistance of Ms. D. Logan in preparing the manuscript is acknowledged with thanks.

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ANTIGENIC TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS Thierry Boon Ludwig Institute for Cancer Research. Brussels Branch. Brussels, Belgium and Universite Catholique de Louvain. Louvain, Belgium

I. Introduction.. ............................................................ 11. Evidence for the Production of Tumor Cells with Increased Immunogeni Mutagen Treatment ................................................. Ill. Immunogenic Variants Obtained in Vitro with Nitrosoguanidine De MNNG .................................... .................................. A. Immunogenic “turn-” Variants ............................................... B. Presence of New Antigens on tum- Variants.. ................................ C. Protection against initial “turn+” Cells. ....................................... IV. Immunogenic Tumor Cell Populations Obtained in Vivo with Triazenylimidazole Derivative DTIC ............................................. A. Production of Immunogenic Lines.. .......................................... B. Presence of New Antigens on DTIC Lines .................................... C. Protective Effects of the DTIC Lines and Association with Chemotherapy.. .. V. Discussion.. ...................................................................... A. Nature of the Antigenic Changes Induced by Mutagens.. ..................... B. Mechanism of the Protection against the Original Tumors.. .................. C. Application to Human Cancer.. .............................................. References ........................................................................

121 123 125 125 127 134 136 136 138 I40 141 141

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Despite vast effort aimed at demonstrating tumor-specific antigens, it is still not possible to state -or deny- that such antigens are generally present on human and animal cancer cells. For human cancer, extensive serological analysis of melanomas and other tumors has revealed antigens whose presence appears to be restricted to single tumors (Carey et al., 1976; Shiku et al., 1976;Pfreundschuh et al., 1978; Ueda et al., 1979;Carrel et al., 1980). It appears, therefore, that some human tumors carry tumor-specific antigens. However, it remains difficult to know to what extent these antigens function as transplantation antigens that elicit a rejection response by the host; one important exception is Burkitt’s lymphoma, against which autochthonous specific killer T cells have been observed (Jondal et al., 1975; Klein and Klein, 1977). In the mouse, it has been clearly demonstrated that experimental tumors induced with oncogenic viruses or with polycyclic hydrocarbons like methylcholanthrene carry tumor-specific transplantation antigens (Gross, 1943; Prehn and Main, 1957; Klein et al., 1960; Klein and Klein, 1964; Prehn, 121 ADVANCES IN CANCER RESEARCH,

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1976). Methylcholanthrene-induced tumors carry tumor-associated antigens that differ for each tumor (Basombrio, 1970). Cytolytic T cells have been demonstrated that are directed specifically against the tumor-associated antigens of virus or carcinogen-induced tumors (Leclerc et al., 1972; Wagner and Rollinghoff, 1973). However the rather encouraging view of tumor immunology and immunotherapy that resulted from the study of these tumors was counteracted by the observations of Hewitt et al. ( 1976) who found no evidence for immunogenicity of 26 spontaneous mouse tumors and who questioned the relevance to human cancer of animal tumor models obtained with viruses or carcinogens (Hewitt, 1978). Spontaneous rat tumors were also found to have little or no detectable immunogenicity (Baldwin, 1966; Middle and Embleton, 1981). Since it is evident that even when rejection responses directed against tumor-specific antigens do occur they are usually too weak to eliminate the tumor cells effectively, the enhancement of these responses has been one of the main objectives of tumor immunology. Among the efforts in this direction, many have involved nonspecific immunostimulation, direct manipulations of the immune system, or immunization with tumor cells which have been modified so as to increase their immunogenicity. A large number of reports have described stimulation of antitumoral responses with nonspecific immunostimulatory agents such as Bacillus Calmette-Guirin (BCG) and Corynebacterium pawum (see reviews by Milas and Scott, 1978, and Baldwin and Pimm, 1978). There is no doubt that for a number of experimental tumors that elicit some degree of a rejection response, these agents often improve the response significantly (Old et al., 1961; Rapp, 1976; Dye and North, 1981). However, a large number of clinical trials have failed to show indisputable therapeutic benefit, perhaps because in most instances of human cancer there is little rejection response to be amplified. Recent advances in the understanding of the regulatory mechanisms of the immune system have lead quite logically to attempts at increasing the antitumoral responses by manipulating regulatory lymphocytes. Inhibition of tumor growth has been observed after treatment of tumor-bearing mice with anti-I-J antibodies reported to affect preferentially suppressor T cells (Greene et al., 1977; Perry et al., 1980).Other results suggest that significant tumor inhibition may be obtained by specific removal of the relevant suppressor T cells by antiidiotypic immunization (Tilkin et al., 198I). Many observations indicate that tumor cells that have been modified so as to acquire new surface antigens often produce better immunity than the original tumor cells (see review by Kobayashi, 1982). Viral infections have been used to produce these additional antigenic determinants. Extracts of mouse tumor cells infected with lytic viruses such as influenza or Newcastle

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

123

disease virus were found to immunize more effectively than extracts of uninfected tumor cells (Lindenmann, 1964; Lindenmann and Klein, 1967; Boone and Blackman, 1972). Viable rat tumor cells infected with the nonlytic Friend virus conferred a better protection against the original tumor than irradiated tumor cells (Kobayashi et al., 1969, 1970, 1975). Another procedure for the addition of new antigens involves somatic hybridization of the tumor cells with allogeneic cells. Enhanced immunogenicity has been observed after injection of such living or X-irradiated hybrids (Kim, 1979; Klein and Klein, 1979). Finally, by coupling chemical groups such as trinitrophenyl to the surface of tumor cells, responses superior to those obtained with the original cells have been obtained (Galili et al., 1976; Fujiwara et al., 1980). We intend to review evidence that mutagenic treatment may represent an additional way to increase the immunogenicity of tumor cells. As will be shown below, tumor cell populations that have been in contact with some mutagens contain stable variants with increased immunogenicity. Most of these variants carry new transplantation antigens. II. Evidence for the Production of Tumor Cells with Increased lmmunogenicity by Mutagen Treatment

Tumor cell lines maintained in vitro sometimes undergo a reduction in their transplantability (Hsu, 1960). It is often unclear whether these unprovoked changes in malignancy are caused by an alteration in the transformed state of these tumor cells or whether they are caused by changes affecting the host - tumor relationship and particularly the antitumoral immune response. A number of independent observations indicate that the latter changes may frequently occur in tumor cells that have been in contact with mutagenic agents. In vitro treatment with mutagens was found to increase the immunogenicity of tumor cells. Koyama and Ishii ( 1969)treated with the mutagenic and carcinogenic compound nitroquinolin- 1-oxide the tumor cell line FM3A/B derived from a spontaneous mouse adenocarcinoma. They cloned the surviving cell population in agar and found that 2 out of 25 clones had become unable to form tumors in syngeneic mice at a dose that was 10-fold higher than the tumor-forming dose of control clones. No such clone was observed among 100 untreated clones. However, it was reported later that the transplantability of the tumor line FM3A/B decreased after long-term culture and that when such a culture was cloned, 1 out of 13 clones examined had a strongly reduced tumorigenicity (Hozumi and Nakamura, 1970). It therefore remains in doubt whether the two initial nontumorigenic variants were the result of the mutagenic treatment. These variants multi-

124

THIERRY BOON

plied in vitro at the same rate as the original FM3A/B cells. Interestingly,the mice that had been injected with living variant cells and had rejected them were partially protected against the original tumor cell line, whereas no protection was observed after injection with irradiated variants. Tsukagoshi and Hashimoto ( 1973)reported that rat sarcoma cell populations that had become resistant to nitrogen mustard after repeated exposures in vitro lost their tumorigenicity in syngeneic rats. Regressions were observed after visible tumor growth. Moreover,these resistant tumor cells were still capable of forming progressivetumors in animals that had received 300 rads of X irradiation. These observations suggested that an immune rejection process was affecting these cells. Boon et al. (1 975) and Boon and Kellerman ( 1977)treated a malignant mouse teratocarcinoma cell line with the mutagen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), with the purpose of obtaining from this pluripotent cell line some variants with altered differentiation properties. They obtained a high frequency of stable variants (tum-) that failed to form tumors in the syngeneic mice. These variants were capable of forming tumors in irradiated animals. In an attempt to obtain variants with reduced metastatic capacity, Kerbel (1979; Kerbel et al., 1981) selected from mouse sarcoma MDAY-D2 variants that were resistant to the lectin wheat germ agglutinin. These variants were obtained after in vitro treatment of the tumor cells with ethylmethane sulfonate, under conditions in which most cells were killed by the mutagen. Two out of 18 lectin-resistant variants were found to be nontumorigenic even at doses that were 10,000-fold higher than the minimal tumorigenic dose of the original cells. The variants multiplied in vitro as rapidly as the tumorigenic cells. They formed progressive tumors in nude mice and in mice that had received 600 rads of X irradiation. Tumors grew but regressed in mice that had received 250 rads. The nontumorigenic variants were found to elicit a much higher cytolytic T-cell activity than the original MDAY-D2 (Dennis et al., 1981). The authors discussed the possibility that the loss of tumorigenicity of these variants could be due to the mutagen treatment rather than to lectin resistance. Resistance to antineoplastic drugs has been shown to coincide occasionally with an increase in the immunogenicity of the tumor cells. Mihich (1969)pointed out that this could explain the phenomenon called collateral sensitivity whereby tumor ceIls that have become resistant to ohe chemotherapeutic agent show an increased sensitivity to other chemotherapeutic drugs in viva He showed that a line of mouse leukemia L12 10 cells that was resistant to methylglyoxalbisguanylhydrazone had acquired an increased sensitivity to arabinosylcytosinein normal but not in irradiated mice. This resistant subline and two other L12 10 lines resistant to other antineoplastic drugs were found to elicit the synthesis of antitumoral antibodies more

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

125

readily thanL12lO(Kitanoetaf., l971,1972;FujiandMihich,1975;Fuji et af., 1977). Whereas initial studies with the first resistant lines suggested that they carried specific antigens that were not present on L1210, subsequent work indicated that the increase of immunogenicity of these resistant cell lines was mainly due to a higher surface density of an antigen already present on L1210 (Fuji et af., 1979). Recent evidence indicates that this antigen is related to a product of mammary tumor virus (Strzadala et af., 1981). Similar results have been obtained by Nicolin et af. ( 1972)who examined a large number of L 1210 cell lines that were resistant to various antineoplastic agents and found that many of them had acquired increased immunogenicity resulting in longer survival time of the host after chemotherapy and increased production of antibodies. These results could be explained by the selection of preexisting immunogenic drug-resistant variants favored by the immunosuppressioncaused by the relevant antineoplasticdrugs (Fuji et af., 1979).Alternatively, since many antineoplasticdrugs have mutagenic properties, the increase in immunogenicity changes may be a direct consequence of the mutagenic action of these drugs. Bonmassar et af. (1970) reported that L1210 sublines that had been transplanted repeatedly in mice in the presence of the antineoplastic drug 5 4 3,3-dimethyl-l-triazeno)imidazole-4-carboxamide(DTIC) became progressively unable to form tumors in syngeneic mice. These sublines remained tumorigenic in cyclophosphamide-treatedmice indicating that the loss in tumorigenicity was the result of increased immunogenicity. The results obtained with the teratocarcinoma cell variants treated with MNNG in vitro and those obtained with the L 1210 cell populations treated with DTIC in vivo have been generalized to other mouse tumor cells. They have led to systematic studies of tumor cells with increased immunogenicity. These studies are described below. 111. Immunogenic Variants Obtained in Vitro with Nitrosoguanidine Derivative MNNG

A. IMMUNOGENIC “turn-” VARIANTS

A very high frequency of clones with strongly reduced tumongenicity was observed with a malignant teratocarcinoma clonal cell line that was treated in vitro with the mutagen N-methyl-N-nitro-N-nitrosoguanidine(MNNG) (Boon et al., 1975; Boon and Kellermann, 1977).Out of 55 clones that were isolated from the surviving population, which amounted to approximately 0.5% of the initial population, 12 failed to form progressivetumors in most syngeneic 129/Svmice at a cell dosage that invariablyproduced tumors with the original malignant cell line. All of 10 control clones isolated without preliminary mutagenesis formed tumors. The clones that failed to form

126

THIERRY BOON

tumors were called “turn-” to distinguish them from the “turn+” initial malignant line and tumorigenic clones. The tum- clones remained incapable of forming tumors after many months of continuous culture. They were nontumorigenic even at doses that were more than 20 times higher than that required to produce tumors with tum+ cells. Representative data obtained with tum- variants are shown in Table I. The lack of tumorigenicity of most tum- variants is not absolute. The mutagenized population actually contained a range of clones extending continuously from those that were only slightly less tumorigenic than the controls to those that formed tumors in only a very small minority of the mice, often with a considerable delay. The tum- clones were found to be indistinguishable from the tum+clones in their rate of multiplication in vitro and in their karyotype. Their inability to form tumors was not due to a reversal of their transformed state, since they were found to be as tumorigenic as the tum+ clone in mice that had received 600 rads of y irradiation. This suggested that the tum- character resulted from an immune rejection process that was prevented in immunodepressed animals (Boon and Kellerman, 1977). The results obtained with teratocarcinoma could be extended to other tumors. When a Lewis lung carcinoma cell line was treated with MNhG, an even higher frequency of tum- clones was obtained (Van Pel et al., 1979). However, many of these clones formed tumors when injected at doses that

TUMORSOBTAINED AFTER

Clone

THE

TABLE I INJECTION OF TERATOCARCINOMA tum- VARIANTS INTO 129/SV MICE” Unirradiated mice

Irradiated mice ~~

PCC4.azal (turn+control) T20 T25 T33 T40 T42 T5 I TI33

40140

(30 days)

30130 (28 days)

2/ I42 101176 3/36 2/16 15/68 2/17 141162

(5 I days) (30 days) (49 days) (35 days) (34 days) (41 days) (32 days)

44/45 50/50 10/1 I 616 13/13 616 12/ I2

(26 days) (28 days) (25 days) (31 days) (29 days) (26 days) (30 days)

Normal mice (unirradiated) and mice given a sublethal dose (600 rads) of whole-body y radiation (irradiated) were injected with 2 X 1O6 living cells ofa control teratocarcinoma tum+ clone and various turn- variants. The results indicate the number of mice forming progressive tumors over the number of injected animals. The number of days given in parentheses represents the mean average time at which mice are sacrificed, showing either a subcutaneous tumor of about 0.5 cm diameter or signs of acute peritoneal swelling due to ascitic tumor (Van Pel and Boon, unpublished).

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

127

were higher than 10 times the minimal tumorigenic dose for tum+cells. All these tum- clones were tumorigenic in irradiated animals. Similar results were obtained with methylcholanthrene-induced mastocytoma P8 15 (Uyttenhove et al., 1980).In this system, the fate of tumor cells that were injected intraperitoneally could be followed by tapping repeatedly the peritoneal cavity of the same mouse and evaluating the number of viable tumor cells by an agar colony assay. The tum- cells were observed to multiply exponentially for 10 days like the tum+ cells. After that the tumcells were eliminated completely within a few days. A large number of lymphocytes and macrophages was observed in the peritoneal cavity at that time. Turn- variants were also obtained from CBA/Ht thymic leukemia TH obtained by Hewitt et al. (1976) (Van Pel and Boon, 1982). A “weak” TH tum- variant that regressed only in about 20% of the mice was submitted to an additional round of mutagenesis. This yielded a cell population containing 70% tum- variants whose tumorigenicity was considerably lower than that of the original variant. Thus, the effect of multiple mutagen treatments on the turn- character can be cumulative. Turn- variants have also been obtained from a spontaneous carcinoma and from three spontaneous leukemias (Van Pel et al., 1983). Thus, tum- variants that are rejected by the syngeneic host have been obtained at remarkably high frequencies from every mouse tumor that was treated with MNNG. Frost et al. (1983) have demonstrated that the mutagen ethylmethane sulfonate can also be very effective in producing tumvariants. A single mutagenic treatment of mammary adenocarcinoma TA3 and of a spontaneous mammary adenoacanthoma produced cell populations that were incapable of forming progressive tumors. Whether variants with increased immunogenicity can also be obtained from tumor cells of other species than the mouse remains to be shown. Incidentally, when a cell line of Trypanosoma cruzi, the agent of Chagas’ disease, was mutagenized in vitro with MNNG, variants that had a reduced virulence in normal mice but were still virulent in irradiated mice were obtained at a high frequency (Boon and Marchand, 1982).Unlike tum- variants from mouse tumors (see below), these parasite variants have not yet been shown to have acquired new antigens. OF NEWANTIGENS ON tum- VARIANTS B. PRESENCE

1. In Vivo Analysis The immunogenic character of tum- variants was confirmed by protection experiments. When animals that had rejected a teratocarcinoma tum-

128

THIERRY BOON

variant were irradiated and challenged with the same vanant, no tumors were formed (Boon and Kellermann, 1977). Adoptive transfer of splenic T lymphocytes from animals that had rejected a tum- variant conferred an effective protection against a challenge with the same variant (Boon and Kellermann, 1977; Boon, Van Pel, and Leclerc, unpublished). Similar results were obtained with tum- variants obtained from Lewis lung, P8 15, and other tumors (Van Pel et al., 1979; Uyttenhove et al., 1980). When mice that had rejected a teratocarcinoma tum- variant were irradiated and challenged with other tum- variants, some protection was observed, but it was invariably weaker than that observed against a challenge with the immunizing variant (Boon and Van Pel, 1978).This was also found with most variants obtained in the Lewis lung and P8 15 systems (Van Pel et al., 1979; Uyttenhove et al., 1980). It could be explained most simply by assuming that tum- variants have acquired new transplantation antigens (tum- antigens) that differ for each variant. A priori, the cross-protection between different variants could have been due to a cross-reactivitybetween tum- antigens. However, as will be seen below, it can be accounted for by the presence of a common determinant already present on the original tum+ cells.

2. In Vitro Analysis The results obtained in vivo suggested that the tum- system offered the opportunity to study the response against a variety of cell surface antigens present on the same background cell so that the specificity of these reactions should be particularly easy to control. Despite a number of attempts involving syngeneic, allogeneic, and xenogeneic immunizations, no antibodies showing specificity for the immunizing tum- variant have been obtained in antisera or in hybridoma supernatants for any of the tumors discussed above. However, a characterization of tum- antigens with cytolytic T lymphocytes (CTL) proved possible. a. Analysis with CTL Populations. CTL showing a definite specificity for the immunizing tum- variant were obtained from peritoneal exudates of syngeneic mice that had rejected P8 15 tum- variants and had been boosted with the same variant a few days before collection (Boon et al., 1980). Very high and specific CTL-mediated lysis was also obtained when spleen cells of mice immunized against P8 15 tum- variants were restimulated in vitro with the immunizing variant. The specificity of the CTL effectors could be confirmed by competition experiments (Boon et al., 1980). Besides the classical methods of using irradiated or mytomycin-treated stimulator cells (Wagner and Rbllinghoff, 1973; Cerottini et al., 1974) it was found convenient to use tumor cell variants that had been rendered resistant to azaguan-

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

129

ine and to carry out the stimulation in medium containing aminopterin (HAT), where these variants are killed (Van Snick et al., 1981). The demonstration of specific T cell mediated lysis confirmed the existence of tum- antigens suggested by the in vivo protection experiments. In addition, an extensive analysis of the tum- antigens of a large number of variants became possible. An example of such a systematic analysis of the specificity of tum- antigens is shown in Table 11. The conclusions derived from this type of analysis performed on 2 1 different P8 15 tum- variants can be summarized as follows (Boon et al., 1980). 1. Most tum- variants ( 14/2 1) carry a strong “private” antigen that elicits a CTL response that is at least two times higher on the immunizing variant than on any other variant or on the turn+cell. The fact that these 14 tumantigens are all different suggests that the repertoire of different tumantigens is very likely to include more than 50 different antigens. 2. All tum- variants share a common antigen with the tum+cell. This is in agreement with previous reports indicating that P8 15 carries a tumor-associated transplantation antigen that elicits a CTL response (Takei et al., 1976; Biddison and Palmer, 1977). 3. No cross-reaction between any pair of tum- variants can be detected except for that due to the tum+antigen. 4. Whereas some tum- variants do not carry a private antigen that elicits a specific CTL response, none of 10 mutagenized clones that had remained fully tumorigenic was found to carry a new antigen detectable by primed CTL. The acquisition of a tum- antigen detectable by CTL is therefore not necessary but may be sufficient for a cell to become a turn- variant.

It is now generally accepted that cell surface antigens are recognized by syngeneic CTL in association with products of the major histocompatibility locus (Zinkernagel and Doherty, 1979). This has been shown for viral antigens, for antigens produced by combining haptens to the cell surface, and for the H-Y antigen (Zinkernagel and Doherty, 1974; Shearer, 1974; Gordon et al., 1975;Gomard et al., 1976). It was therefore of some interest to examine whether mutagen-induced antigenswere following the rule. The H-2 restriction pattern of six P8 15 tum- antigens was examined by measuring the inhibition of specificcytolysisby various anti-Kdor anti-Dd reagents. The CTL activity directed against three of these antigens was inhibited by a variety of anti-Kdsera or monoclonal antibodies, that against another was inhibited by anti-Dd sera, while that against two others was inhibited neither by anti-Kd nor by anti-Dd reagents (Van Snick et al., 1982). Normal amounts of H-2Kdand Dd antigens were found on the P8 15 tum- variants. The pattern of specific CTL reactions elicited by various P815 tum-

TABLE I1 ANTIGENICSPECIFICITIES OF P8 15 turn- VARIANTSDEFINED BY CYTOLYTIC SPLENIC LYMPHOCYTES'

Lysis of target cellsb PI Anti-P21

P32

P35

P59

P60

P76

P89

1.31 (18.6)

(1.04)

1.95 (1.22)

0.96 (1.03)

1.42 (1.31)

2.09 (1.29)

1.9

2.36 (1.29)

11.19 (0.93)

13 (0.94)

0.W

(1.06)d

c

P2 I

P91

pi98

1.26 (1.14)

1.08 (0.98)

(0.81)

1.81 (0.98)

3.24 (1.19)

1.75 (0.94)

1.81 (0.97)

2.18 (0.94)

2.9 I (0.94)

20.64 (0.99)

14.9 ( 1.05)

17.5 (1.24)

20 (1.14)

22.33 (0.95)

1.74

0.67 (0.8)

1.04

( 1.07)

(1)

1.69 (1.21)

0.68 (0.8)

2.27 (2.66)

1.07 (1.02)

I .33 (0.94)

1.11

P215

25! Anti-P32

1.43 (1.02)

0.69 (0.97) I

Anti-P35

Anti-P59

Anti-P60

12.98 (1.22)

4.69 (0.86)

17.69 (1.09)

0.65 (0.97)

0.36 (1.12)

0.97

0.65 (1.01)

0.3 I (0.96)

0.68 (0.7)

I 1 (:817)

1.58 (1.28)

(y:;:)

I (i:!; I

I .4 (1.12)

0.9

Anti-P76

Anti-P89

Anti-P9I

Anti-PI98

Anti-P215

3.2 (1.14)

I 1

3.13 (1.15)

4.29 (1.06)

0.15 (1.06)

0.15 (2.23)

0.17 (1.63)

0

2.5 (0.95)

1.84 (1.68)

3.39 (1.03)

6.66 (1.61)

2.92 (1.2)

2.08 (0.99)

1.38 (1.3)

2.95 (0.93)

4.44 (1.11)

2.5 (1.06)

0.24 (2.39)

0

0

'

;:.I)

2.57 (0.89)

-

0 -

1.31 (0.9)

0.54 (0.74)

2.48

3.08 (1.11)

1.71 (1.04)

2.09 (1.11)

3.07 (1.08)

1.49

( I. 12)

0.66 ( I .02)

0.29 (0.91)

1.16 (1.18)

I .48 (1.2)

0.8 1 (1.12)

0.95 (1.14)

1.4 (1.12)

0.99 (1.16)

-

1.7 (0.59)

3.46 (0.97)

3.6 I (0.76)

3.98

4.07 (0.88)

0.2

-

( I .65)

4.52 (1.41)

0.82 (0.97)

1.03 (0.98)

I (x.';;)1

DBA/2 mice received ip 2 X lo5living cells from P8 15-derivedtum- clones. Three weeks later, 3 X lo7splenic lymphocytes were restimulated in vitro with 6 X lo5cells of the immunizing variant. The cytolytic activity of these effectorcells was measured 5 days later against appropriate target cells in a 4-hour chromium release assay. hP1, a tum+clone of P815. P21, P32, P35, P59, P60, P76, P89, P91, P198, P215, tum- clones derived from P815. Lytic units. d Normalized lytic index taking into account the difference in general sensitivity of the targets and the differences in cross-reactive lysis of the effectors(see Boon et al., 1980).Values significantlyabove one indicate that the effectorcells lyse the targets preferentially (Marchand, Uyttenhove, Van Snick, Van Pel, and Boon, unpublished).

I32

THIERRY BOON

variants showed a strong but not complete correlation with that obtained in vivo in cross-protection experiments (Uyttenhove et af., 1980; Boon et al., 1980).This and the high level of CTL activity that was observed suggest that CTL play an important but not exclusive ;ole in the rejection of these variants. For Lewis lung carcinoma tum- variants, CTL effectors directed specifically against the immunizing variant were also obtained. However, the CTL activitieswere weaker than those observed against P8 15 variants (Vessikre et af., 1982). b. Analysis with CTL Clones. Unlike the analysis of cell surface antigens with antibodies, the analysis with CTL populations suffers from the lack of monospecific and readily available permanent reagents. However, considerable progress has been achieved in recent years with the recognition that normal or primed CTL precursors can be stimulated in clonal conditions, provided a source of the T cell growth factor interleukin-2 is added to the medium. The proliferation and cytolytic activity of single T cell clones can then be observed (MacDonald et af., 1980; Gillis and Watson, 1981). Moreover, Gillis and Smith (1977) have shown that active and specificCTL can be maintained and expanded in culture for several months in medium containing T cell growth factors. Since then, long-term CTL clones have been obtained that are directed against a variety of cell surface antigens (Nabholz et al., 1978; Weiss et af., 1980; Fathman and Hengartner, 1978; Glasebrook and Fitch, 1979; von Boehmer and Haas, 1981). The retention of high activity and specificity by long-term CTL clones is favored by the addition of irradiated spleen cells and stimulator cells (Glasebrook and Fitch, 1979; Weiss et al., 1980). The limiting dilution approach was applied to tum- variants derived from P815 using as a source of interleukin-2 the supernatant from allogeneic mixed lymphocyte cultures (Maryanski et af., 1982a). Estimates of the frequency of CTL precursor cells in spleen from mice that had rejected these to 2 X variants ranged from These frequencies are of the same order of magnitude as those reported for precursors present in spleen from mice that have rejected tumors induced by murine sarcoma virus (MSV) (Brunner et af., 1980) and in spleen from female mice immunized against syngeneic male cells (Kanagawa et al., 1982). It is only slightly lower than that observed in spleen from mice primed against allogeneic cells (Ryser and MacDonald, 1979). As observed in the anti-male and anti-MSV response the frequency of CTL precursors was at least 10-fold higher after in vivo priming. The frequency of CTL precursors directed against P815 tumvariants increased at least 100 times when immune spleen cells were stimulated in vitro with the immunizing variant before stimulation in clonal conditions. Among the CTL clones obtained after immunization with P8 I5

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

TARGET CELLS P1

133

CTL CLONES

P35

P198

ANTI-P8 15

"80"1 ANTkP35

I

a

ANTI-P9 1

60

ANTI-P 198

40

"$4

10

"84

10

O o 80 4

10

008 2 04

,o

LYMPHOCYTE TARGET RATIO

FIG.I. Lymphocytes of syngeneic DBA/2 mice that had rejected P8 15 turn- variants P35, P91,or PI98 were stimulated in limiting dilution conditions with the immunizing variant under conditions described by Maryanski ef al. (1982a). Cytolytic T cell clones lysing specifically the immunizing variant were obtained. They could be maintained for many months in medium containing T cell growth factorsobtained from allogeneicmixed lymphocytecultures. The CTL clone labeled anti-m I5 was obtained after immunization and restimulation with P35. Result ofchromium releasetestson tum-targetsP35,P91, P198,andtum+P815cIonePl are shown. Results obtained by Maryanski and Boon reproduced with permission from Boon ef a/. ( 1982).

134

THIERRY BOON

tum- variants, some showed a strict specificity for the immunizing variant and others lysed equally well all P8 15-derived targets including the tum+ cell. Long-term CTL clones of both kinds could be obtained. Examples showing the strict specificity of these clones are shown in Fig. 1. Long-term CTL clones specific for the tum- antigens of Lewis lung carcinoma derived variants have also been obtained (Vessikre et al., 1982). Production of antigen-loss tumor cell variants by selections with CTL directed against tumor-associated antigens has been reported by Fidler et al., (1976; Fidler and Bucana, 1977) and by Schirrmacher and Bosslet (1980). Specific CTL clones were used to apply immunoselection to turn- variants (Maryanski and Boon, 1982). Antigen-loss secondary variants could be obtained for some P8 15 tum- variants at frequencies ranging from to For other turn- clones no antigen-lossvariants were obtained, suggesting that they occur at a frequency that is lower than loT6.Selections carried out with one P815 tum- variant showed that it carries at least two new antigenic specificities that can be lost independently. Specific CTL clones against each specificity could be obtained (Maryanski and Boon, 1982). When injected into DBA/2 mice, these antigen-loss variants proved definitely more tumorigenic than the original tum- variants, confirming that the turn- antigens recognized by CTL play an important role in the rejection of the tum- variants. It is expected that the occurrence of these antigen-loss secondary variants will be of considerable utility for the biochemical characterization of the tum- antigens. Since most tum- variants occasionally form tumors, escaping P8 15 tumcells were tested for their sensitivity to CTL clones directed against the original tum- variants. Most of the escaping tumors were found to have lost at least one variant-specificdeterminant (Maryanski et al., 1983a).A similar situation was studied by Urban et al. (1982) who found with strongly antigenic UV-induced mouse tumors that in the rare instances in which one of these tumors grew progressively in a normal mouse the tumor cells had become largely resistant to CTL directed specifically against this tumor.

C. PROTECTION AGAINST INITIAL “turn+” CELLS Whereas teratocarcinoma cell lines elicit a potent humoral response against embryonal antigen F9 in syngeneic mice (Artzt et al., 1973), no evidence for a rejection response directed against malignant cell line PCC4 has been observed either in normal mice or in mice that received multiple injections of irradiated PCC4 cells (Boon and Van Pel, 1978). Even in animals that formed a subcutaneoustumor that was removed surgically, no protection against a subsequent challenge was observed (Boon et al., 1979). Nevertheless, a definite protection against PCC4 cells was obtained in mice that had rejected teratocarcinoma turn- variants (Boon and Van Pel, 1978).

TUMOR CELL VARIANTS OBTAINED WITH MUTAGENS

135

The protection extended to the original transplantable teratocarcinoma OTT6050, indicating the presence of an otherwise undetected tumor-associated transplantation antigen (Georlette and Boon, 1981). This protection conferred by tum- variants lasted more than 100 days and was radioresistant. It was however relatively weak since it broke down when the challenging dose exceeded the minimal tumorigenic dose by a few fold. Better protection was observed when very large numbers of living tumor cells were inoculated for immunization. No significant protection was observed after injection of irradiated tum- variants. Protection was also observed against Lewis lung carcinoma and mastocytoma P8 15 tum+ cells after immunization with living cells of the relevant tum- variants (Van Pel et al., 1979;Uyttenhove et al., 1980).For Lewis lung carcinoma this is less remarkable than the protection against teratocarcinoma since irradiated tum+cells also confer some degree of protection. For P8 15, no protection was obtained with irradiated tum+cells. However, this tumor clearly elicits a partial rejection in vivo and a CTL response (Takei et af.,1976;Uyttenhove et al., 1980).A characteristic feature of the protection obtained with both Lewis lung and P8 15 tum- variants is that variants differ considerablyin their ability to confer protection (see discussion below). One P8 15 tum- variant conferred nearly complete protection when living cells were injected and still conferred significant protection when injected as irradiated cells, under conditions where none was conferred by irradiated tum+cells (Uyttenhove et al., 1980). The protection obtained against turn+cells is concordant with the results obtained in cross-protectionexperimentsand with those obtained with CTL clones. It indicates that all the tum- variants share a common antigenic specificity already present on the tum+cell. For teratocarcinoma this determinant appears to lack any immunogenicity when it is located on the tum+ cell. It appears therefore that tumors that appear nonimmunogenic may nevertheless carry specific transplantation antigens that can be revealed by tum- variants. If this observation could be extended to human tumors, this would clearly provide new opportunities in cancer immunotherapy. It was therefore considered worthwhile to examine whether the protection observed with tum- variants could be extended to other weakly or nonimmunogenic mouse tumors in conditions approaching those applying to primary tumors. This implied avoiding as much as possible artifactual antigenicity which can result either from genetic differences between the primary tumor bearer and the mice used for the experiments or from antigenic evolution of the tumor cells in vivo or in vitro. We resorted to tumors that H. Hewitt et af. (1976) had obtained and transplanted in a carefully maintained inbred CBA colony. A tum- variant, that was obtained from thymic leukemia TH soon after its adaptation to culture, was found to confer significant protection against the tum+ cell line and against the

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TABLE Ill PROTECTION INDUCED BY tum- VARIANTSAGAINST T w o SPONTANEOUS LEUKEMIAS' Percentage of mice with tumors after challenge withb Immunizing cells

LEB I

LEC I

-

LEBl (turn+)irradiated LEB26 (turn-) living LEB28 (turn-) living

95 ( I 9/20) 43 (9/21) 62 (20/32)

LECl (turn+)irradiated LEC30 (turn-) living LEC37 (turn-) living

100(12/12) -

33 (3/9)

-

100 (l9/19)

100 ( I9/ 19)

i

100 (10/10) 100 (12/12) 100 ( I2/ 12) 42 (5/12)

CBA/Ht mice were immunized with cells of two spontaneous leukemias, LEB and LEC. These are leukemia 1 and IV obtained by Hewitt and Blake (1978). The tum+ cells ( 5 X lo6) were killed by y radiation (5000 rads). LEB tum- variants ( lo6)and LEC tum- variants ( loJ) were injected intrapentoneally as living cells. Control mice received the same amount of injection medium. After 50 days, the mice were injected intraperitoneally with cells of tum+ clone LEBl (3 X lo2)and LECl ( lo2).Mice living 60 days after the challenge were considered negative. Control mice died after an average time of 32 days for LEB I and 13 days for LEC I. Values in italics indicate significant protection.

original transplantable tumor (Van Pel and Boon, 1982).CTL clones lysing specifically tumor TH were obtained from spleen cells of animals that had rejected TH tum- variants. More recently, similar results have been obtained with two spontaneous leukemias isolated by H. Hewitt (Van Pel el al., 1983). Results showing the protection by tum- variants are shown in Table 111. This and other results have confirmed that protection against a tumor is obtained only with tum- variants derived from the same tumor. We think that these results considerably increase the likelihood that weak antigenic structures will be found on most cancer cells. It would be interesting to find out whether similar results can be obtained with spontaneous tumors that have undergone only a very small number of transplantations although even this situation will not completely exclude artifactual antigenicity. IV. Immunogenic Tumor Cell Populations Obtained in Vivo with Triazenylimidazole Derivative DTlC

A. PRODUCTION OF IMMUNOGENIC LINES

On the basis of evidence suggesting that treatment with antineoplastic drugs may affect the antigenicity of tumor cells, E. Bonmassar ef al. (1970)

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treated cells of leukemia L1210 in vivo with the cytotoxic drug 5-(3-3-dimethyltriazenoimidazole-4-carboxamide)(DTIC). The tumor cells were transplanted serially in (DBA/2 X BALB/c)F, mice (CDF,) treated for 10 consecutive days with DTIC. In the course of these passages, the L12 10 cells progressively lost their malignancy in normal mice while maintaining their ability to form progressive tumors in DTIC-treated mice. After 12 passages in DTIC-treated animals one L12 10 line had become almost completely unable to form tumors in CDF, mice at a dose at which a control L12 10 line killed all animals. Such lines will be referred to as DTIC lines. Even though the DTIC lines had become resistant to this drug, their lack of malignancy in normal mice was not due to DTIC dependence since they formed tumors not only in mice treated with this drug but also in those treated with cyclophosphamide (Bonmassar et al., 1970). This and the observation that DTIC has immunosuppressive effects (Giampietri and Bonmassar, 1978)suggested that the DTIC lines formed progressive tumors only in immunosuppressed mice but were rejected in normal mice. In agreement with this, it was possible to establish by [lZSI]IUdRuptake experiments that DTIC lines were rejected by normal mice approximately 10 days after the injection (Fioretti and Riccardi, 1975; Riccardi et al., 1978). The DTIC lines appeared to have acquired a stable heritable variation since their lack of malignancy persisted after multiple passages in cyclophosphamide-treated mice. The authors suggested that the DTIC lines had acquired strong antigenicity causing their rejection either because antigenic variants had been selected in the DTIC-treated animals or because the drug had induced somatic mutations resulting in strong antigenicity (Bonmassar et al., 1970). The somatic mutation hypothesis was plausible because DTIC has alkylating properties and binds nucleic acids (Skibba et al., 1970). In support of this hypothesis, the antimutagenic compound quinacrine, that inhibits neither the immunosuppressive nor the cytotoxic action of DTIC, prevented the formation of DTIC lines with reduced malignancy (Giampietri et al., 1980). DTIC most probably needs to be transformed in the liver into a mutagenic metabolite since the formation of the DTIC line was inhibited by CCl, and activated by phenobarbital treatment that respectively decreases and increases liver metabolism (Contessa et al., 1981). The results obtained with L1210 were extended to mouse lymphoma L5 178Y (Nicolin et al., 1976b).Cell lines that were unable to form tumors in normal syngeneic mice are obtained after five in vivo passages in DTICtreated mice. These cell lines remained capable of forming tumors in mice that were immunosuppressed with 400 R of whole-body irradiation. Similar results were obtained with C57BL/10 sarcoma 51033 and virus-induced leukemias LSTRA and RBL5. (Bonmassar et al., 1975; Houchens et a!., 1976). Representative results obtained with L1210 and LSTRA are shown

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THIERRY BOON LSTRA

1

3

L 12 10/Ha

5

7

1

3

5

7

9

TRANSPLANT GENERATION

FIG.2. Median survival time and percentage of long-term survivors ofCDZF, mice injected with cells of leukemia LSTRA or L1210 Ha that had been transplanted in DTIC-treated mice for the number of times indicated on the abscissa.The L1210 Ha and the LSTRA DTIC lines were injected into mice (0)and immunodepressed mice that had been treated with DTIC (0) or cyclophosphamide (A) (Bonmassar, unpublished).

in Fig. 2. From an AKR lymphoma it was possible to obtain only a weakly immunogenic DTIC line even after a large number of passages (Bonmassar et al., 1979). Perhaps this was due to a permeability resistance that prevented the penetration of the drug. Nontumorigenic DTIC lines were also derived from L1210 and LSTRA by passage in DTIC-treated nude mice (Campanile et al., 1975).These lines formed tumors in nude mice indicating that their rejection was mediated by T lymphocytes. Even though all the work that is discussed below has been carried out with DTIC lines obtained after in vivo treatment, it has recently been reported that immunogenic lines can be obtained from L 1210by repeated treatments with DTIC in vitro (Contessa et al., 1981). This procedure requires the presence of a liver microsomal fraction to transform the drug into an active metabolite. B. PRESENCEOF NEWANTIGENS ON DTIC LINES 1. In Vivo Analysis

Adoptive transfer into irradiated mice of spleen cells from mice that had rejected an L12 10 DTIC line conferred protection against the DTIC line but not against the original tumor line (Nicolin et al., 1974~).Similarly, immu-

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nization with viable cells of a L5 178Y DTIC line or adoptive transfer with spleen cells immunized against it conferred protection only against the DTIC line (Nicolin et al., 1976b). Protections in Winn type assays or after adoptive transfers were also observed in irradiated mice injected intracranially with L5 178Y DTIC lines and normal lymphocytes sensitized in vitro with this DTIC line. Here also, no protection was observed against L5 178Y (Romani et al., 1982). In one instance, irradiated cells from an L5178Y DTIC line resulted in enhancement of the growth of the same line (Riccardi et al., 1977).Surprisingly,it was found that a DTIC line derived from L 1210 and another derived from L5 178Y conferred significant protection against each other (Nicolin et al., 1976a). These results suggest that the DTIC lines were carrying new antigens not present on L 12 10 or L5 178Y. However, they do not exclude the possibility that the DTIC lines have a much increased density of the transplantation antigen present on the original line. More compelling was the observation that mice could be made tolerant to an LSTRA DTIC line by injection of irradiated cells of this DTIC line followed by a cyclophosphamideinjection, whereas no tolerance against the DTIC line was obtained by injecting irradiated LSTRA cells and cyclophosphamide (Houchens et al., 1976). 2. In Vitro Analysis The presence of new antigens on the DTIC lines could be demonstrated conclusively with CTL derived from mice that had rejected these immunogenic lines. Spleen cells immunized against an L1210 DTIC line lysed this line much more effectively than the original tumor cells (Nicolin et al., 1974b; Testorelli et al., 1978). Moreover, competition with L12 10 cells reduced only slightly the lytic reaction against the L 1210 DTIC line. Similar results were obtained with DTIC lines derived from lymphomas L5 178Y and LSTRA (Nicolin et al., 1974a; Santoni et al., 1978). Oligoclonal sublines were derived from an L5 178Y DTIC line by injecting four cells into immunosuppressed mice. The CTL analysis of two of these lines indicated that both carried specific antigens absent from L5 178Y and from the other DTIC sublines (Fioretti et al., 1978). This suggests that the DTIC lines consist of a large number of variants with different antigens. For all these leukemias, the CTL obtained after immunizations with DTIC lines crossreacted with the original tumor line, indicating that the DTIC lines had conserved the relevant tumor-associated transplantation antigens. In contrast to the antigenic differences between the DTIC lines and their original tumors, one report indicated that CTL resulting from immunization with an L 1210 DTIC line lysed an L5 178Y DTIC line equally well and vice versa. Furthermore, antisera obtained by immunizing rabbits with an L5178Y DTIC line and absorbed with L5178Y bound not only to the

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immunizing DTIC line, but also bound equally well to a DTIC line derived from L1210 (Nicolin et af.,1976a). This would imply that the diversity of specific DTIC “drug-mediated tumor antigens” is limited. However, no cross-reaction was found between an L5 178Y DTIC line and an LSTRA DTIC line nor with other tumor combinations (Taramelli et a/., 1981). When normal syngeneic lymphocytes were stimulated in vitru with an L1210 DTIC line, the thymidine uptake was three- to fourfold higher than that observed in primary mixed lymphocyte tumor cell (MLTC) cultures camed out with L1210 cells. A similar difference was observed in stimulation assays performed with lymphocytes immunized against the DTIC lines (Testorelli et af., 1978). After primary MLTC, a CTL activity showing specificity for the immunizing DTIC line was obtained for leukemias L 1210, EM, and L5 178Y. The specificity of this reaction was confirmed by cold target competition (Romani et af., 1979). In view of the reports that some methylcholanthrene-induced sarcomas carry foreign H-2 antigen (Invernizzi and Parmiani, 1979, the possibility that DTIC antigens represent modified H-2 antigens was examined with a LSTRA-DTIC line obtained in H-2dmice. A normal amount of H-2dand no evidence for the presence of the foreign H-2band H-2k antigens was found (Taramelli et af.,1981). Similar results have been obtained with P8 15 tumvariants (Van Snick, Van Pel, and Parmiani, unpublished). C. PROTECTIVE EFFECTSOF THE DTIC LINESAND ASSOCIATION WITH CHEMOTHERAPY Leukemias L12 10, L5 178Y, and LSTRA elicit transplantation immunity when injected as irradiated cells. The corresponding DTIC lines, whether injected as living or as irradiated cells, conferred against the original tumor a protection that was similar to that obtained with irradiated tumor cells (Nicolin et af.,1976b; Riccardi et af.,1977). However, viable cells from one L1210 DTIC line had a protective action that was superior to that of irradiated L 12 10 cells when inoculated a few hours after an L12 10 injection (Nicolin et af.,1981). A definite protection against LSTRA but only a very weak protection against RBL5 was obtained after immunization with viable cells of the corresponding DTIC lines (Houchens et af., 1976). For a dimethylbenzanthracene-inducedlymphoma that did not have a detectable transplantation immunogenicity, no protection was observed with DTIC lines that were obtained from this tumor (Fioretti et a/., 1980). Interesting experiments were camed out to examine whether a combination of chemotherapy and injection of DTIC lines after tumor challenge could induce cures more effectively than chemotherapy alone. Viable cells from an LSTRA DTIC line injected 1 day after the tumor challenge

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improved considerably the fraction of long-term suMvors obtained with an antineoplastic drug (Houchens et af., 1976). Similar results were obtained with an L12 10 DTIC line. Viable cells from this line improved the outcome of chemotherapy more effectivelythan irradiated tumor cells (Nicolin et af., 198 1). V. Discussion

A. NATUREOF THE ANTIGENICCHANGES INDUCED BY MUTAGENS The results summarized here are compatible with the view that the tumvariants obtained with MNNG and the DTIC line represent two examplesof the same phenomenon. The DTIC lines probably contain a large number of different antigenic variants (Fioretti et al., 1978). Considering the observations of Koyama and Ishii (1 969), Kerbel (1979), and others it is likely that mutagens other than MNNG or DTIC also produce stable tumor cell variants that carry new antigens. An intriguing problem is presented by the extremely high frequency of antigenic variants in mutagenized populations. This frequency is many orders of magnitude higher than that of most metabolic mutations (Sat0 et al., 1972; Chu, 1974). Trivial explanationslike the possibility that MNNG and DTIC act as chemical modifiers of the cell surface can be readily excluded by the stability of the tum- variants and the DTIC lines. Another possibility that can be ruled out is the selection of preexisting immunogenic variants. This would imply that increased immunogenicity would somehow be associated with resistance to MNNG or DTIC. However, the tumvariants are not resistant to MNNG (Emma, Boon, Van Snick, and Marchand, unpublished). For the DITC lines, this hypothesis was more plausible since the lines are resistant and the drug is immunosuppressive. However, immunogenic lines were also obtained by treatment of L1210 lines that were resistant to the cytotoxic action of DTIC, indicating that selection is not necessary (Bonmassar et af., 1972). The variants obtained with nitroquinolin-1 -oxide were not resistant to the drug (Koyama and Ishii, 1969). Thus, it appears that antigenic variants arise by a truly heritable change resulting from a direct action of the mutagens on the tumor cells. Such a change could be mutational or epigenetic. Following Siminovitch (1976), we shall consider as mutational events those that involve any heritable nucleotide base change, deletion, or rearrangement in the primary structure of DNA, and as epigenetic events any other cause of hereditary variation. A priori, the high frequency of variants seems more compatible with an epigenetic event. For instance, the genes coding for the tum- antigens could

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normally be repressed by molecules that would occasionally be inactivated as a result of mutagen treatment. This simple model is not compatible with results showing that somatic cell hybrids resulting from the fusion of different P8 15 tum- variants or of tum- and tum+ clones express the tumantigens of both fusion partners (Maryanski et a/., 1983b). However, this codominant expression of tum- antigens could be accounted for by more elaborate epigenetic models. For instance, the repressor gene, which controls the gene coding for the tum- antigenic protein, could itself be repressed by this protein (R. Thomas, personal communication). A mutational origin of the new antigenicity would fit well with the stability of the tum- variants and the low frequency of antigen-loss variants. Assuming that the mutagen produces at random similar types of changes on the genetic regions that determine cell survival in vitro and on the regions that determine antigenicity, one can estimate the relative size of these two genetic targets. This can be calculated with the Poisson formula from the frequency of tum- variants and from the fraction of the cells that survive mutagen treatment. This leads to the estimate that the genetic target related to antigenicity is of the order of 5% of that related to cell growth. This enormous value would imply that a very large number of genes, probably more than 1000, would produce strong transplantation antigens when their normal product would be altered. Considering that a large fraction of mouse proteins shows polymorphism (Roderick et al., 197l), this would imply that the number of histocompatibility genes would be of the same order. This is much higher than what has been observed (Cloudman, 1932; Amos et af., 1955; Prehn and Main, 1958; Snell and Stimpfling, 1968). To avoid this difficulty, one could postulate that a large number ofgenes that are normally silent would be activated by the mutagens and produce the new antigens. A wide variety of new antigens could also be created by DNA rearrangements similar to those involved in the formation of the immunoglobulin sequences (Hozumi and Tonegawa, 1976; Leder et al., 1980). These rearrangements might be particularly sensitive to the action of mutagens so that only a small number of genes forming a hypermutable region could be implicated. Myeloma variants that produce antibodies with an altered heavy chain have actually been obtained at very high frequencies (Birshtein et af., 1974; Preud’homme et af.,1975). It will be very difficult to understand the nature of the antigenic variants before information can be obtained regarding the structure of the antigens or the location of the putative tum- mutations. The isolation of tumantigens has been prevented by the lack of specific antisera. The difficulty of obtaining specific antibodies against strong transplantation antigens is actually not unique to tum- variants. A similar situation has been encountered with a number of H-2 mutations that provoke a strong skin graft rejection

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but little or no antibody response (Klein, 1978.). This is also found with most methylcholanthrene-induced tumors with the exception of sarcoma Meth A where antibodies have been obtained that are specific for this tumor (DeLeo et al., 1977). In the absence of immunoprecipitation, transplantation antigens have to be fractionated on the basis of their immunogenicity in vivo (Law et al., 1978, 1980) or their ability to restimulate specific CTL precursors in vitro (Fast and Fan, 1978). Preliminary experiments suggest that a particulate fraction prepared from one P8 15 tum- variant is capable of restimulating CTL (Beyer, Lambotte, Van Snick, and Boon, unpublished results). The use of interspecies somatic cell hybrids has proved very useful for the chromosomal assignment of many human and mouse genes (Ruddle and Creagan, 1975; Swan et al., 1979). Recently, this technique has permitted the assignment of the gene that determines the tumor-associated antigen of Meth A to chromosome 12, which also cames the gene clusters encoding the immunoglobulin heavy chains (Pratcheva et al., 1981). However, the application of this technique to tum- variants would not be straightforward because of the lack of antibodies. It is unlikely that CTL clones would recognize tum- antigens on mouse- hamster hybrids unless H-2 antigens were expressed on these hybrids. One would expect this to complicate the chromosome assignment. However, the existence of secondary antigen-loss variants derived from tum- variants by CTL selection raises new possibilities. If tum- variants were obtained in tumors from heterozygous F, mice, and if antigen-loss variants could be induced by agents such as y irradiations that provoke the loss of large chromosomal regions, the relevant regions might be identified with DNA probes showing restriction-size polymorphism (Wyman and White, 1980). An interesting alternative to somatic cell genetics could be based on the finding of Mintz and Illmensee (1975) that teratocarcinoma cells implanted in early mouse embryos can become integrated in normal mouse tissue. In some chimeric mice teratocarcinoma cells have been shown to contribute to the germ line (Cronmiller and Mintz, 1978). If this could be achieved with a teratocarcinoma tum- variant and if the new antigen could be detected on normal tissue, this might lead to the mapping of the relevant genes by classical genetics. By their diversity the antigenic variants induced by mutagens are similar to the tumors induced by UV radiation, which are so immunogenic that they do not produce progressive tumors in normal mice. They grow progressively in UV-treated mice because of the induction of specific suppressor T cells (Pasternak et al., 1964; Kripke, 1974;Fisher and Kripke, 1977).A large variety of tumor-associated antigens that show little or no cross-reactivity is also found on methylcholanthrene-induced tumors (Basombrio, 1970). Lennox ( 1980) has proposed that these antigens may in fact be formed by

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recombinant gp70 glycoproteins of murine leukemia viruses. However, this hypothesis is not yet supported by convincing evidence and fractionations of some tumor-associated antigens have demonstrated that they are carried by molecules that are distinct from gp70 (Rogers et al., 1978). Even though tum- variants derived from P8 15 elicit anti-gp70 antibodies more readily than the tum+ cell that also carries gp70, there is not yet any evidence that gp70 molecules constitute the tum- antigens (Jacquemin, 1982). Considering that most chemical carcinogens such as methylcholanthrene and of course UV are mutagenic and considering the extremely high frequency of mutagen-induced antigenic variants, there is a definite possibility that many tumor cells transformed by these carcinogens have acquired two independent mutations. The first would provoke the tumoral transformation while the second would produce the tumor-associated transplantation antigen. This would imply that the tumor-associated antigen bears no functional relation to the transformation process. Prehn ( 1975) has proposed this hypothesis to account for the difference in antigenicity between the spontaneous and the carcinogen-induced tumors. In support of the requirement for two independent mutations, he found that tumors produced by high doses of carcinogens were more immunogenic than those produced by low doses. However, no such correlation was observed in similar experiments performed by Stutman ( 1975). To conclude, almost everything remains to be learned about the nature of the mutagen-induced cellular changes that lead to the expression of new antigens. One can only remark that if some form of immune surveillance against mutagen-induced tumors exists (Ehrlich, 1909; Burnet, 1964), then a hypermutable region producing strong transplantation antigens provides an appropriate mechanism. B. MECHANISM OF THE PROTECTION AGAINST

THE

ORIGINAL TUMORS

The protection conferred by immunogenic variants is specific in the sense that protection is observed only against the parental tumor (Georlette and Boon, 1981; Van Pel and Boon, 1982). The efficacy of this protection is however rather low. As described above, the tum- variants confer protection even against tumors that appear to be nonimmunogenic. One rather obvious explanation is that immunization with tumoral cells is usually carried out with irradiated cells whereas for immunization with tum- variants living cells are used. As a consequence, intact immunogenic tum- cells are present in the animals in very large numbers for a period that usually exceeds 10 days (Uyttenhove et al., 1980). This may account for part of the tum- effect. However, other factors must be involved since living teratocarcinoma cells later removed by

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surgery failed to confer any protection whereas tum- variants do, and since one P8 15 tum- variant protects even when immunization is carried out with irradiated cells (Boon et af.,1979; Uyttenhove et al., 1980). It is therefore probable that tum- antigens serve as helper antigens for the response against the tumor-associated (turn+)antigen (Mitchison, 1970). This could be due to nonspecific effects of the response against the tumdeterminant. For instance a local immunostimulation could be caused by fa'ctors released by lymphocytes and macrophages involved in the anti-tumresponse. More specific cooperative effects in the immunogenicity of the tum- and the tum+ determinants could also occur, as found in the hapten carrier effect. This has been called associative recognition (Lake and Mitchison, 1976). In the antibody response against the Thy-1 antigen, it has been shown that alloantigens function as helper antigens. For help to occur, the Thy-1 and the helper antigen must be expressed on the same cell, suggesting that as in the hapten carrier effect some degree of physical association between the two antigens is required (Lake and Douglas, 1978). It remains to be found whether the help between the tum- and the tum+ antigen involves the cooperation of two lymphocytes that each recognizes one determinant or whether these determinants combine to form an efficient immunogenic complex. Large differences in protective ability are observed among tum- variants derived from the same tumor. For tumor P8 15 and TH, present evidence suggests that variants that carry weak tum- antigens, in the sense that they elicit little specific CTL response, protect better than others having strong tum- antigens (Uyttenhove et af., 1980; Boon el af., 1980; Van Pel and Boon, 1982). One interpretation is that there exists a competition between the responses against the tum- and the tum+ determinants, for instance because strong tum- antigens result in rapid elimination of the immunogen. Another interpretation is that tum- variants arise by antigenic modification situated between two extremes, one being the acquisition of new antigenic specificities that show little or no associative recognition with the tum+ antigen and the other consisting of a modification of the tum+ antigen resulting in increased immunogenicity without acquisition of new specificities, Variants corresponding to the second possibility would of course be better immunogens against the tum+cells. For application of immunotherapy, one advantage of mutagen-induced antigenicity over that provoked with viruses or haptens is that, since many different variants can be compared for their protective effect, efficient protectors can hopefully be selected. It will be interesting to establish by what mechanism tum+cells sometimes escape immunity conferred by tum- variants. There is mounting evidence that antigen-lossvariants play an important role in the escape of tumor cells

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(Bosslet and Schirrmacher, 1981). We have found that populations of P8 15 cells that have emerged after a nearly complete rejection contain variants that have lost one or more determinants of the tumor-associated antigen (Uyttenhove, 1981; Uyttenhove et al., 1983).

c. APPLICATION TO HUMAN CANCER It is quite obvious that at the present time many obstacles prevent a systematic attempt to apply mutagen-induced variants to cancer immunotherapy. Let us consider the scheme whereby tumor cells removed by surgery could be adapted to culture and treated with the mutagen so as to obtain immunogenic cells. These cells could then be injected to the patient with the purpose of eliciting a rejection response directed against the residual tumor cells that would have escaped classical treatment. A first difficulty of this procedure is the necessity of obtaining long-term cultures of human tumor cells. It is common knowledge that the success rate of adaptation of human tumor cells to culture is still very low for most tumors. Moreover, the establishment of cultures often requires a long time so that the cells might be available too late to be of any use to the patient. We see no reason why mutagen treatment applied to human tumor cells would not produce immunogenic variants as it does when applied to mouse tumor cells. While this still remains to be proved, it should be noted that mutagen-induced antigenic changes on human cells have actually been reported (Pious and Soderland, 1974). However, a major problem will be the necessity of detecting in vitro which clones are immunogenic. Even in the mouse system, it has not yet been possible to develop a discriminatory test involving unprimed lymphocytes. The approach of using repeated mutagenesis as for the DTIC lines would dispense from recognizing immunogenic clones. However, if viable cells were to be used as the immunogen, it would be necessary to obtain indications that the mutagenized population had lost its tumorigenicity. Will immunogenic variants induce a significant elimination of residual tumor cells? The fundamental requirement is of course that primary tumor cells carry some specific antigenic structure that could serve as a target for the response induced by the immunogenic cells. As stated above, it is difficult to know to what extent human tumors carry such determinants. However, the protection conferred by tum- variants against spontaneous mouse leukemias suggests that the use of tum- variants may extend considerably the class of tumors on which the presence of specific transplantation antigen can be ascertained. Even so, the protection conferred by tumvariants is rather weak and one cannot compare the protection against a challenge with a very small number of tumor cells injected after immunity is

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installed to that against residual tumor cells that were present in the organism before the immunizing variants. There residual cells may be much more difficult to eliminate. One could also fear that the presence of a large primary tumor would render the host tolerant with respect to the putative tumor-specific transplantation antigen. However, we observed that in the teratocarcinoma system, this does not occur. Tum- variants were rejected and conferred a protection in animals in which a large primary subcutaneous tumor had been removed surgically (Boon et al., 1979). In view of these many difficultiesit appears that a reasonable first goal will be to demonstrate that new transplantation antigens can be acquired by human tumor cells as a result of mutagenic treatment. We hope that progress made in the fundamental analysisof mutagen-inducedvariants will reach a point where these variants can contribute to treatment of human cancer. REFERENCES Amos, D., Corer, P., and Mikulska, Z. (1955). Br. J. Cancer 9,209 - 2 15. Artzt, K., Dubois, P., Bennett, D., Condamine, H., Babinet, C., and Jacob, F. (1973). Proc. Natl. Acad. Sci. U.S.A.70,2988-2992. Baldwin, R. (1966). Int. J. Cancer 1,257-264. Baldwin, R., and Pimm, M. (1978). Adv. Cancer Res. 28,91- 147. Basombrio, M. (1970). Cancer Res. 30,2458-2462. Biddison, W., and Palmer, J. C. (1977). Proc. Natl. Acad. Sci. U.S.A.74, 329-333. Birshtein, B., Preud’homme, J., and Scharf, M. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 3478-3482. Bonmassar, A., Frati, L., Fioretti, M., Romani, L., Giampietri, A., and Goldin, A. ( 1 979). Eur. J. Cancer 15,933-939. Bonmassar, E., Bonmassar, A., Vadlamudi, S., and Goldin, A. (1970). Proc. Natl. Acad. Sci. U.S.A.66, 1089- 1095. Bonmassar, E., Bonmassar, A., Vadlamudi, S., and Goldin, A. (1972). Cancer Res. 32, 1446- 1450. Bonmassar, E., Testorelli, C., Franco, P., and Cudkowicz, G. (1975). Cancer Res. 35, 1957- 1962. Boon, T., and Kellermann, 0. (1977). Proc. Not/. Acad. Sci. U.S.A. 74,272-275. Boon, T., and Marchand, M. (1982). Ann. Soc. Belg. Med. Trop. 62,69 - 7 1 . Boon, T., and Van Pel, A. (1978). Proc. Natl. Acad. Sci. U S A . 75, 1519-1523. Boon, T., Kellermann, O., Mathy, E., and Gaillard, J. (1975). “Teratomas and Differentiation,” pp. 161 - 166. Academic Press, New York. Boon, T., Van Pel, A., and Warnier, G. (1979). Colloq., 27th Protides Biol. Fluids pp. 173- 177. Boon, T., Van Snick, J., Van Pel, A., Uyttenhove, C., and Marchand, M. (1980). J. Exp. Med. 152, 1184- 1193. Boon, T., Vessiere, F., Van Snick, J., Van Pel, A., and Uyttenhove C., (1982). Arch. Biol. 93, 111-125. Boone, C., and Blackman, K. (1972). Cancer Res. 32, 1018- 1022. Bosslet, K., and Schirrmacher, V. (1981). J. Exp. Med. 154,557-562.

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CHROMOSOMESAND CANCER IN THE MOUSE: STUDIES IN TUMORS, ESTABLISHED CELL LINES, AND CELL HYBRIDS

Dorothy A. Miller Department of Human Genetics and Developmentand the Cancer Center College of Physicians and Surgeons, Columbia University,New York. New York

Orlando J. Miller Department of Human Genetics and Development. Department of Obstetrics and Gynecology. and the Cancer Center College of Physicians and Surgeons, Columbia University,New York. New York

1. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI.

Introduction . . . . .. .. . ... . . . . . . . ... . ., . ............... . .. .. . . . ... ... .. . ... .. . . . T-cell Leukemias. . . . . ......,............................................. B-Cell Leukemias . . . .. . . . ... . .. .......... ... . . . . .. .. . .. .. ... ..... . .. . ... ...... Plasmacytomas.. . . .. .. ... . .. .. ...... ..... . .. . . . .. . ... .. .... . .. . .. . ... . . ... . ... Evidence from Cell Lines for the Role of Chromosome 15. . .. . . . . . . . . . . . . . . . . Viruses and Genes Related t o Tumorigenesis ......... . Do All Tumors Have Specific Chromosome Changes?. . . . . .. . . . . ., ...... . . . . . Gene Amplification: Homogeneously Staining Regions and Double Minute Chromosomes .... ... ... ... ... .. . .... ...... ......... ... .. General Characteristics of Mouse Cell Lines . .. . .. .. . .. ... ... ..... .., ......... Suppression of Tumorigenicity in Hybrid Cells . . . .. . . ... . ..... ... .. . .. . ... . . . Chromosome Loss in Tumorigenic Hybrids .. ... . . . ... ... ... . . .. . . . . . . . . . . . . . Chromosome 4, in V i m Growth and Tumorigenicity .. . ..... . . . .. . .. . . . . . . . . Complementation Analysis and the Number of Genes Involved in Tumorigenicity ..., ........, .. . . . . . ........ . ..... ... . . . . . . . . ....... . ... ..... Trisomy 15 in Tumorigenic Hybrids.. ....... ...... .. .. . .. . . . ..... .... ........ Suppression of Tumorigenicity by DNA Fragments.. .. ... ........ ... ...... .. . SV40-Induced Transformation and Tumorigenicity in Hybrid Cells.. . . ... . . . Common Mechanisms of Tumor Suppression in Interspecific Hybrids . . . . . . . Genetic Basis of Transformation and Tumorigenicity. . .. ... . . . .. . . . . . ... . . . . . Altered Hexose Transport in Malignant Cells . ........ .. . ... . . .. . .. ... .... . . . . Host Cell Recruitment into Tumors via Cell Fusion.. .. ... . .. ... , . ... ... ... .. Conclusions.. . ... . . .. .. .. . .. .. . ... . .. ........ ........ . .. .. ................. ...

.....,.......................................................

153 154 156 157 158 158 159 160 163 167 167 168 170 171 172 172 175 176 177 177 178 179

1. Introduction

Determination of the karyotypic changes related to mouse tumorigenesis has had to await the development of banding techniques to identify normal and abnormal chromosomes, and the accumulation of enough data to sort out significant from fortuitous changes. Two general approaches have been used study of tumors per se, particularly leukemias, and study of somatic 153 ADVANCES IN CANCER RESEARCH, VOL. 39

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cell hybrids in which one or both parents is a tumorigenic cell line. These experimental approaches are dealt with separately; however, the same genes may control tumorigenicity in both cases. II. T-cell Leukemias

Inbred strains of mice were developed to provide model systems for studies of complex disorders such as cancer so that the genetic factors of the organism could be kept constant while environmental factors were varied. The AKR strain, which was developed by Furth et al. (1933), has provided such a model for leukemia studies. AKR mice routinely develop leukemias by about 10 months of age; the thymus, spleen, and lymph nodes become greatly enlarged in these mice and the cancer leads to death. Early studies showed that these thymomas usually had a modal number of 40 chromosomes, but almost always included some cells with 4 1 chromosomes (Kurita and Yosida, 1961 ; Cailleau and Munro, 1964). Ascites transfers or tissue cultured lines derived from the tumors showed more variable chromosome changes, both in number and structure (Cailleau and Munro, 1964).These, and other studies, made it appear advantageous to study tumors as early as possible if one were to detect the significant chromosome changes. The karyotypic results with spontaneous AKR tumors were confirmed when banded chromosome studies became possible. Dofuku et al. (1975) showed, by identifying the G-banded chromosomes in direct tumor preparations, that I0 of 1 1 tumors had three copies of number 15 (plus additional numbers 12 and 17 in two of them), while one tumor had only trisomy 12. Since trisomy 15 was the predominant change in seven of the tumors, this change appeared to be of particular importance, though perhaps not essential. Kodama et al. (1978) confirmed these findings using quinacrine and Hoechst 33258 banding; 13 of 20 tumors had some cells with trisomy 15 and the remaining 7 had a normal karyotype. AKR mice have Gross (N-tropic) murine leukemia virus (MuLV) integrated into the genome (Rowe el al., 1972). Wiener, Klein, and their colleagues ( 1978a) investigated the chromosome changes in tumors produced by a different (B-tropic) virus. Radiation leukemia virus (RadLV) was injected into C57BL/6 female mice and either the preleukemic bone marrow cells or leukemic spleen cells from these animals were injected into C57B1/6 males. Of 5 1 female-derived tumors that developed, 27 had a stem line of 41,XX,+ 15; in a few cases an additional number 17 was present. Leukemias induced by MuLV and RadLV must have arisen from the same minor T-cell population of the thymus, since they both had low levels of Thy-I (theta) antigen and high levels of H-2 alloantigen (Chazan and Haran-Ghera, 1976).

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X Rays and chemicals also induce leukemias which are associated with aneuploidy. Early studies on X-ray-induced leukemias in C57BL or RF mice (Kurita and Yosida, 1961) showed that there were 4 1 chromosomesin some of the tumor cells. The extra chromosome was later identified as a number 15 (Chan et al., 1979). Leukemias produced by injecting dimethylbenzanthracene (DMBA) or benzpyrene (BP) in CRW or C57BL mice resulted in a large proportion of the tumors (37/45 and 14/15, respectively) having trisomy 15, in addition to many other types of trisomy (Chan et al., 1979). In another study, female C57BL/6 mice were given DMBA by stomach tube and the tumor cells transplanted (iv or ip) to males. All analyzed leukemia cells were trisomic for number 15 (Wiener et al., 1978~). In this study, cells from the first and third transplantations virtually all had a modal number of 4 1 chromosomes, whereas those in the second transplantation were more variable in number. Thus even early passage tumors may be difficult to analyze. As with the viral-induced leukemias, the target cell was Thy-1 positive, but the authors point out that it is unlikely that the DMBA was merely activating a virus, because the viral induction of leukemia led to increased size of the lymphoid organs, whereas DMBA did not, nor did DMBA require an intact thymus. In order to determine whether only part of number I5 must be triplicated in leukemic cells, studies were carried out using the T( 14;15)6Catranslocation (Wiener et al., 1978b).In this translocation, chromosome number 15 is divided between a 1415chromosome that includes most of number 14 and the distal two-thirds of number 15, and a 1514chromosome that includes the centromeric third of-number 15 with, perhaps, a small amount of the distal end of number 14. Five of six leukemias induced by DMBA in CBA mice homozygousfor the T6 translocation(CBA T6T6) had two copies of the 1415 and a single copy of the 1514 chromosome; i.e., only the distal two-thirds of the 15 tended to be present in triplicate (Wiener et al., 1978b).When DMBA was used to induce leukemia in (C57BL X CBA T6T6)F1 mice, all six tumors had a modal number of 4 1 or 42 and this included two copies of the 1415chromosome plus a single 15. In this case there was selective trisomy of the number 15 derived from the CBA parent rather than the C57BL parent, even though the two strains were said to be equally susceptible to DMBA leukemogenesis (Wiener et al., 1979). In contrast, in similar studies with (CBA X CBA T6T6)F, mice there was no preference for one or another number 15 (Klein, 1981a). Thus the T6 translocation chromosome was not being selected for, but rather chromosome 15-linkedalleles characteristic of CBA appeared to be more favorable than those of C57BL for the development of the leukemia. The tendency of trisomy 15 cells to predominate in leukemia was so marked that leukemic cells tolerated trisomy of other chromosomesif these

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were attached to the number 15. T-cell leukemias were DMBA induced in animals homozygous for Rb(l.13, Rb(5.15), or Rb(6.15) centric fusion (biarmed) chromosomes. In each case, the leukemic cells had 36 telocentnc chromosomes plus three copies of the biarmed chromosome. In animals heterozygous for any one of these Rb chromosomes, either the Rb(-. 15) chromosome or the normal 15 could be duplicated (Spira et af., 1979). These results have been confirmed in spontaneous tumors in animals homozygous for the AKR/Rb(6.l5)1Ald translocation (Herbst et al., 1981). This biarmed chromosome normally is very stable. However, it was not always stable in the leukemic cells and an acrocentric number I5 (but not number 6), an Rb( 1 1.13, or an iso( 15.15) was observed occasionally (Herbst et af.,1981). In [AKR X AKR Rb(6. I5)]F, mice either the normal 15 or the Rb(6.15) chromosome was duplicated in the leukemic cells (Klein, 1981a). Thus, here, too, the important factor was not whether a translocation chromosome was duplicated in the leukemic cells, but whether there was trisomy 15. Although duplication of chromosome 15 was a very common early chromosome change in T-cell leukemias, it was not a uniform finding, and diploid tumors have been found (Kodama et al., 1978; Chan et af.,1979; Klein et af., 1980). Comparison of Moloney virus-induced diploid and trisomic T-cell leukemias showed that these did not differ in morphology or Thy- 1 type, but the mitotic rate in vivo was greater for the trisomic tumors (Spira et al., 198la). It is thus possible that the trisomic cells more or less rapidly outgrew the diploid tumor cells. However, Kodama et al. ( 198I ) followed the karyotypic changes of four initially diploid AKR tumors during in vivo passage. One tumor remained diploid after I5 passages; the three other tumors had some cells with trisomy 15 or 17, but these did not replace the cells with a normal karyotype. A similar conclusion was reached by Chan et a/. (1 981), who used N-methyl-N-nitrosourea to induce leukemias in newborn CFW or adult CFW or AKR mice. Trisomy 15 was more common in tumors of newborn than adult mice and in adult CFW than in adult AKR mice; although the hyperdiploid cells all had an extra copy of number 15, these cells coexisted in the same tumor with diploid cells. 111. B-Cell Leukemias

Evidence obtained from rats indicated that DMBA produced the same type of chromosome changes in sarcomas, leukemias, and carcinomas (Levan eta/., 1977a);that is, the agent rather than the target cell determined the type of chromosome changes. DMBA induces both T-cell and B-cell leukemias in SLJ mice, providing a test system to determine whether in mice trisomy 15 is correlated with the type of target cell or with the kind of

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inducing agent. Of the nine tumors that developed, six were T cell derived (Thy-1 positive).Four of these six were trisomic for the distal half of number 15, in this case translocated to the distal end of the X (thus defining more precisely the segment of number 15 that is involved in tumorigenesis), and two were normal (Spira et al., 1980). The remaining three tumors, which were not T cell in origin (Thy-1 negative), and five others produced in T-cell-depleted animals, were all trisomic for number 12, in some cases with an associated trisomy 3 or 18. None of the none-T cell tumors was trisomic for 15. Thus, a single agent produced different chromosome changes in different target cells. This view is deceptively simple, however, and trisomy 15 has been demonstrated in some B-cell leukemias. Radiation-induced tumors in BALB/FR(N2) or spontaneous tumors in (BALB X feral)F, mice were characterized by enlarged spleen and lymph nodes but normal thymus. The tumor cells were of B cell origin; they were not killed by antisera to Thy- I, as expected for thymocytes, and they expressed immunoglobulin (Ig) on the cell surface. Only a few cells were analyzed, but trisomy 15 (plus trisomy 17 in one case) was found in leukemias in both types of mice (Fialkow et al., 1980). Similarly, most spontaneous B-cell tumors in old BALB/c mice (Thy- 1 negative, surface Ig positive) were trkomic for number 15 (Wieneret al., 1981). A spontaneous B-cell leukemia (BCL,) was characterized not by trisomy, but by monosomy (or nullisomy) (Schroder et al., 1979).The claim that a T( 12;15)chromosome similar to that seen in plasmacytomas (Section IV) was present in this spontaneous B-cell leukemia (Voss et al., 1980)was not convincingly illustrated in the published figures. IV. Plasmacytomas

Mouse plasmacytomas can also show a change in chromosome 15, but in this case it is not trisomy; instead, the distal segment of number 15 is translocated to another chromosome. The original observations were made by Shepard et al. (1976), who compared cells from lines MOPC-2 1 and MOPC-3 15 (which were originally induced by mineral oil injection into BALB/c mice) with the X5563 line (a spontaneoustransplantable tumor in C3H mice). There were many chromosome changes, but the common finding in all three cell lines was an elongated 12 and a shortened 15 (or 18) chromosome. Since there is no evidence at this time that this is not a reciprocal translocation, we will refer to this as T( 12;15). The MOPC-3 1C line did not have such a T( 12;15 ) translocation but had a different reciprocal translocation, T(6;15) (Shepard et al., 1978). Since the breakpoint in the number 15 was at band D3/E in each case, the authors suggested this chromosome change might be characteristic of mouse plasmacytomas.

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DOROTHY A. MILLER A N D ORLANDO J. MILLER

Studies from other laboratories have supported the idea that plasmacytomas induced by a variety of agents show the same changes. Yoshida et al. (1978) found a T( 12;I 5) or T( 10;15) translocation in other established plasmacytoma lines. Pristane-induced plasmacytomas in BALB/c or CBB-22 mice had either the T( 12;15) or the T(6;15) translocation (Ohno et al., 1979). Tumors that had the T( 12;15) translocation produced either IgA/kappa or IgA/lambda chains, but those that had the T(6;15) produced only IgA/ kappa chains (Wiener et af.,1980). The genes for the immunoglobulin heavy chain have been mapped to chromosome 12, for the kappa light chain to number 6, and for the lambda light chain to number 16 (D’Eustachio et al., 198 1). Rowley ( 1982) pointed out that in human Burkitt lymphoma and B-cell acute lymphocytic leukemia the chromosomes (number 2, 14, and 22) that carry the immunoglobulin genes were specifically involved in translocations with number 8, and she suggested that a promoter from the latter was placed close to a locus involved in proliferation of this cell lineage. The same idea was expressed earlier (Klein, 198 la) to account for some of the mouse data. Recent findings in rat plasmacytomas support this same view (Wiener et al., 1982). V. Evidence from Cell Lines for the Role of Chromosome 15

A role for chromosome 15 in the expression of malignancy is supported by evidence that the most striking difference between the A9 cell line and a highly tumorigenic derivative of it, A9HT, was an increase from four to six copies per cell of chromosome 15 (Allderdice et al., 1973). Comparison of two independently derived Friend virus-induced leukemia cell lines maintained in vitro showed that the only chromosome change in common was the presence of a marker chromosome containing the two proximal Qbright bands of number 15 (D. A. Miller et al., 1979). Like plasmacytomas, these cells had a rearranged chromosome with a breakpoint in the distal third of the chromosome. VI. Viruses and Genes Related to Tumorigenesis

Although a number of factors are involved in the development of murine leukemia, it is not clear how any of these are related to specificchanges in the number of copies of chromosome 15, or to a specific break in this chromosome. Number 15 does not usually have a viral integration site. Two viral genomes have been mapped to specific chromosomes in the AKR mouse: Akv-I is located near the centromeric end of number 7 (Rowe et al., 1972) and Akv-2 is near the centromeric end of number 16 (Kozak and Rowe, 1980). Viruses are integrated at different locations in other strains (Rowe

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I59

and Kozak, 1980) and there is evidence that there can be stable germ line reinsertion of virus at additional sites in female AKR mice that are producing virus (Rowe and Kozak, 1980; Herr and Gilbert, 1982). Presumably, leukemic clones might show reinsertion into a site on chromosome 15, but there is no evidence on this point. In addition to integrated proviral sequences, there are several genes that help determine viral susceptibility, including the H-2 haplotype (mapped to number 17) (Miller and Miller, 1972) and Fv-2" Or alleles (mapped to number 4) (Rowe et al., 1974). Studies on chimeric mice showed that AKR CBA animals failed to develop leukemia at the rate or time expected for AKR, even though AKR and CBA have H-2k and Fv-1" in common (Barnes et al., 1973). The results with (CBA X AKR)F, animals and their first and second generation backcrosses to the AKR strain suggested that CBA-derived alleles of at least two other loci are involved in the suppression of leukemia in this system (Tuffrey et al., 1980). Interaction between retroviral oncogenes and the host genome can lead to suppression of the transformed phenotype (Section XVIII).

-

VII. Do All Tumors Have Specific Chromosome Changes?

Specific chromosome changes have been reported in a variety of tumors other than those already described. Dofuku et al. (1979) reported that spontaneous mammary tumors from GR, C3H, and noninbred Swiss mice all had trisomy 13in most of the cells, although the modal number remained 40 because there was random loss of other chromosomes. The identification of the chromosomes in these tumor cells has been, correctly, questioned by Spira (1980). Deletion of the distal region of number 2 was implicated in radiation-induced myelocytic leukemia (Azumi and Sachs, 1977; Hayata et al., 1979). Bladder epithelial cell lines transformed in vitro were near diploid; some had trisomy 6 or, more rarely, trisomy 15 and an abnormal 3 (Cowell, 1980a). More commonly it has been impossible to single out specific significant karyotypic changes in a tumor because there were multiple changes in chromosome number and/or marker chromosomes present. For example, karyotype analysis of a fetal colon carcinoma failed to define a specific change, even though there were only 40-42 chromosomes and the karyotype was stable (Manolov et al., 1979). Similarly, salivary gland epithelial cell lines transformed in vitro or in vivo were near tetraploid and had both structurally changed chromosomes and loss of one or two numbers, 1,4,7, 9, and 14 (Cowell, 1981a). Some tumors appear to have no chromosomal aberrations. This was true for some spontaneous leukemias (Kodama et al., 1978), as well as those

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induced by treatment with achemical (Chan et al., 1979),radiation (Chan et al., 1979), or a virus (Klein et al., 1980). Klein et al. (1980) postulated that the large cellular insert carried by the Abelson virus could carry out the same function as gene duplication does in tumors induced by other agents. However, it has remained difficult to rule out the presence of very small chromosome changes in apparently diploid tumor cells. Studies on very extended prometaphase chromosomes from bone marrow cells of human acute lymphocytic leukemia (ALL) patients showed that subtle structural chromosome changes were present in the tumor cells that could not be detected in the usual preparations (Yunis et al., 1981). Such studies have not yet been carried out in the mouse. Another explanation for a normal karyotype in tumor cells was offered by Cowell ( 1981b), who derived an apparently diploid line from a carcinoma-producing mouse bladder epithelial cell line that had previously had three copies of number 15. He speculated that a normal number 15 was lost and that the two numbers 15 that remained were duplicated abnormal chromosomes. Teratocarcinomas are considered by some investigators to differ from almost all other cancers in lacking any mutational change (deletion, translocation, base substitution, etc.) (Mintz and Fleischman, 1981). Cell lines with normal karyotypes have been derived from teratocarcinomas (Martin et al., 1978; McBurney and Strutt, 1980; Mintz and Cornmiller, 198I), presumably representing undifferentiated stem cells. One of these lines, which retained a stable karyotype even after freezing, was successfully injected into blastocysts and subsequently appeared in some cells in all the somatic tissues, including the oocytes (Mintz and Cornmiller, 1981). No karyotypic changes were detected. VIII. Gene Amplification: Homogeneously Staining Regions and Double Minute Chromosomes

Gene amplification, an increase in the number of copies of a gene or other DNA sequence, occurs under a variety of circumstances. It is particularly common among retrovirus-related oncogene sequences (Callahan and Todaro, 1978), which may be significant as far as mechanism is concerned. Up to 100 copies of a viral LTR-related sequence were present on the Y chromosome in normal mice of one species, with smaller numbers observed in other species (Phillips et al., 1982). Gene amplification may play a role in tumorigenesis, and cytogenetic methods have been important in demonstrating the presence of amplified sequences in tumor cells. Two types of chromosome abnormality have been found almost exclusively in transformed or malignant cells: homogeneously staining regions (HSRs) and double minute chromosomes (DMs).HSRs were first described

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in Chinese hamster cell lines that became resistant to methotrexate by markedly increasing the number of copies of the dihydrofolate reductase gene (DHFR) (Biedler and Spengler, 1976).This HSR was pale stainingwith Giemsa and showed little or no differentiation along its length, hence the term homogeneous. However, not all HSRs are homogeneously stained (George and Francke, 1980;Cowell, 1980b),presumably because the nature of the DNA in the repeating unit is different in each HSR. One type which contained secondary constrictionswas referred to as a DSR, or differentially staining region (0.J. Miller et al., 1979). HSRs may be located interstitially or terminally, or they may comprise an entire chromosome arm. The elongated segment may be at the site of the normal gene; for example, an HSR containing amplified rRNA genes was present on the short arm of a number 14 in a phenotypically normal human (D. A. Miller et al., 1978). This is not always the case, however, and HSRs may appear in sites not normally associated with the gene in question (0.J. Miller et al., 1979). HSRs have been found in tumors or tumor cell lines from various rodents (Biedler and Spengler, 1976; Bostock et al., 1979; 0. J. Miller et al., 1979; George and Francke, 1980; Cowell, 1980b; Berenson et al., 1981; Kopnin, 1981; Levan and Levan, 1982). The only amplified gene yet identified in untreated tumor cell lines is the 18s 28s ribosomal RNA gene (0.J. Miller et al., 1979; Tantravahi et al., 1982). Although the total number of copies of the rRNA genes was increased 3-fold in the rat XC sarcoma line and 10-fold in the rat H4 hepatoma line, with up to 3000 or more copies of the gene per cell in the latter, most of the amplified genes were transcriptionally inactive. and highly methylated (Tantravahi et al., 1981, 1982). Nevertheless, the numbkr of active genes was somewhat increased over the number found in normal cells. Whether rRNA gene amplification is a common feature of tumor cells remains to be determined. An increased size of nucleolus organizer regions (which contain active rRNA genes) was observed on one chromosome 19 in the FSD-IF4 Friend erythroleukemia line (D. A. Miller et al., 1979), on one number 18 in the S49.1 lymphoma line (Francke and Gehring, 1980), and on both numbers 15 in the P10 teratocarcinoma cells, where one of the NORs was nearly twice the size of the other (McBurney and Strutt, 1980). Cell lines made resistant to methotrexate contained multiple copies of the DHFR gene in an HSR (Dolnick et al., 1979;Berenson et al., 1981). Since in both kinds of HSR there was much more DNA than could be accounted for by the estimated number of amplified rRNA or DHFR genes, additional DNA must have been amplified. The nature of the DNA is not known, nor is the nature of the sequences amplified in the HSRs in other cell lines. Not every elongated marker chromosome arises by gene amplification, nor is gene amplification always associated with a visible HSR or DMs. One

+

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DOROTHY A. MILLER A N D ORLANDO J. MILLER

case illustrates both these principles. Melton et al. (198 1) found a 25- to 50-fold increase in production of the X-linked enzyme hypozanthine-phosphoribosyltransferase (HPRT) in the mouse neuroblastoma NBR 4 line. There was one very long chromosome in the cells, containing three copies of the X attached in tandem to a number 12. The cells did not contain an HSR despite an approximately 50-fold amplification of the gene (Brennard et af., 1982). Double minute chromosomes (DMs) were initially described by Cox et af. ( 1965) in human childhood malignancies and by Mark (1967) in murine Rous sarcomas. DMs are small, acentric, double fragments of chromosome. They can vary in size, even within the same cell, and sometimes appear ring shaped (Levan and Levan, 1980). The number of DMs varies from cell to cell, presumably because they lack a centromere and are segregated randomly to daughter cells. DMs replicate only once early in the cell cycle (Levan et af.,1978). In several tumors DMs showed an inverse relationship to HSRs (Levan et af.,1978; Cowell, 1980b; George and Francke, 1980; Kopnin, 1981) and it can be anticipated that the staining of DMs will be as variable as that of HSRs. The mechanism by which the transformation from HSR to DMs or DMs to HSR takes place is not known and the circumstances under which such transformations were observed have varied. HSRs were found in stable methotrexate-resistant mouse or Chinese hamster cell lines and DMs in unstable resistant lines (Kaufman et al., 1979), implying the HSRs were less easily lost than were DMs. Levan et af.( 1977b) correlated the presence of DMs in the SEWA line of polyoma-induced osteosarcoma in ASW mice with growth in vivo, particularly in A strain as compared to ASW mice. During one period of in vitro culture, the DMs were replaced by C-bandless metacentric chromosomes (CMs) which were pale staining and early replicating (Levan et af., 1978). The CMs disappeared during in vivo growth, being again replaced by DMs. In more recent studies, the DMs were replaced by HSRs in various terminal locations (Levan and Levan, 1982). George and Francke (1980) studied two sublines from a Y 1 mouse adrenocortical tumor cell line; one subline had HSRs and the other had DMs. Although both lines had the same doubling time, when they were cocultivated the cells with the HSR predominated. Amplified DNA sequences isolated and cloned from the DMs hybridized to the HSRs as well as the DMs, indicating that the DMs and HSRs shared at least some amplified DNA sequences (George and Powers, 1982). Some mouse salivary gland epithelial cell lines produced by DMBA treatment in vitro had HSRs and DMs in the same lines but not the same cells (Cowell, 1981a). In one case, DMs were present initially but were replaced by an interstitial HSR (Cowell, 1980b).

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Despite their erratic transmission to daughter cells, DMs can remain in a cell line for an extended period (Salum and Larripa, 1975). DMs were not eliminated by fusion of DM-containing cells with normal or transformed cells (Balaban-Malenbaum and Gilbert, 1980; Kano-Tanaka et al., 1982).

IX. General Characteristicsof Mouse Cell Lines

Established cell lines, many of them derived from tumors, have been used to study tumorigenicity both of the cell lines themselves and of their hybrids. A list of mouse cell lines is presented in Table I. These have been selected because the published banded karyotypes were clear enough to be of use to the reader, although not every chromosome has been correctly identified in some of them. Most cell lines are heteroploid with karyotypic variation from cell to cell: although the modal number of chromosomes is given in the table, in some cases this represents a relatively small proportion of the cells and it is not uncommon for each of the 10-20 karyotyped cells to have a slightly different chromosome constitution, including abnormal, or marker, chromosomes. Thus, even cell lines that have the diploid modal number, 40, may not have a normal karyotype. All 20 pairs of mouse chromosomes are acrocentric. Abnormal chromosomes can be formed in a variety of ways: reciprocal translocation, centric fusion, isochromosome formation (both arms identical), deletion, inversion, insertion, amplification of a chromosome region, and so on. All these types of abnormalities have been observed in one or more cell lines, but not every type may be present in a particular line. The origin of a biarmed chromosome is easy to determine if each arm is derived from a complete normal mouse chromosome. The origin of acrocentric markers may be much more difficult to determine, especially if there have been multiple chromosome interchanges or if small segments are involved. Such abnormal chromosomes are often referred to as unidentified markers. Cell lines show strikingdifferencesin the types of abnormal chromosomes present. For example, in the L cell derivatives A9 and B82,359/0or more of the chromosomes were biarmed, including a number of isochromosomes (Allderdice et al., 1973); in MSWBS cells, 35Yo of the chromosomes were biarmed, but none were isochromosomes (Hashmi et al., 1974); and in Ehrlich- Lettre diploid (ELD) cells, only 2-4% of the chromosomes were biarmed (Nielson, 1976). The number of acrocentric marker chromosomes was similarly variable: only 1 of 40 chromosomes was abnormal in the S49 line compared to 19 of 39 in EL4 (Francke and Gehring, 1980). It is well to keep in mind that cell lines with the same name or designation or sublines of these that have been maintained in different laboratories may differ in

TABLE I KARYOTYPIC CHARACTERISTICS OF MOUSECELLLINES Number marker chromosomes Cell line C3H/lOTj P 1OC-2-5 C57BL melanoma Clone me1 2C Clone PG 19 Clone mmc 38 Cloudman S9 1 CCL53.1 (PCHPRT-) CSG lines Ehrlich ascites ELD, 7 strains EL4 Friend virus Clone 745 F S D 1F4 bld L cell A9 A9 B82 A9HT

Modal number

Biarmed (iso-)

Acrocentric

Other

71

-

2

Centricfrag.

1-2(2)

1-2

61-65

5

6 4 10

12 65-72

-

DMs. HSRs 3dots

2

Many

41-46 39

1

12 19

39 38 37

4 3(1) 4(2?)

med 59 m53.6 m50.4 med 55

23(many) 19(4) 34(14) 26(many)

7 4 2 12 11

5 10

C3H

Centric frag., DMs C57BL/6J DBA/2

Few

43

1

Tumor Strain

C57BL

2-4dots -

type

How grown

Reference

Fibrosarcoma

Plastic discs Boone ez al. ( 1979) in vivo Attached Boone ez al. ( I 979)

Melanoma

Attached Attached Attached Attached Attached

Melanoma

Salivary gland Attached carcinoma Mammary car- Ascites cinoma Ascites Lymphoma Suspension

Jonasson el al. (1977) Jonasson ef al. (1977) Jonasson ef al. (1977) Jonasson ez al. (1977) Halaban el al. ( I 980) Cowell ( 198I a) Sasaki ez al. (1974) Nielson ( 1976) Francke and Gehring ( 1980)

HSR=MI

Leukemia DBA/2 DBA/2 (BALB/c X BALB/B)F,

Suspension Suspension Suspension

D. A. Miller er al. (1979) D. A. Miller el al. (1979) Rajan (1 977)

2 dots I-2dots 1-2 dots 2dots

C3H

Attached Attached Attached Attached

AUderdice et al. (1973) M. H. Russell et al. (1977) M. H. Russell ef al. (1977) Allderdice et al.(1973)

L1210

38

L1210 L1210 L1210 L5 I78Y LBN/a2 LBN/b3 MB lines

40 41 39 40 41 42 39-41

MM-46

71

TMT- I ,2 MOPC-2 1 SLU-5 MSC MSWBS P3-X63-Ag8 RAG S49.1 SEWA WEHI-3

Y 1-DM, CCL74

68 73 74-75 Hetero 28-29 63-64 m6 I

3

8

1 dot

DBA

1 dot

HSR(M T X )

-

l(1)

2 1

+

11 l(1) 32)

6 9 9

+

5 18-20 9

BN

-

Leukemia

C57BL

-

C3H

-

BALBIcd

-

BALB/c

-

A.SW BALBIc

Osteosarcoma Leukemia

LAF,

Adrenocortical Attached

-

2(1)

2 2

39

3

3

DMs

38

2

7

2 HSRs

40 82

6(4)

45

Ascites Suspension Suspension Suspension Ascites Ascites Attached

Bladder carcinoma Mammary car- Ascites cinoma Suspension Plasmacytoma Solid Suspension Sarcoma Attached Sarcoma Ascites Suspension Myeloma Renal adenoAttached carcinoma Lymphoma Suspension

HSR = m2 1-2 dots -

40 76-79

Ascites

-

BALB/c BALB/c (AxA.SW)F,

Ascites Suspension

Dowjat and Wlodarska (1981) Kusyk (1978) Kusyk ( 1 978) Trowsdale ef al. (1980) Berenson et al. (198 1) Bregula er al. (1979) Bregula et al. ( 1 979) Cowell (1980a) Tsuboi e?al. ( 1980) Tsuboi er al. (1980) Shepard er al. (1974) Shepard er al. ( 1 979) S. W. Russell er al. (1974) Hashmi er al. (1974) SchrMer ef al. ( 1980) Hashmi er al. (1974) Francke and Gehring ( 1980) Jonasson ef al. (1977) Bornstein and McMorrow ( 1980) George and Francke ( 1980)

carcinoma

Y I-HSR YACIR X5563 a

3 Many

-

Attached A.Sn C3H

Lymphoma Myeloma

Suspension

In some cases the mean (m) or the median (med) is given instead of the modal number of chromosomes.

George and Francke ( 1980) Jonasson ef a/. (1977) Shepard et al. (1976)

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DOROTHY A. MILLER A N D ORLANDO J . MILLER

chromosome makeup (e.g., ELD, Nielson, 1976; L1210, Kusyk, 1978; Trowsdale et al., 1980; Dowjat and Wlodarska, 1981). Dot chromosomes presumably reflect deletion of most of the chromosome arm, leaving an intact centromeric region; these chromosomes are distributed regularly at mitosis and dots have remained in L cells and ELD cells for years. Double minute chromosomes lack centromeres and since they are not distributed regularly may be amplified or lost from a culture; under certain conditions they may be replaced by homogeneously staining regions (see Section VIII). Some cell lines have gone through a tetraploid stage, with subsequent reduction in chromosome number, e.g., L cells and RAG cells. In these cases, four copies of some normal chromosomes are seen, and sometimes two copies of marker chromosomes that were present at the time of chromosome duplication without cytokinesis. Many transformed cell lines show no evidence of tetraploidization (Friend leukemia cells, D. A. Miller et al., 1979; S49, Francke and Gehring, 1980), which is thus not an essential step in cell transformation. The presence of mutiple karyotypic changes in cell lines, as well as the progressive changes that occur with time (see, e.g., Nielson, 1976), make it difficult to identify critical changes related to tumorigenesis in these lines (Allderdiceet al., 1973). Nevertheless, the karyotypes of cell lines have been used in a variety of investigations. Long established lines of the same type of tumor have been compared and a common change noted (plasmacytomas, Shepard et al., 1976; Friend leukemia, D. A. Miller et al., 1979). In other cases, evolution with time has been evaluated [conversion ofDMs to CMs in SEWA, Levan et al., 1978; decrease in tumorigenicity without loss of a T(12;15) chromosome, Shepard et al., 1979; conversion in vitro from normal to malignant state, Cowell, 1980al or after altered growth conditions (growth in vivo on a plastic disc, Boone et al., 1979; ascites derivative of a solid tumor, Tsuboi et al., 1980). The identification of marker chromosomes has been used to assure that emergent tumor cells were of the purported origin (S. W. Russell et al., 1974; Boone et al., 1979; Weinhold et al., 1979; Tsuboi et al., 1980). In a few studies, intraspecific mapping has been camed out using mouse cell lines (HPRT, Hashmi and Miller, 1976; glucocorticoid receptors, Francke and Gehring, 1980).Much effort has gone into the derivation of diploid cell lines from teratocarcinomas (see Mintz and Cornmiller, 1981); these cells have been used to study such diverse problems as the time of X-inactivation (Martin et al., 1978; McBurney and Strutt, 1980)and the ability of tumor cells that have been incorporated into blastocysts to behave as normal cells in the tetraparental progeny (Mintz and Cornmiller, 1981). The extensive use of cell lines in somatic cell hybrid studies is discussed below.

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X. Suppression of Tumorigenicity in Hybrid Cells

The ability of malignant cells to grow progressively in a syngeneic, athymic, or immunosuppressed host is called tumorigenicity. It is a late step in the development of malignancy, and is not identical with earlier stages in carcinogenesis such as initiation, promotion, or transformation. (For an interesting discussion of transformation, see Puck, 1979.) The tumorigenicity of virtually all types of tumor cells is suppressed by hybridization with normal diploid cells, other nontumorigenic cells, or even some types of tumor cells, except when the hybrid cells have undergone significant and specific chromosome loss from the nontumor parental genome (Harris, 1971; Wiener et al., 1974a,b,c; Jonasson et al., 1977; Evans et al., 1982). This rule applies no matter what the cell or tissue origin of the tumor (sarcoma, carcinoma, melanoma, lymphoma, teratocarcinoma) or the initial carcinogen (spontaneous, methylcholanthrene, polyomavirus, SV40 virus, Moloney virus), and holds even in crosses between cells of different mammalian species such as mouse X human (Jonasson and Harris, 1977), mouse X rat (Lyons and Thompson, 1977), or mouse X hamster (Howell and Sager, 1979). There are still no proven exceptions to this rule despite various claims to the contrary, usually in studies lacking the precise cytogenetic analysis now possible using both cytological and genetic markers. XI. Chromosome Loss in Tumorigenic Hybrids

The clear-cut association of chromosome loss with the emergence of tumorigenic hybrid cells from much less tumorigenic hybrid parents was first noted by Hams et al. ( 1969) in studies on intraspecific mouse hybrids. This was confirmed in several laboratories (e.g., Ruddle et al., 1970; Belehradek and Barski, 1971) but especially by Hams, Klein, and their associatesin a large series of hybrids between various highly tumorigenic cell lines and the much less tumorigenic A9 cell line or diploid cells such as fibroblasts and lymphocytes (Bregula et al., 1971; Klein et al., 1971;Wiener et al., 1971, 1973, 1974a,b). The hybrids fell into several classes. One class retained a high number of chromosomes, sometimes equal to the sum of the parental contributions. These hybrid cells were generally nontumorigenic and the occasional malignant segregant which arose from them generally had a reduced number of chromosomes. Another class of hybrids underwent more rapid and extensive chromosome loss, and showed a much higher level of tumorigenicity. Hybrids between tumor cells and freshly isglated diploid cells were frequently found in this class, e.g., Ehrlich ascites tumor cells X embryonic fibroblasts (Bregula et al., 1971) and this was especially true of tumor X lymphocyte hybrids (Wiener et al., 1974a).

168

DOROTHY A. MILLER A N D ORLANDO J. MILLER

Crosses between malignant polyomavirus-transformed cells and T cell lymphocytes produced hybrids which, by the time of examination, had already undergone extensive chromosome loss; they had an average of 56.5 chromosomes instead of the approximately 93 expected (Berebbi et al., 1981). Their high degree of tumorigenicity is thus no surprise. It is interesting that the hybrid cells not only retained the polyoma genome and expressed polyoma antigens but also expressed various T-cell properties: morphology, growth as a suspension culture, and Thy 1-2 differentiation antigens. Several workers have suggested that the differentiated state of a cell may be an important variable in the tumorigenicity of hybrids. This is clearly not the case in this or most other hybrid systems. The tumorigenicity of a wide variety of tumor cells is suppressedwhen they are hybridized with fibroblasts (Wiener et al., 1971)or lymphocytes (Wieneret al., 1974a). In such hybrids, tumor suppression requires retention of one or more chromosomes from the diploid parent, regardless of the epigenetic state of the cell. It should be emphasized that chromosome loss need not be extensive for the reappearance of tumorigenesis. R. A. Miller and Ruddle (1976) studied four hybrid clones of PCC4azal teratocarcinoma cells (strain 129/Sv origin) X C3H/HeJ thymus cells. All four had 4- 6 fewer chromosomes than expected of a 1 : 1 fusion of modal cells, and all four were tumorigeneic. Four tumors which arose from three clones had 0, I , 1, and 7 fewer chromosomes than the hybrid cells injected, i.e., 5 - 1 1 fewer than the average expected. Below, evidence will be summarized that a gene or genes on chromosome 4 plays an important role in the expression or suppression of tumorigenicity, while genes on chromosome 15 and perhaps other chromosome are also involved. XII. Chromosome 4, in Vitro Growth and Tumorigenicity

Evans et al. ( 1982)found that PG 19 melanoma X A/Sn diploid fibroblast hybrids grew very slowly when both copies of chromosome 4 from the diploid parent had been retained, and usually could not be cloned. The single clone obtained had almost complete sets of both parental chromosomes and did not form tumors. Jonasson et af. (1977) showed that similar melanoma X CBA diploid fibroblast hybrid clones regularly lost one copy of chromosome 4 from the diploid parent. Furthermore, all tumors which arose from in vivo passage of the hybrids lost the second copy of the diploid number 4 as well. The loss of both copies ofthe diploid-derived number 4 in tumors arising from these hybrids was demonstrated by loss of the glucose6-phosphate dehydrogenase-lb (Gpd- 1") variant found in CBA cells (Jonasson et af., 1977). Similarly, tumors which arose from A9HT X diploid C57BL hybrids had lost the Gpd- 1" (C57BL) variant and both copies of the C57BL chromosome 4 (Fenyo et af.,1980). Of seven tumors produced by

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melanoma X CBA T6T6 hybrids, Jonasson et al. (1977) found both copies of chromosome 4 derived from the CBA parent had been lost but that two copies of chromosome 4 were present in three of the tumors and three copies (giving trisomy 4)were present in four. The extra copy of number 4 must have arisen by nondisjunction in the melanoma parent. This is an important point, because it emphasizes the necessity of using genetic markers or chromosome markers to distinguish the origin of specific homologs; simply counting the number of copies is not enough. This point was overlooked by Halaban et al. ( 1980),who concluded, because there were several copies of chromosome 4 and a virtually unchanged modal number of chromosomes in the excised tumors compared to the hybrids from which they arose, that the malignancy of these hybrids was not due to the selective growth of a subpopulation of cells that lost this or any other particular chromosome. This conclusion is not justified in any case because the two hybrid lines in question arose in vivo by fusion of injected tumor cells with host cells and were thus already selected as being composed of cells capable of in vivo growth, i.e., tumorigenicity. Not all malignant hybrids appear to have eliminated both copies of the normal chromosome 4. Croce et al. (1979) studied three tumorigenic hybrids between the human spontaneous fibrosarcoma line, HT 1080, and mouse peritoneal macrophages. The presence of mouse PGM2 in tumor cells derived by animal passage of all three hybrid clones indicated that at least one copy of mouse chromosome 4 had been retained by some of the tumor cells. A parallel cytological study of cells in two of the three hybrid lines showed that each had less than a haploid mouse complement, with no copy of either chromosome 4 or 14 in most cells. (0.J. Miller et al., 1976). This fits with the earlier work showing the importance of chromosome 4 in slowing in vitro growth. The analysis of hybrids between tumor and diploid cells with different cytological markers on chromosome 4 (they show a marked difference in the size of the heterochromatic C-band region) has confirmed the importance of specific loss of chromosome 4 derived from the normal parent and the fact that both copies of the normal 4 need not be lost for a hybrid to be tumorigenic (Evans et al., 1982). Furthermore, the number of copies of number 4 from the malignant parent appears to have a reciprocal effect, enhancing tumorigenicity. This was observed in YACIR lymphoma X CBA fibroblast hybrids, SEWA sarcoma X Rb( 16.17)7Bnr fibroblast hybrids, A9HT sarcoma X CBA/H-T6T6 fibroblast hybrids, and TA3HaB mammary carcinoma X CBA/H-T6T6 fibroblast hybrids. The enhancing effect of extra copies of the tumor chromosome 4 was manifested both when it occurred as an isolated nondisjunctional product and when it occurred as part of a 2 tumor: 1 normal cell hybridizational event (Evans et

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al., 1982). It is possible that the same gene dosage effect accounts for the general observation that asymmetric hybrids with 2 tumor cells: 1 normal cell are more likely to be tumorigenic (e.g., Schafer et al., 198I ; Evans et al., 1982). Thus, Murayama-Okabayoshi et al. (197 1) found that the number of hybrid cells necessary to produce tumors in 50% of the animals inoculated was lowest in 2 Ehrlich : 1 L-cell hybrids, 1O-fold higher in 1 Ehrlich : I L-cell hybrids, and 100-fold higher in 1 Ehrlich :2 L-cell hybrids. Chromosome loss also occurred in these hybrids, but it is likely that a dosage effect was also involved, with the percentage of tumorigenic cells in the hybrids reflecting the relative numbers of one or more chromosomes contributed by the Ehrlich ascites tumor cells or by the low or nontumorigenic L cells. In hybrids involving all kinds of malignant tumors examined by Harris and his associates (carcinoma, teratocarcinoma, sarcoma, lymphoma, and melanoma), suppression of tumorigenicity required the activity of some gene located on the normal number 4. This supports the notion that all these tumors have some still undefined genetic lesion in common. XIII. Complementation Analysis and the Number of Genes Involved in Tumorigenicity

Hybrids between malignant cells of two different types were sometimes highly malignant despite retention of approximately the complete sets of parental chromosomes. Since the tumorigenicity of each type of tumor was suppressible by hybridization with nontumorigenic cells, this suggests that the same genetic loss or impaired gene function was present in the various types of tumor cells. Examples of this were hybrids between methylcholanthrene-induced MBA sarcoma cells and Moloney virus-induced YACIR lymphoma cells (Harris, 1971) and hybrids between tumorigenic A9HT cells and Ehrlich, MSWBS, SEWA, or YACIR tumor cells (Wiener et al., 1973). Sometimes, however, complementation occurred between two types of tumor cells, and their hybrids were nontumorigenic (chromosome loss in these hybrids could, of course, regenerate tumorigenicity). MSWBS sarcoma X YACIR lymphoma hybrids were considerably less tumorigenic than either parent (Harris, 1971) and the same was true of HTC rat hepatoma cells X B82 mouse L cells (Lyons and Thompson, 1977). TA3B mouse mammary carcinoma X Bl Syrian hamster sarcoma cell hybrids were almost totally nontumorigenic in nude mice (Marshal and Dave, 1978). Hybrids between tumorigenic SV40-transformed mouse embryo cells and mouse melanoma cells were also nontumorigenic (Gee and Harris, 1979). In principle, this kind of complementation analysis should make it possible to define, from the number of complementation groups, the number of separate genetic defects which can lead to the expression of tumori-

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genicity. It would be interesting to apply this approach to tumors known to have specific chromosome imbalances, e.g., 15-trisomic B-cell and T-cell leukemias. XIV. Trisomy 15 in Tumorigenic Hybrids

Jonasson et af. (1977) found that hybrids between melanoma cells and diploid cells (fibroblasts or lymphocytes) showed a profound suppression of their tumorigenicity. Tumors did arise in a small proportion of inoculated mice, but only hybrids in which one of the chromosomes 15 had undergone duplication to produce trisomy 15 were capable of such proliferation in viva This same requirement for increased dosage of some gene or genes located on chromosome 15 was seen in hybrids between other malignant cells and diploid cells as well. This change was in addition to the loss of chromosome 4 already noted in these tumorigenic hybrids. Hybrids between SEWA polyoma-induced sarcoma cells and diploid fibroblasts had several interesting features (Jonasson et af., 1977). SEWA cells gave a 100% tumor incidence with inocula of 5 X lo4 cells while the hybrid cells gave a much lower take incidence, as noted earlier in SEWA X CBA T6T6 hybrids (Wiener et af.,1971). With larger inocula, the SEWA X fibroblast hybrids gave much higher take incidences than melanoma X fibroblast hybrids. This ease of escape from suppression of tumorigenicity may reflect the preexisting trisomy 15 of the SEWA tumor cells themselves, and the fact that both copies of the diploid chromosome 4 did not need to be eliminated from the emergent tumor cells (Jonasson et af., 1977). Since fewer specific changes in chromosome complement were thus required for expression of tumorigenicity, a larger proportion of the SEWA X diploid hybrid cells was tumorigenic. The amplified genes presumed to be present in the DMs present in the SEWA cells (Section VIII) might also have played some role. Lymphomas which arise in AKR mice are frequently 15 trisomic (Section 11). Hybrids of AKR-derived lymphoma line TlKAUT with CBA T6T6 cells were of two types: highly tumorigenic lines in which there were more than the expected number of the AKR-derived number 15 and fewer copies of the CBA-derived number 15, and low tumor lines in which there were slightly fewer than the expected number of AKR 15 (Spira et af.,1981b). This suggests that a gene on the AKR number 15 can enhance tumorigenesis, and that it is not simply a matter of gene dosage, since it is not the total number of chromosomes 15 but the number derived from the malignant parent that is important. It is possible that this chromosome may have undergone a rearrangement or other change which specifically activated a normal cellular gene whose product leads to a transformed phenotype

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Klein, 198 la). A trans-acting control, emanating from the normal homolog, may be involved, as noted earlier in the somatic cell hybrid studies. XV. Suppression of Tumorigenicity by DNA Fragments

Several laboratories have reported suppression of tumorigenicity in mouse- human hybrids which have minimal or no detectable human chromosomes (Jonasson and Hams, 1977; Chopan and Kopelovich, 198 1). Rodgers (1 979) showed, by molecular hybridization, that the equivalent of about 0.6% of the human genomic DNA was still present in PG 19 mouse melanoma X irradiated human fibroblast hybrids which Jonasson and Hams (1977) found had no detectable human chromosome. Presumably, some of the human DNA which had been fragmented by the intensive irradiation was retained in the hybrid cells and was able to suppress tumorigenicity. In this regard, it is interesting that cytoplasmic transmission of a factor suppressing tumorigenicity was demonstrated in cybrids produced by fusing enucleated cytoplasts of nontumorigenic cells with hamster tumor cells (Howell and Sager, 1979), mouse teratocarcinoma cells (Shay et af., 198 l), or mouse mammary tumor cells (Giguere and Morais, 198 1). Howell and Sager (1980) reported that SV40 DNA, flanked at each end by at least 1 kilobase of host genomic DNA, could be transferred with the cytoplasm of enucleated SVT2 mouse cells when these were fused with 3T3 cells to form cybrids. They also found that three of four of the reciprocal cybrid clones, produced by fusing enucleated 3T3 cells with tumorigenic SVT2 cells, were nontumorigenic; they concluded that a gene or genes from nontumorigenic cells could be transferred via the cytoplasm and thus restore a normal phenotype to tumorigenic cells (Howell and Sager, 1981). This finding opens the door to molecular cloning of the gene(s) involved, analogous to the cloning of oncogenes using transformation of cells of one species with DNA from transformed cells of another species (Padhy et af.,1982; Pluciani et af.,1982). It is interesting that DNA from normal mouse BALB/3T3 or NIH/3T3 cells could transform NIH/3T3 cells, but only if the DNA was sheared to fairly small lengths, presumably freeing a transforming gene from a cis-acting controlling region (Cooper et al., 1980). Chromosomal rearrangements may act in the same way, leading to increased expression of normal cellular genes (Klein, 198 la). XVI. SV40-Induced Transformation and Tumorigenicity in Hybrid Cells

It has been known for many years that while the addition of a viral genome to a cell can lead to a transformed phenotype, the transformed cells may not be tumorigenic, as shown by progressive growth. For example, Kit

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et al. (1969) transformed mouse kidney cells with SV40 virus and maintained them in cell culture for many passages. Cells at passage 26 were not tumorigenic but cells at passage 7 1 were. The tumorigenicity of polyomavirus-induced sarcomas and Moloney virus-induced lymphomas could be suppressed by hybridization with A9 cells, even though the hybrids retained the viral genome and expressed viral antigens (Harris et al., 1969; Harris, 1971), indicating that tumorigenicity could be dissociated from transformation and expression of viral antigens. This was true of SV40 virus too. Because of the great amount of work done with SV40-transformed cell X normal cell hybrids, and the striking discrepancies between the conclusions reached by workers in different laboratories, we have reviewed the data and figures in the numerous published reports with particular care. This analysis indicates that there are virtually no conflicting observations; from a cytogenetic point of view, the results are all consistent with the principle enunciated above (Sections X and XI) concerning tumor suppression and chromosome loss in hybrid cells. Howell and Sager ( 1979)made hybrids between tumorigenic SV40-transformed SVT2 mouse cells and a nontumorigenic derivative of 3T3 cells. The hybrids retained the transformed phenotype and expression of T antigen but had much reduced tumorigenicity in athymic nude mice. Three of 14 hybrid clones were nontumorigenic and 11 were tumorigenic in the usual in vivo assay; however, in each of the two tumorigenic hybrid clones tested, 100- 1000 times as many hybrid cells as SVT2 parental cells were required to produce tumors when tested in a very sensitivecoinjection assay (lo7 lethally irradiated 3T3 cells were injected into each nude mouse in addition to the test cells). Further evidence that the tumorigenic cells contained a mixture of a few tumorigenic and many nontumorigenic cells came from the production of subclones which were no longer tumorigenic or anchorage independent although they still expressed SV40 T antigen (Howell and Sager, 1979).In 3T3 X SVT2 hybrids there was noncoordinate suppression of tumorigenicity and other properties of the transformed state as well (Howell and Sager, 1981). Gee and Hams (1979) hybridized early passage (nontumorigenic) and later passage (tumorigenic) SV40-transformed mouse embryo cells with cells of the malignant mouse melanoma line, PG19. Cells of both types of hybrids had chromosome numbers approximating the sums of the two parental sets; they expressed viral T antigen and a transformed phenotype but did not form tumors when lo5- lo7cells were inoculated into syngeneic newborn mice. Some product of the retained host chromosomes of the nontumorigenic and the tumorigenic SV40-transformed cells was apparently able to suppressthe tumorigenicity of the melanoma cells, while some product of the chromosomes of the melanoma cells suppressed the tumori-

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genicity of the late passage SV40-transformed cells. Thus, the tumorigenicity of SV40-transformed cells behaved as a recessive in intraspecific mouse hybrids, reflecting a loss of genetic information. In contrast, the transformed phenotype itself was dominant, as shown in many other studies. These results have been interpreted as differing considerably from those of Croce et al. ( 1975) and Koprowski and Croce (1977), who consistently observed a high take incidence in nude mice inoculated with 2 X lo6- lo7 mouse - human hybrid cells containing an SV40 genome. However, injection of such a large number of cells may conceal the nontumorigenicity of a large proportion of the injected cells. Use of the more sensitive coinjection assay of Howell and Sager (1 979) would provide a more reliable way to determine whether most of the hybrid cells were indeed tumorigenic in nude mice. Nilsson et al. (1977) have emphasized that growth in nude mice is not a reliable criterion of malignancy in human lymphoma, leukemia, or myeloma lines, and one wonders whether this might be the case in mouse peritoneal macrophage- human cell hybrids. Whether or not this is the case, these hybrids differ significantly in other ways from the intraspecific hybrids studied by others. Croce and his associates produced hybrids by fusing SV40-transformed human cells, e.g., of the LN-SV or GM54A lines, with mouse peritoneal macrophages. These interspecific hybrids rapidly lost most of their human chromosomes although retaining SV40 T-antigen production; some of the hybrids contained only the human chromosome into which the SV40 genome was integrated: chromosome 7 in hybrids derived from LN-SV (Croce et al., 1975) and chromosome 17 in hybrids derived from GM54A (Koprowski and Croce, 1977). The hybrids formed tumors in nude mice despite the presence of a near-diploid (in 25%) or near-tetraploid (in 7590) complement of chromosomes contributed by the nonmalignant mouse parent. This apparently unusual behavior may simply reflect the fact that these hybrids were also strikingly different from the intraspecific hybrids previously studied in having lost all but a tiny fraction of the chromosomes contributed by the malignant parent: frequently a single human chromosome was retained. Under these conditions, loss of a significant number of mouse chromosomes from the near-diploid hybrids would produce monosomic or nullisomic cells which might be incapable of growth in vitro or in vivo. Consequently, only limited chromosome changes may have been tolerated. It is interesting that the near-tetraploid tumorigenic hybrids failed to undergo some degree of chromosome loss. Does this reflect their having been studied soon after hybridization, or some more biologically interesting factor? Whatever the reason, the argument for the authors’ conclusion that malignancy of these two classes of hybrids is due solely to the presence of the viral genome is not convincing, because that is

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not true of virally transformed cells in general, and, more important, an altered gene dosage effect based on an increased or decreased number of copies of specific mouse chromosomes, or structural changes in the chromosomes, was not ruled out. In fact, one malignant near-diploid hybrid cell recovered from the hybrid tumors was trisomic for chromosome 11 (Croce et al., 1975), indicating that significant changes in the mouse karyotype did occur. Mouse chromosome 11 is one of the most readily identifiable chromosomes. The G-banding illustrated in the figures accompanying these reports was not adequate to permit exact identification of some of the other chromosomes. Study of these intriguing hybrids with more precise cytogenetic methods, using both cytological and genetic markers, might yield further insight into chromosomes (and genes) which regulate the expression of tumorigenicity. XVII. Common Mechanisms of Tumor Suppression in Interspecific Hybrids

The use of interspecific hybrids to study the cytogenetics of mouse carcinogenesis has hardly begun despite growing evidence that suppression of malignancy can operate across species barriers. All of 84 independently derived clones of A549/8 human lung cancer X 3T3-4E mouse fibroblasts were nontumorigenic (Carney et al., 1979). Jonasson and Harris (1977) made hybrids between PG 19 mouse melanoma cells and diploid human fibroblasts and lymphocytes. The tumorigenicity of the melanoma cells was suppressed in the three hybrids studied despite the preferential elimination of many human chromosomes. Interspecific hybrids offer the advantage of greater ease of identification of the species of origin of each chromosome. One can confidently predict that interspecific hybrids which preferentially lose mouse chromosomes will provide new insights into mouse chromosomal factors important in the expression and suppression of tumorigenesis. Chief among these may be Chinese hamster-mouse hybrids (e.g., see Howell and Sager, 1978; Schafer et al., 1981) but hybrids between tumorigenic human cells and mouse cells (Carney et al., 1979; Croce et al., 1979) might also be useful in looking for mouse chromosomes or chromosome fragments carrying genes which suppress malignancy. Such genes may play important roles in regulating growth and cell differentiation, some perhaps having evolved more specifically to prevent the uncontrolled growth of cells in particular cell lineages. Interspecific hybrids which lose chromosomes of the tumorigenic nonmouse parent (Croce et al., 1975) might be informative concerning mouse chromosomes involved in malignancy. On the other hand, mouse- human hybrids in which the mouse cell is tumorigenic could be used to yield

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information about homologous human genes involved in the expression of the tumorigenic mouse phenotype. Kucherlapati and Shin (1979) studied nine PG 19 mouse melanoma X human fibroblast hybrids and found them to be highly tumorigenic. The tumors had fewer human chromosomes than the hybrid clones from which they arose, and both copies of five human chromosomes were absent in over 80% of the hybrid tumor cells. If the tumor-suppressing effect could be assigned to a specific human chromosome which has a segment carrying genes homologous to a segment of mouse chromosome 4, this could help in localizing more precisely the mouse gene involved.

XVIII. Genetic Basis of Transformation and Tumorigenicity

Based on the poor in vitro growth of tumor X diploid cell hybrids which had retained both copies of diploid number 4,and the loss of both copies of diploid number 4 from hybrids able to grow in vivo, Jonasson et al. (1 977) and Evans et al. (1982) suggested that chromosome 4 contains a locus which codes for some function regulating cell growth in vitro as well as in vivo, and that impairment of this function is involved in producing the transformed state. This might imply that tumorigenicity involves a genetic lesion on the same chromosome (the same locus?) as that involved in transformation. This is an interesting idea, which needs to be considered in conjunction with growing evidence that the genome of many organisms contains more than a dozen cellular oncogenes (Coffin et al., 1981; Klein, 1981b), which are normally not expressed but can be activated by insertion of a viral promotor 5’ to the gene (Hayward et al., 1981) or a viral enhancer which can be inserted either 5’ or 3’ to the gene (Payne et al., 1982; Yaniv, 1982). This results in a 10- to 100-fold increase in transcription of the gene over that controlled by its own promoter, and leads to transformation. The increased level ofgene product is comparable to that achievable by gene amplification in tumors containing DMs or HSRs (described above). Activation of cellular oncogenes is an alternative to direct introduction of a viral oncogene (Coffin et al., 1981). Both represent additions of an active gene and appear to be the opposite of the genetic loss brought about by loss of one or both copies of the normal chromosome 4 found by Harris and his associates in tumorigenic hybrids between spontaneous, virus-induced, or chemically induced tumor cells and nontumorigenic cells (Jonasson et al., 1977; Fenyo et al., 1980; Evans et al., 1982). However, each may reflect the action of the same transacting control. The expression of the src gene, like tumorigenesis, is suppressed when transformed cells are hybridized with nontransformed cells (Marshall, 1980; Dyson et al., 1982).

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XIX. Altered Hexose Transport in Malignant Cells

Bramwell and Harris (1978a,b) found that the ability of mouse cells to grow progressively in vivo was linked to the presence of a structurally altered polysaccharide moiety on a particular membrane glycoprotein. This abnormality was found on many different types of tumors and cosegregated with tumorigenicity in crosses between malignant and nonmalignant cells, being absent in nontumorigenic hybrids and present on emergent tumorigenic hybrids (Atkinson and Bramwell, 1981). This protein plays a role in glucose transport; its alteration in malignant cells may be responsible for the systematic decrease in the Michaelis constant (K,) of the membrane hexose transport system of malignant cells recently demonstrated by White et al. ( 1981). This change renders the malignant cells more efficient scavangers of hexose than the competing normal cells are, giving them a selective advantage. It remains to be seen whether the gene on chromosome 4 which determines the expression or suppression of tumorigenicity acts via an effect on the polysaccharide moiety of this specific membrane glycoprotein, either indirectly or by way of an altered glycosyltransferase or sialidase. Such a mechanism might account for the gene dosage dependence postulated by Evans et al. ( 1982)on the basis of their observations on the opposing effect of the number of copies of chromosome 4 derived from the malignant parent and those from the nonmalignant parent on the tumorigenicity of hybrid cells. XX. Host Cell Recruitment into Tumors via Cell Fusion

Cytogenetic analysis has made it possible to demonstrate that tumor cells can fuse with host cells (Wiener et al., 1972; Fenyo et al., 1973; Aviles et al., 1977; Halaban et al., 1980). Such tumor- host hybrid cells were present at very low frequencies in the tumor cell population, but provided a pool of cytogenetic variability which could be important in the progression of some tumors, because such cells could undergo extensive chromosome segregation. Based on the studies already described, one would expect tumor- host hybrid cells to be nontumorigenic until elimination of specific chromosomes derived from the normal diploid parent occurs. This need not be associated with extensive chromosome loss. Halaban et al. (1980) studied hybrids between PCHPRT- Cloudman S9 1 melanoma cells (modal chromosome number, 83) and DBA/2 host cells (2n = 40). Three tumorigenic hybrids had modal chromosome numbers of 109,123, and 124, as expected with 1 : 1 fusions and minimal loss of chromosomes from either set. The investigators were unable to distinguish chromosomes of DBA/2 origin from those of melanoma origin; consequently, loss of a specific DBA/2 chromosome, e.g., a number 4, or gain of a specific chromosome, e.g., a number 15, could not be evaluated.

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XXI. Conclusions

Cytogenetic analyses of mouse tumors, tumor-derived cell lines, and tumor X nontumor cell hybrids have shown that specific chromosomes play a role in the expression of tumorigenicity. The chromosome most regularly involved was number 15, followed in frequency by number 12. Additional copies of chromosome 15 were found in T-cell and B-cell leukemias, in a highly tumorigenic L-cell-derived line, and in tumorigenic cell hybrids; in a number of these studies trisomy 12 or 17 was also found, eitber alone or in combination with trisomy 15. Breaks of chromosomes 12 and 15 brought about by translocation were commonly seen in plasmacytomas. The range of tumors that have been studied by modern cytogenetic techniques is still very small. In a limited variety of solid tumors, changes in structure or number of various chromosomes other than number 12, 15, and 17 have been described. Gross chromosome changes are thought to act via changes in gene dosage or level of gene expression. An increase in copy number of one or a small number of genes was found in various tumors. Gene amplification can sometimes be recognized cytologically by the presence of HSRs (homogeneously staining regions) or DMs (double minutes) which contain the amplified genes. In only a few cases was the amplified gene identified, and in no case was it shown whether only a single gene had been amplified. Although an increased number of copies of chromosome 15 was observed in tumorigenic somatic cell hybrids, the characteristic change in hybrids was loss of one or both chromosomes 4 derived from the nonmalignant parent. Such a change was not observed in tumor cells, but this is not unexpected if monosomy (or nullisomy) 4 leads to decreased viability or cell death. It is possible that deletion, inactivation (by insertion, rearrangement, or DNA methylation), or mutation of a gene locus on chromosome 4,rather than loss of an entire chromosome, precedes tumor development in vivo. The use of prometaphase chromosome banding techniques might shed light on this question and help in localizing the gene involved.

w LEDGMENTS ACKNO The research of the authors is supported by grants from the American Cancer Society (CD66), the March of Dimes-Birth Defects Foundation (I-361), and the U.S. Department of Health and Human Services through the National Cancer Institute (CA27655) and the Institute of General Medical Sciences (GM25 193).

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POLYOMAVIRUS: AN OVERVIEW OF ITS UNIQUE PROPERTIES Beverly E. Griffin and Stephen M. Dilworth' Imperial Cancer Research Fund. Lincoln's Inn Fields. London. England

1. Introduction ...................................................................... II . Background ............................................................ III . Structural Organization of the Viral Genome .................................... IV . Comparison of the Polyomavirus and SV40 Genomes ........................... V . The Tumor Antigens ............................................................. A . Definition..................................................................... B. Characterization .............................................................. C. New Approaches.............................................................. D. Location within the Cell ...................................................... E . In V i m Functions of the T-Antigens ......................................... F. In Vivo Functions of the T-Antigens.......................................... VI . Mutants of Polyomavirus......................................................... A . tsa Mutants ...................................................................

............................................................

C. mlt Mutants .................................................................. D. ncr Mutants ................................................................... E. Late Mutants.................................................................. VII . Cellular Transformation .......................................................... A. Models for Transformation ................................................... B. Integration of Viral Genes into Host Chromosomes .......................... C. Free Viral DNA .............................................................. D. Roles of T-Antigens in Transformation ....................................... VIII . The Lytic Cycle .................................................................. 1X . Conclusions ...................................................................... References........................................................................

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None but those who have experienced them can conceive of the enticements of science. In other studies you go as far as others have before you. and there is nothing more to know; but in a scientific pursuit there is continual fund for discovery and wonder (Mary Shelley. 1818).

I. Introduction

An excellent account of the early studies on polyomavirus appeared in 1969 in a long review by Eddy. one of the pioneers of study on oncogenic DNA viruses. Anyone initiating studies on polyomavirus would be well advised to read this review carefully and anyone actively working on the virus to consult it for its wealth of background information. For example. * Present address: MRC Laboratory of Molecular Biology. Cambridge CB2 2QH. England. I83 ADVANCES IN CANCER RESEARCH. VOL. 39

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Copyright 0 1983 by Academic Press Inc. All rights of reproduction in any form . ISBN-0- 12-006639-4

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results on chromosomal changes in transformed cells, a topic largely ignored in subsequent studies, are considered. From a variety of data, it was concluded that there was no obvious correlation between polyomavirus induced transformation and cellular chromosomal changes. Although transformed cells appeared to accumulate chromosome abnormalities, these could not be related in any specific way to the initial viral infection. Thus, whatever the mode oftransformation by polyomavirus, it does not appear to involve specific chromosomal translocations. From the point of view of our article, one of the most noteworthy aspects of thk Eddy (1969) review is its consideration of large variations observed among different strains of polyomavirus. For instance, in early studies, the “Toronto” strain of virus, derived from a mouse mammary tumor, caused rapid development of bilateral kidney sarcomas in hamsters with high frequency, whereas the Stewart- Eddy (SE) strain only occasionally produced such tumors. Recognizing that some of the differences observed among strains of polyomavirus, such as the above, might be due to hybrid viruses (although this has not been proved), pure stocks were made from viruses selected using the plaque assay technique. From SE virus, cultures derived from small plaques or from large plaques were studied and found to vary in a number of aspects (GotliebStematsky and Leventon, 1960). Small plaque virus was more readily adsorbed to cells than large plaque virus, and was found to be, in general, more oncogenic as judged by the rate and frequency of transformation of hamster, mouse, or rat cells. Stoker (1 960) used the Toronto (small plaque) strain of virus to study the oncogenic activity of polyomavirus because of its enhanced potential for tumor production. IE work on interferon induction, on the other hand, large plaque virus was superior to small plaque virus. As we note subsequently, some of the apparent discrepancies among present day studies with polyomavirus may be due to the different properties associated with different strains of virus and the different levels of viral antigens induced by individual strains within a single host species or between hosts. Eddy concluded that “whereas the lytic phase of polyoma virus is not unlike that of a number of non-oncogenic viruses [to this one could add, ‘or some oncogenic viruses such as SV40 or BK virus’], the transformation phase remains an enigma.” Our article attempts to extend these statements and to explain why we think that functions involved in the lytic phase of polyomavirus can be correlated mainly with the expression of one viral protein, the large T-antigen, and those of the transformation phase (in fibroblast cells) with another, the middle T-antigen. Moreover, we would maintain that whereas comparative studies between polyomaviruses and several other members of the papovavirus group, notably SV40 and BK

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virus, will continue to be of considerable value in understanding the lytic phase of these viruses, in transformation, polyomavirus must be considered somewhat apart. An alternativeanalogy may lie with a group of RNA tumor viruses, the retroviruses. These latter viruses have been shown to have acquired their transforming function from their host cells, and via this function induce changes at the cell surface which are probably associated with transformation (for review see Bishop, 1982, and references therein). It is not known where polyomavirus acquired the information for its transforming function(s),but it seems reasonable to suggest that it was acquired from its host (Soeda et al., 1980a). It is known that this transforming function can be found associated with cellular membranes (It0 et al., 1977b). The analysis of Pontkn in 1971 of the morphological differences between cells transformed by polyomavirus and by SV40 led him to suggest that the control exerted by SV40 appeared to result in unrestrained growth, possibly by an alteration of the regulation of cell division, whereas that exerted by polyomavirus resulted in irregular cell growth, the key alterations probably lying within the cell membrane. To Ponth, the differences in cell changes produced by these two viruses suggested different modes of oncogenic action. This hypothesis remains a very valid point of departure from which to consider transformation by polyomavirus. As will be apparent, this article is a subjective one and somewhat simplistic in that it attempts to draw general conclusions about the behavior of polyomavirus, often at the expense of detail. It has made no attempt to be completely comprehensive, in that some topics do not seem quite ready for review and some have been or are being extensively reviewed elsewhere. In this regard, we wish to call attention to articles published within the last 5 years that are particularly relevant to our review. These include (1) Organization of the genome of polyomavirus (Fried and Griffin, 1977);(2)Recent advances in polyoma virus research (Consigli and Center, 1978); (3) Viral “tumor antigens.” A novel type of mammalian regulatory protein (Weil, 1978);(4) Organization and expression of the genome of polyomavirus (Ito, 1980); (5) The tumor antigens and the early functions of polyomavirus (Turler, 1980); (6)Polyoma T-antigens (Eckhart, 1981); (7) Functions of T-antigens of SV40 and polyoma virus (Hand, 1981); (8) The transformation of cell growth and transmogrification of DNA synthesisby simian virus 40 (R. Martin, 1981); (9) Transforming genes and gene products of polyoma and SV40 (Schafiausen, 1982); (10) Organization and replication of papova virus DNA (DePamphillis and Wassarman, 1982); (1 l) The HR-T gene of polyoma virus (Benjamin, 1982); (12) chapters in “DNA Tumor Viruses” (Tooze, 1981); and (1 3)a projected review by N. Acheson and J. A. Hassell on transcription (this series).

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II. Background

Polyomavirus is a member of the papovavirus family. Its genome consists of a small, double-stranded, closed circular DNA molecule that exists in virions as a complex with cellular histones surrounded by capsid proteins. In contrast to the other members of the papovavirus family which it most closely resembles, that is, SV40 and BK virus, polyomavirus appears to encode a transforming function distinct from the function responsible for ’lyticinfection. The origin of this transforming function is not clear although it has been suggested that it was acquired from its host (mouse) at some stage after the evolutionary separation of mouse from man and monkey (Soeda et al., 1980a). In tissue culture, rodent cells have been arbitrarily classified into two types depending upon their ability to support a productive infection by polyomavirus or to undergo the changes that result in cellular immortalization or “transformation.” “Permissive” cells are those that can be infected by the virus, support viral DNA replication, and produce the proteins necessary for virus packaging; in these cells the host eventually lyses and releases large quantities of virus particles. A number of new properties are expressed by the infected cells prior to lysis, many of them common to the “abortive transformation” of cells (Stoker and Dulbecco, 1969, see below). Only mouse cells fulfill all these criteria for polyomavirus. Other rodent cells vary in their degree of permissivity. Hamster cells are “semipermissive,” with a limited number of cells producing low yields of virus, and whereas some rat cells are almost nonpermissive, with little or no virus production, others are not (P. Bourgaux, personal communication). Mouse, rat, and hamster are the main species that have been widely studied as hosts for polyomavirus, although many other mammalian cells appear to have receptors for the virus (PontCn, 1971). The degree of permissivity seems to be regulated at the level of genetic control over viral gene expression, since fused permissive and semipermissive cells are capable of being productively infected (Basilico et al., 1970). On infection of nonpermissive cells with polyomavirus, the virus is absorbed by the cell, uncoated, and the “early region” (coding for the T-antigens) transcribed. The host cell is stimulated to undergo DNA synthesis and division, and a number of properties associated with transformed cells are transiently expressed. Little or no viral DNA is synthesized, however, and most members of the cell population soon cease to transcribe viral RNA, lose viral DNA, and revert to their uninfected state. This process has been termed “abortive transformation” (Stoker and Dulbecco, 1969). A small proportion of the cells, however, acquire and stabilize the viral genome in such a way that they continue to transcribe viral DNA and express the early proteins. These cells permanently acquire novel properties,

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including, among others, a change in cell morphology (becoming more stellate in appearance), a loss of contact inhibition, an ability to grow in semisolid medium without surface attachment, an ability to form tumors in syngeneic animals, and a disrupted cytoskeletal array; they are thus said to be “transformed.” The small size of polyomavirus, and its ability to grow and transform cells efficientlyin vitro, has made it an extremely useful model system both for the study of viral replication and virally induced tumorigenesis. The complete DNA sequence and comprehensive details of viral gene expression have been determined (see Tooze, 1981). With current molecular biological techniques, this has now enabled the genome to be altered and studied almost at will, thus providing much information on viral functions and their interaction with the host cell. This interplay of viral and cellular components provides a starting point for understanding normal cellular regulatory mechanisms and will hopefully lead to a fuller recognition of the controls involved in viral, and perhaps other, forms of tumorigenesis. The in vitro growth properties of the virus enables these mechanisms to be studied under well-defined experimental conditions. Detailed considerations of the action of polyomavirus in vivo, with scattered notable exceptions, are largely confined to the older literature (e.g., see Rowe et al., 1959; Eddy, 1969; PontCn, 1971). In an interesting recent study, McCance ( 1981) showed that the ability of the virus to persist in its host was highly sequence dependent. That is, in a comparison of the persistence of a wild-type strain of virus (A2) and numerous viable deletion mutants, with a single exception only the former had the capacity to survive in the animal, suggesting that the finer aspects of viral DNA replication in vivo and in vitro might be different, if not in mode then at least in degree. Most of the early experimentalevidence has suggested that the resistance to polyomavirus shown by its natural host (mouse) is immunological in nature. Studies on athymic (nude) mice bear this out; adult nude mouse are very sensitive to the oncogenic activity of this virus (Allison et af., 1974; Vandeputte et al., 1974; Stutman, 1975; Arya and Galarraga, 1982). Recently, a demyelinatingand wasting disease has been shown to be associated with the presence of polyomavirus (A2 strain) in nude mice (Sebesteny et af., 1980; McCance et af.,1983). Intracerebral inoculation of uninfected mice produced hind quarter paralysis in all surviving mice 10- 23 weeks postinfection; extensive myelin disruption was observed within the spinal cord and brain of sacrificedanimals. Similar diseases have been previously shown to be associated with the presence of other members of the papovavirus group such as the JC virus of man (Padgett et af.,1971) and SV40 (Holmberg et al., 1977), but they had not been previously reported for polyomavirus. Attention is drawn to this work because of the potential danger of

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papovaviruses to man, particularly worth consideringalongside the increasing use of immunosuppressant therapy. 111. Structural Organization of the Viral Genome

In a similar way to the small prokaryoticvirus 4x1 74 (Sangeret al., 1977), the coding capacity of polyomavirus has been extended by the use of overlapping reading frames for encoding different proteins (Hunter et al., 1978;Smart and Ito, 1978;Soeda et al., 1980b)(see Fig. 1). Most noticeably, the entire coding capacity of four of the known viral proteins (small and middle T-antigens), and the minor viral capsid proteins (VP2 and VP3), as well as the N-termini orthe other two (largeT-antigen and the major capsid protein, VPl), and the noncoding “control” region is encompassed within slightly over half of the viral genome while the other half codes in a linear fashion for the rest of large T-antigen and VP 1. The obvious question is why has the virus been so parsimonious in part of its genome only? One possible explanation is that this is not the case, but other viral proteins may exist that have not yet been discovered. In line with this suggestion, it has been noted (Soeda et al., 1979b, 1980a,b; Griffin et al., 1981) that fairly extensive, apparently “unused” open reading frames exist within the genome, notably ones in the region coding for the C-termini of large T-antigen and VP 1. In this regard, studies with monoclonal antibodies raised against the viral T-antigens suggest that the large and middle T-antigens are not individual proteins but rather “families” of proteins (Dilworth and Griffin, 1982), although there is no evidence to date that these originate by the use of any other than the “normal” reading frames. Similar conclusions may be deduced from the results of It0 and Spurr ( 1980). Further, Bolen et al. ( 1981) and Hare and King ( 1982)have separated polyoma virion VP 1 into a family of distinct subspecies, only three or four of which appear to be present in purified capsids. Although we do not suggest that other undiscovered viral proteins necessarily exist, we suggest that even excluding protein modification and posttranslational processing, they could exist. Moreover, with our present technology and emphasis, it would be easy to understand how proteins made in small quantities, or those not concerned with transformation, could be overlooked. For example, all “early” proteins studied to date have been identified by their interactions with antibodies in antitumor sera (see below); any protein not involved in tumorigenesis might therefore be overlooked. At the level of transcription, most studies on “early mRNAs” have been camed out not with wild-type virus but with tsa mutants, and therefore carry the hazard of the thermolabile constraint (Cogen, 1978; Kamen et a!., 1980; Treisman et al., 1982a). Present knowledge of the organization of the viral genome has been

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FIG. 1. Landmarks on the DNA of polyomavirus (A2 strain). The conventional Ifpall physical map (Griffin et al., 1974)is shown. It has been divided into 100 units, with the single EcoRl cleavage site at position O/lOO; all other numbers refer to corresponding nucleotide numbers in the primary DNA sequence (Soeda et al., 1980b). The postulated location on the genome of the viral origin of replication and the six known virally coded proteins (the three “early” proteins, small, middle, and large T-antigens, and the “late” capsid proteins, VPI , VP2, and VP3) are indicated, with data taken from Griffin eta/.(1974), Smart and Ito ( 1 978), Kamen et al. (1980), and Soeda et al. (1 980b). The unique coding region for middle T-antigen, discussed in the text, lies within nucleotides 809- 1497, using a reading frame different from that which encodes large T-antigen in the same area of the genome (adapted from Soeda et a/., 1980b).

discussed in detail elsewhere (Ito, 1980; Soeda et al., 1980b; Griffin et al., 1981) and is shown schematically in Fig. 1. It is interesting to note that one major stretch of sequence difference between polyomavirus and SV40 (see below) lies in the region most important to cellular transformation by polyomavirus. If these two viruses do transform cells by different mechanisms, as seems likely for fibroblasts, at least, then one key to this difference resides within this region. Other aspects of genome organization are considered in the following comparison with SV40. IV. Comparison of the Polyomavirus and SV40 Genomes

During most of the 1970s, insight into the functions of polyomavirus was masked by attempts to force an SV40 mould onto both viruses. Polyoma-

I90

BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

virus was released from this constraint chiefly by the discovery of its novel middle T-antigen (It0 et al., 1977b)and the elucidation of the primary DNA sequences of both viruses (Fiers el al., 1978; Reddy et al., 1978; Soeda et al., 1980b; Deininger et al., 1980).Nonetheless, these two viruses (as well as BK virus) have enough homology to support the notion that they evolved from a common ancestor (Soeda et al., 1980a). Attempts to demonstrate homologies directly have been only partly suc’cessful. Studies of heteroduplex formation by electron microscopy showed limited homology in the region coding for the viral major capsid protein, VPl, when dimeric mixed species of the two viral DNAs were allowed to reanneal (Wu et al., 1979). DNA hybridization studies which could detect only high degrees of homology were negative, but, when stringency was relaxed, nearly 509/0 homology between the two viral DNAs was observed (Howley et al., 1979), as illustrated in Fig. 2. These data and those aimed at uncovering immunological cross-reactivityare consistent. Thus, cross-reactivitiesto date were observed first with the major viral capsid proteins, the VPI s (Shah et al., 1977; Benton et al., 198I), then with the large T-antigens (McCormick et al., 1982). With the appropriate antisera, one would predict that cross-reactivity with the other viral capsid proteins, and possibly with the small T-antigens, could also be observed (see Fig. 3). The major regions of nonhomology between the two viral genomes lie in the apparent noncoding (“control”) regions, the region that codes for the unique portion of middle T-antigen in polyomavirus, and the region encompassing the C-termini of both the large T-antigens and the major viral capsid proteins. All three of these regions may be of unique importance, the first with its obvious effect on gene expression and two of the others in connection with cellular transformation by the individual viruses. Postulated “control” elements within the noncoding regions of polyomavirus and SV40 are illustrated in Fig. 4. Since the viruses have different hosts with which they interact, both with regard to productive and, in part, nonproductive expression, it is perhaps not surprising that these regions, which must rely heavily on viral - cellular interactions, should have different primary sequences. Comparative studies may ultimately reveal that these sequences show more homology between the virus and its host than between the two viruses themselves, suggesting either a mimicking response by the virus to its host or, more interestingly, the direct acquisition of these sequences and their activities from the host cell (see Soeda et al., 197913; Queen et al., 1981; Ding et al., 1982a). The major similarities between polyomavirus and SV40 (or BK virus) in the control region are in sequences thought to specify the viral origins of replication (Soeda et al., 1979b); the major differences are the absence of long repeat sequences, such as those found in SV40 DNA, in a number ofwild-type (notably large plaque) strains

A

>69% HOMOLOGY

~ 6 4 % HOMOLOGY

FIG.2. Organization of the genomes of (A) SV40 and (B) polyomavirus (see Fig. 1) and a comparison of their coding potentials and DNA sequence homologies. The putative origins of replication (OR) for both viruses are indicated, for polyomavirus, the letters K and H represent cleavage sites of the restriction endonucleases KpnI and HindII, respectively. The similarities in viral organizations are apparent. One major difference between the two viruses lies in the fact that SV40 apparently codes for no protein which corresponds to the middle T-antigen of polyomavirus, whereas the “agno” gene product of SV40 (Jay el al., 198 I ) has no known counterpart in polyomavirus. (Data taken in large part from Fiers et al., 1978, Reddy er al., 1978, and Soeda er a/., 1980b). For polyomavirus, the regions that hybridize with SV40 DNA under moderately stringent conditions, in which about 70%homology is observed, and that which hybridizes under less stringent conditions which allows the detection of about 65% homology, are indicated by heavy black lines (data taken from Howley er al., 1979; see Fig. 3).

A

SMALL T -ANTIGEN 20

- YE

1

B

60

40 I I

100

80 I I

I I I

m II I =

120 I

L

I = lamino acid

FIG.3. Amino acid homologiesbetween polyomavirus (A2 strain) and SV40 (strain 776) proteins as predicted from DNA sequence. with gaps left where necessary in order to maximize similarities (see Soeda ef al., 1980b;Griffin el al., 1980a).Black areas (W) are used to designate homologies: K and H are cleavagesites of the restriction endonucleases KpnI and HindII, respectively(see Fig. 2). The size of each protein, in terms of its amino acid sequence as predicted from the DNA sequence, is indicated by the numbers above each antigen. In general, sequences at the N-termini are more conserved than those at the C-termini of the proteins. (A) The large and small T-antigen homologies and the location of some of the functional mutations in defined tsa mutants (see text) are shown. Line 2, and parts oflines 1 and 3, in the figure ofthe large T-antigen, include the region which encompasses the unique sequence of middle T-antigen, and one of the major areas in the large T-antigen that lacks homology between polyomavirus and SV40. (BK virus resembles SV40; see Tooze, 198I ) . It has been argued (Soeda et al., 1980a)that polyomavirus might have acquired the sequence found in this region from its mouse host. If this is so, one would predict that it is extremely unlikely to be a functionally essential region for both antigens. Studies on the mlt mutants suggest that qualitatively, at least, this is the case. rThese mutants have deletions within thisregion and areallcompetent for growth,although some, like dl-8 (seeFig. 5 ) , do not grow well.] The C-terminus of middle T-antigen, including the very hydrophobic sequence (Fig. 3,is located in the region (line 3) where extensive homology between the two species resumes. Thus, if a mouse coding sequence has been acquired by polyomavirus during evolution, it need not have been specifyinga membrane-associatedprotein. To speculate, it may be the juxtaposition ofa membrane-specifying sequence and a phosphotransfer (protein kinase) activity that has given polyomavirus a unique “transforming gene.” If so, there may exist in wild mice a primordial variant of polyomavirus that is nontransforming. Line 7 shows the main difference predicted for the T-antigens from two strains of virus, the A2 strain (Soeda ef al.. 1980b)and strain 3 (top line) (Deininger et al., 1980),which lies in the other major nonhomologous area between the two viruses. (B) The homologies between the minor capsid proteins VP2, VP3, and the major capsid protein, VPI. VP2 has all the sequences present in VP3, but in the case of polyomavirus at least, is not a precursor of the latter, since the proteins are translated from different mRNAs (Sidell and Smith, 1978).

A SV40 P

e CAT{

Rl

RI

R2

R2

AT

HP

MP

ATG

545

-t

’5146

0 POLVOMA

I DNaSe 1

I

DNaK I

FIG.4. Comparisons of the organization of the so-called “control regions” in the DNAs of (A) SV40 and (B) polyomavirus that contain signals, thought to be essential for viral DNA replication and transcription, which lie between the coding regions for the major “early” and “late” gene products (see Fig. 2). The figure has been aligned such that the putative origins of replication for the two viruses (HP sites, see below) lie on top of each other; these sites have considerable primary DNA sequence and structural homologies (Soeda ei a/., 1979b).The location of the major cap sites lies between the lategene initiation signals and the PvuII (P) sites nearest them; the numbers shown at right correspond to the initiation signals for the early gene products, and at left, the late gene products (data taken from Buchman ef a/.. 1981; Griffin et a/., 1981). Recently, SV40 has been shown to encode a small DNA binding protein, the “agnogene” product, within the region immediately preceding position 545 in the DNA sequence (Jay et al., 1981). No comparable protein has yet been identified for polyomavirus. R, direct sequence repeats; HP, cruciform structures, that is intramolecular “hairpin loops” or other structures, allowed by the sequence (Soeda et al., 1979b); Tag, D 2 protein (large T-antigen-related) binding sites (Tjian, 1980); P, PVuII restriction endonuclease sites; B, the single EclI restriction site in polyomavirus (see Novak ef al., 1980); A,T, the A,T-rich sequences; TATA, potential transcriptional “promoter” signals; A2, an 1 I base pair insertion found in the A2 strain of polyomavirus; E, “enhancer” sequences (reviewed, Dynan and Tjian, 1982);DNase I, sensitivity sites to DNase I (Herbomel et al., 1981). Recently, it has been shown that polyomavirus large T-antigen in lytically infected cells binds preferentiallyto sites that include the region labeled A2 (Tag I) and the potential early “promoter” (TATA box) signal (Gaudray et a/.. 1981; Cowie et al., 1983);in addition, usingmonoclonal antibodies, a third site has been identified which lies between these two (our unpublished observations).It was previously noted (Soeda ei al., 1979b)that the hexanucleotide AGAGGC appeared six times in the “noncoding” sequence of polyomavirus (A2 strain), twice on the early strand, and four times on the late strand, and was not found elsewhere over a large stretch of DNA. This finding led to the suggestion that the hexanucleotide must be functionally significant,possibly acting as a regulatory signal in transcription. Gaudray ef a/. (1981) suggest that it is involved in the specific binding to large T-antigen.

POLYOMAVIRUS AND CELLULAR TRANSFORMATION

195

of polyomavirus. In SV40, these long repeating sequences have been shown to be quantitativelyimportant in the expression of viral genes and have been called “enhancers”; polyomavirus may have a functionally similarsequence (Banerji et al., 1981;de Villiers and Schaffner, 1981; Moreau et al., 1981; Levinson et al., 1982;Byrne et al., 1983;reviewed, Dynan and Tjian, 1982). Polyomavirus DNA has two short repeat sequences for which we have postulated a role in the modulation of replication (see below). A comparison, at the nucleotide and protein level, appears to us to support the notion implied earlier that correlations between polyomavirus and SV40 are of most value in the study of the lytic cycle and encapsidation, and may be of limited value in defining cellular transformation. In this regard, it should be noted that for polyomavirus there is no apparent analogy with SV40 in regard to the host protein, 53K. This protein binds to the large T-antigen of SV40 and is in some as yet unidentified manner associated with transformation (Lane and Crawford, 1979; Linzer and Levine, 1979; Linzer et al., 1979), but no equivalent binding to any of the T-antigens of polyomavirus has been observed (Crawford, 1983). These findings may be consistent with the notion that cellular p53 may play a regulatory role during normal cell growth (Campisi et al., 1982) which is disturbed by the presence of SV40, leading ultimately to transformation in a manner postulated by PontCn (1 97 1). V. The Tumor Antigens

A. DEFINITION

The T (tumor)-antigenswere first recognized and defined by the presence of antibodies directed against them in the sera of animals bearing polyomavirus-induced tumors (Habel, 1965). The use of syngeneic virally transformed cells to produce a tumor ensures an immune response to antigens related to the transformed state of the cells, since the viral proteins expressed in the transformed cells are likely to be recognized as foreign and antibodies thus produced against them. This has been shown in many cases to be the major antibody response of animals carrying virus-induced tumors. However, a number of antibody activities against nonviral proteins have also been observed (It0 et al., 1977a;Hutchinson et al., 1978;Schaflhausenet al., 1978; Simmons et al., 1979; Rundell et al., 1981). This may be a consequence of alteration of normal cellular proteins in such a manner that they become immunogenic during the events leading to transformation. It could also be the result of the presentation of a cellular protein to the animal in an abnormal form (for example, modified in some way or complexed to another protein) or in an unusually high quantity, or presentation possibly even of a protein not normally seen by the immune system of the animal (for

196

BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

example, an embryonic antigen). It is not clear at present whether any of these alternatives account for the properties of antitumor sera. Most polyomavirus antitumor sera have been raised against rat or hamster fibroblast cells that have been transformed by the virus in vitro and subsequently cloned, propagated, and introduced into syngeneic hosts. Under these conditions, the predominant antibody response is against the “early” proteins (T-antigens) encoded by polyomavirus; the terms “T-antigens” and “early” proteins have become almost interchangeable. Usually, such “antiT sera” contain only low levels of antibodies directed against nonviral proteins. In addition to the three viral antigens so far identified, other viral proteins may exist, but because of their poor immunogenicity in tumor cells, possibly as a result of production in low quantities, or their presence only in lytically infected cells, have not yet been observed. These problems need to be recognized but they have not hindered the initial characterization of the polyomavirus T-antigens.

B. CHARACTERIZATION The T-antigens were originally identified by detection of complementfixing activity in antitumor serum which reacted with both polyomavirusinduced tumors and traqformed cells (Habel, 1965). This activity was separate from antivirion activity, and its major component was located within the nuclei of lytically infected cells by immunofluorescent techniques (Takemoto et af.,1966). More recent characterization of the T-antigens has involved the use of antitumor sera to immunoprecipitate T-antigens from lysates of cells labeled with radioactive amino acids, precipitated polypeptides subsequently being electrophoretically separated on polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). The three main polypeptides identified by this approach from mouse 3T6 cells infected with the A2 wild-type strain of polyomavirus have been designated large, middle, and small T-antigens (see Fig. 1). Their apparent and predicted molecular weights, compared with those of SV40, are given in Table I. Many groups, using a variety of approaches, have established the physical arrangement of the coding areas of the early region of the virus. These approaches include peptide mapping analyses of the T-antigens (Hutchinson et al., 1978; Smart and Ito, 1978; Hunter et af.,1979; Simmons et af., 1979) which have defined the relationship among them; DNA sequence analysis of the complete polyomavirus genome of two strain of virus (Soeda et al., 1980b; Deininger et af., 1980, and references therein) which has located open reading frames capable of coding for proteins and allowed tentative predictions of their amino acid sequences; immunoprecipitation of T-antigens from genetically and physically defined mutants (Ito et al., 1977a,b, 1980; Silver et af., 1978; Schamausen et af., 1978) which has

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

197

TABLE I MOLECULAR WEIGHTSOF VIRALPROTEINS” Pol yomavirus

Large T-antigen Middle T-antigen Small T-antigen VPI VP2 VP3

Calculatedb

Observedc

SV40b

88K 49K 23K 42K 35K 23K

88-1OOK 55K 22-23K 45-48K 35K 23K

82K -

20K 40K 38.5K 27K

a Data taken from Buchman el af.( I 98 1) and Griffin et al. (I98 1). From DNA sequence. On SDS- PAGE.

established the regions of the genome coding for each T-antigen; and characterization of the splice positions within the early messenger RNA molecules of polyomavirus lytically infected and transformed cells (Kamen et al., 1980; Treisman et al., 1981a) to establish the regions of the genome deleted within the messenger RNA for each antigen. The physical arrangement arrived at by these various techniques is summarized in Fig. 1. It can be seen that the genome organization is extremely economical, encoding three different polypeptides within less than 3000 base pairs of DNA. This economy is most striking in the region of the genome used to code for the unique parts of both large and middle T-antigens, the mRNAs being translated from two different reading frames. The T-antigens can be seen to be closely related to each other: All three appear to have the same N-termini; middle and small T-antigens have a large sequence in common; and each T-antigen has a unique region at its C-terminus. The establishment of this physical arrangement, together with the DNA sequence, has enabled complete amino acid sequences to be predicted for each of the three T-antigens (Griffin et al., 1980b; Tiirler, 1980). That for the middle T-antigen is reproduced in Fig. 5. A number of interesting features have been observed in the sequences predicted for the T-antigens of polyomavirus (reviewed in Griffin et al., 1980b; Eckhart, 1981; Novak and Griffin, 1981b), most notably in the unique sequence of middle T-antigen. For example, the latter consists of approximately 30% hydrophobic amino acids, with charged residues clustered into areas of like charge. Near the C-terminus (amino acids 394 to 4 15) (Fig. 3,middle T-antigen has a stretch of 22 continuous hydrophobic amino acids which are arranged in a sequence characteristic of those found

198

BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

H2N-MEl'

ASP ARG VAL LEU SER ARG ALA ASP LYS GLU ARG LEU LEU GLY LEU LEU LYS LEU PXI

ALA TYR LYS

20

LEU LEV

40

LEU HIS PRO ASP LYS GLY GLY SER H I S ALA LEU MET GLN GLU LEU ASN SER LEU TRP GLY L.M S THR PHE LYS THR GLU VAL TYR ASll LEXJ ARG MET ASN LEU GLY GLY THR GLY PHE GLN/VAL

60

ARG CXN LEU TRP GLY ASP PHE GLY ARG MET GU4 GLN

GLN GU4 SER

80

THR PHE GLY ASP ARG

lYR

100

TYR GL.N ARG PHL. CYS ARG MET PRO LEU THR CYS LEU VALvASN VAL LYS TYR SER SER

CYS

120

HIS Aw; GLU LEU LYS ASP LYS CYS ASP ALA

140

ARG ARG LEU H I S /uJ\ ASP GLY TRP ASN LEU SER THR LYS ASP NG-18

SER CYS ILE LtU

C7IS LEU LEU ARG LYS GLY

ARG CYS LEU VAL LEU GLY GLU CYS PHE CYS LEU GLU CYS

TYR M E T

GLN TRP PHE GLY THR NG-18 NG-59

PIX3 'ER ARG ASP VAL Lni ASN LEU "R ALA ASP PHE LLE. ALA SERvMET PRO

160

LLE %P TRP

180

LEU ASP LEU ASP VAL HIS SER VAL TYR ASN PRO/LYS ARG APG . SER GW GW LEU ARG ARG

200

TYR THR MET Tt!R THR GLY H I S SER ALA MEY GLU ALA SER THR SER

220

THR PRO AIA THR SER ARG A X LEU ARG

240

MS

AIA ALA 'IIIR VAL H I S

c;LN GLY ASN GLY MET ILE SER SER GLU SER GLY

d1.8

LEU PRO SER LEU LtU SER ASN PRO THR TYR SER V?LM . EX

ARG SER HIS SER TYR PI(0 Pw)

MOP 1033

THR

&dl.8VAL LEU GLN C41.22 U d ILE H I S d1.27

PRO H I S ILE LEU LEU GLU GW ASP GLU ILE LEU V?Lv

280

a sw

300

PRO LEU GW GW GLU GW Gw GW TYR MEY PRO MEY GLULUIAsP

320

P m ILE ILE

340

TRP GLU GLY LtU LLE LEU ARG ASP LEU GLN ARG ALA H I S

360

LEU LEUISER PRO MFI' m.ALA d1.23 45

d1.27

ASPIGLNIASP GIN LEUG . Lu

LEU

m LEU

TYR PRO ARG THR PXI Pw) GLU

m

LEU TYR PRO

41-22

41-23 1015

ASP ILE LEU PRO GLY GLU GLN C a L PRO GIN LEU ILElPRO.PRO 1015

PRO ARG ALA GLY LEU SER.PRO PHE ASP PXI

LLE LEU ASP ALA SEX P 1387-T

260

45

GLN Aw; MET Aw; AIA

Y7 M A H I S SER M I 2 GIN ARG H I S LEU AIG Aw; LtU GLY ARG

THR HIS AFG ALA

ALA LEU ARG

-4q!

380 400 420 421

-59

ASP-ILEASN

bDP 1033

ARG-

Fy 1387-T GLN-TRA

FIG.5 . The proposed amino acid sequence of polyomavirus middle T-antigen as deduced from the DNA (A2 strain) sequence (Griffin, 1981). The sequence common to the three T-antigens (L,M, S) as well as that common to the middle and small T-antigens (M, S) are indicated; beyond the latter site is the sequence unique to the middle T-antigen (see Fig. I). The location ofa number ofdeletion mutants, that is, the hrt mutant NG-18, the mlt mutants dl-8, dl-22, dl-27, mutant 45, dl-23, 1015, an hrt insertion mutant NG-59, and two point mutants MOP 1033 and Py 1387-T (for references, see text), are also shown. The 22-long hydrophobic sequence, flanked by polar amino acids, present near the C-terminus ofthe middle T-antigen is underlined. The importance of this region in transformation has been shown using cloned fragments of DNA that express decreasing amounts ofthe C-terminus (p42 to pl2, as shown). Whereas p42, p7, p52, and p46 retain the ability to transform rat- I cells, although with less than

POLYOMAVIRUS AND CELLULAR TRANSFORMATION

199

in many transmembrane proteins. That is, they lack charged amino acids, histidine, or proline, and are flanked by lysine or arginine residues (reviewed in Warren, 1981). Discrepancies of about 10% exist in the measured and predicted molecular weights for the large and middle T-antigens (see Table I). With large T-antigen, this possibly is a reflection of posttranslational modification, since it is known to be heavily phosphorylated in vivo (It0 et al., 1977a; Turler and Salomon, 1977; Schafhausen et al., 1978). In support for this notion, large T-antigen synthesized in vitro has a lower apparent molecular weight than the in vivo product. The disparities of middle T-antigen molecular weights, however, are less easy to understand. Middle T-antigen synthesized in vitro and in vivo has the same apparent molecular weight (Hunter et al., 1978) and does not appear to be extensively modified in vivo (Schaffhausen et al., 1978; Schafhausen and Benjamin, 1979; Smith et al., 1980; our unpublished results). A possible explanation may lie in its conformation; a large amount of polypeptide chain bending could result from proline residues in the unique region of middle T-antigen and produce a low mobility on electrophoresis. This is consistent with the abnormal behavior of middle T-antigen on SDS-PAGE and with the observation that the apparent molecular weight appears to alter markedly with varying acrylamide concentrations (our unpublished results). In Fig. 5, amino acids deleted or altered in a number of mutant middle T-antigens are also shown. Although it is difficult to deduce much from these changes, it is interesting to note that while much of the middle T-antigen polypeptide can be deleted without destroying its role in transformation, a small change at a key position, see NG-59, can completely alter its activity. The hydrophobic sequence at the C-terminus is shown from studies with the mutant Py 1387T to be necessary for the association of middle T-antigen with membranes and the expression of the phosphorylation activity observed in vitro (see below) (Carmichael et al., 1982), and from studies with cloned fragments to be crucial in transformation (Novak and ~~

10% of the efficiency observed with the clone of a fragment that retains the complete sequence of middle T-antigen (as well as its normal termination codon), p12 was nontransforming (Novak and Griffin,1981a).Another sequence shown to be relevant for transformation is that encompassed within the deletion mutants dl-22, dl-23, and 1015 (Griffin and Maddock, 1979; Magnusson ef al., 1981; Ding et a/., 1982b); the latter has lost an especially proline-rich sequence which may be important in specifying the conformation of middle T-antigen. Mutants dl-22 and dl-23 have lost a particularly acidic region which is followed by a tyrosine residue (amino acid 3 15) that may be needed in the protein kinase reaction associated with the middle T-antigen (Schallbausen, 198I). In the latter context, it is noteworthy that the middle T-antigen specified by dl-23, although it shows only a low level of activity in the protein kinase assay (Smith et a/., 1979), nevertheless contains a phosphotyrosine residue (Dilworth, 1982).

200

BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

Griffin, 1981a). Middle T-antigens of many of the other mutants appear to be associated with membranes (our unpublished data). The sequence deleted in the mutant dl-23 appears to be largely responsible for the abnormal mobility of middle T-antigen on SDS-PAGE, since by similar analysis the protein expressed by cells infected with this mutant has an apparent molecular weight of43K, consistent with prediction from the DNA sequence (Ito et al., 1980; Smolar and Griffin, 1981). The single base change alterations that result in the temperature-sensitive phenotype of a number of large T-antigen mutants (see Fig. 3) are also interesting, in that the single amino acid replacements have such a marked effect on the thermostability of the protein. C. NEWAPPROACHES Further work on the properties of the polyomavirus T-antigens has been limited by difficultiesassociated with the use of antitumor sera. Among such difficultiesare (1) the broad range of antibody activities present in the serum which means that not only is there no specificity for any particular T-antigen but antibodies to nonviral proteins are also present; (2) the low activity of antibodies present in the serum which is a result of the method used in its production. The small quantities of T-antigens expressed in both lytically infected and transformed cells, together with the lack of a specific assay for any of the T-antigens, have meant that purification of sufficient quantities of protein for use in a conventional immunization procedure has hitherto not been possible. To overcome these problems, two alternative approaches have been used. It has been recently reported (Sutcliffe et al., 1980; Walter et al., 1980) that specific antibodies can be raised against defined chemically synthesized peptides. The advantage associated with this approach is that reasonably large quantities of antibodies can be prepared. Moreover, they can be purified by affinity chromatography and therefore have a high specific, as well as well-defined, activity. The disadvantages of such an approach are many, however. For example, the antibody levels and activities can vary widely from animal to animal, and frequently extensive cross-reaction occurs with apparently unrelated polypeptides. This cross-reaction may reflect primary sequence similarities, or contamination of the immunizing peptide with other peptides arising from the synthetic procedure or through conjugation to the carrier protein. Moreover, the heterogeneity associated with the T-antigens (see later) poses another problem when using such antipeptide antibodies for their study, in that the antibodies may not respond to posttranslational modifications or structural alterations that can occur within the protein in viva A number of antipeptide antibodies may

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

20 1

consequently be required to avoid unknowingly selecting a subpopulation of the T-antigen molecules. The data obtained with such sera, in the light of these reservations, may be incomplete. At present, this approach has been used to make specific antibodies to the middle T-antigen of polyomavirus (Walter et af.,1981). These antibodies were raised against a peptide found near the C-terminus of middle T-antigen and are capable of immunoprecipitating the antigen. In their study, Walter et al. (1 98 1) used the deletion mutant dl-8 as a source of the antigen; studies with this mutant (Griffin and Maddock, 1979;Griffinet al., 1980a)that induces a protein containing 93% of the sequence of the wild-type middle T-antigen suggest that it is a fully functional species. An alternative approach to the T-antigen immunological problem has been to make use of the monoclonal antibody secreting hybrid cell line technology introduced by Kohler and Milstein ( 1975). This approach enables existing immunization procedures to be adapted to produce unlimited quantities of a single antibody with a known activity. The production of 10 cell lines that secret antibodiesdirected against the polyomavirus T-antigens has recently been described (Dilworth and Griffin, 1982). These antibodies fall into three groups accordingto the T-antigens with which they react (Fig. 6 and Table 11). The aPy C (or “common”) antibodies bind to all three known viral T-antigens, and therefore must react with the N-terminal region of the proteins. The aPy LT antibodies bind specificallyto the large T-antigen@.), and the aPy MT antibodies bind to the middle T-antigen(s). Mapping of the binding sites for the aPy LT and aPy MT antibodies has been carried out using truncated forms of the T-antigens induced by defined mlt mutants of polyoma virus or (for aPy LT antibodies) virally transformed cells (Dilworth and Griffin, 1982) (see Fig. 7). Use of monoclonal antibodies has led to the detection of antigenic heterogeneity among both large and middle T-antigens. As mentioned previously, large T-antigen is known to be substantially modified in both lytically infected and transformed cells (It0 et al., 1977a; Turler and Salomon, 1977;Schaffhausen et a/.,1978).Not only is it aphosphoprotein, but it may also contain other modifications; on SDS- PAGE, it can be separated into two different mobility species (Turler and Salomon, 1977; Schaffhausen et al., 1978; Ding et al., 1982b).Large T-antigen has also been reported to elute from isolated cell nuclei at varying ionic strengths (Gaudray et al., 1983), which supports the notion of different forms of large T-antigen, possibly with different in vivo activities. That the proteins collectively described as large T-antigen are multifunctional might be expected from the numerous functions associated with this antigen (see Table I11 and subsequent discussion). Whether different forms of large T-antigen are responsible for separate reactions has not been demonstrated, but it appears not

FIG. 6. Autoradiograph of the SDS-PAGE separation of proteins immunoprecipitated from an [35S]methionine-labeledextract of 3T6 mouse cells infected with wild-type polyomavirus, using the supernatants from 10 cloned hybridoma cell lines Track B, a supernatant blank. Subsequent tracks, left to right: proteins immunoprecipitated with supernatants from hybridoma lines aPy(antipo1yoma) C l and C4, aPy LTI, 4 , 7 , and a P y MTS, 7, 16, 10,and 13, respectively. aPy C antibodies recognize and precipitate all three T-antigens, aPy LT only large T-antigen, and aPy MT only middle T-antigen. The positions of large (LT), middle (M-T), and small (S-T) T-antigens, as well as the viral capsid protein VPI which is frequently found associated with large T-antigen (see text) are indicated (on the left). The positions of the two host proteins specifically irnmunoprecipitated in two cases are indicated on the right (unmarked arrow and 80K). Many of the minor bands which are immunoprecipitated have been shown to be truncated versions of the T-antigens, and one that migrates only slightly slower than middle T-antigen, a 60K protein, is related to it. From Dilworth and Griffin (1982).

203

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

TABLE 11 PROPERTIES OF MONOCLONAL ANTIBODIESUSED FOR IMMUNOPRECIPITATION STUDIES

P.Ab. NO.^

T-Antigen recognized

aPy c1 aPy c 4

70 I 702

Large, middle, small Large, middle, small

aPy LT I aPy LT4 aPy LT7

71 1 712 713

Large Large Large

aPy MT5 aPy MT7 aPy MTlO aPy MT 13 aPy MT 16

72 1 722 723 724 725

Middle Middle Middle Middle Middle

Antibodya

a

Other polypeptides recognized

> 200K -

From Dilworth and Griffin (1982). According to recommended nomenclature, Crawford and Harlow ( 1982).

Basepairs Hpa N

900

"po

1000

1200

1100 I

4

1

I

8

1400

I

1500

I

7

I

L -T

Frame2

M -T

Frame 3

*,

dl-8

r:/1

1300

dl-23

1014

LT-4

MT-16

+

MT-10 MT-13

+

+ -

+ +

+ + +

1 +: ''

+

;; -

-

+

FIG. 7. Schematic diagram showing the location of the polyoma virus mlt mutants (with deletions in HpaII fragments, 4, 7,or 8, see Fig. I ) Data on the mutants were taken from the followingsources: dl-8, dl-23 (Smolar and Griffin, 198I), dl-22, dl-27 (Ding et al., 1982b), and dl-1013, 1014, and 1015 (Magnusson et al., 1981). The table inset indicates the ability of the supernatants from the various aPy hybridoma lines to immunoprecipitate the appropriate protein from infected cell lysiites, with plus (+) representinga positive reaction and minus (-) a negative reaction (see Fig. 6). The blanks left for aPy MTlO and 13indicate uncertainty (either very weakly positive or negative). All of the monoclonal antibodies were found to be secreting rat IgG,, immunoglobulins. From Dilworth and Griffin ( 1 982).

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BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

TABLE 111 PROPERTIES ASSOCIATED WITH THE VIRALT-ANTIGENS

In virro

In vivo

Large T-antigens (located in nucleus) Polyoma DNA “ongin region” binding Induction of cellular DNA synthesis ATPase activity Initiation of viral DNA synthesis Repression of early mRNA synthesis Raise levels of late mRNA synthesis Lower cellular serum requirement Establish cell line permanency Middle T-antigens (located in membranes) Tyrosine-specific kinase activity Induction of High saturation density of cell growth Anchorage independence growth in agar Tumor formation Lower cellular contact inhibition Phenotypic changes Increased agglutinability of lectins Increased nutrient transport Increased protease secretion Cytoskeletal disruption

unlikely. Moreover, there is some evidence for this with SV40 (Gidoni et al., 1982). The aPy Cmonoclonalantibodies,as well as aPy LT4, are capable of precipitating all of the detectable large T-antigen in a cell, whereas aPy LT 1 can precipitate up to 95%, and data on aPy LT7 vary between 50 and 90%, depending upon the source of large T-antigen. The exact nature and significance of these variabilities remain to be determined, but the masking of antigenic determinants by protein interactions, or by posttranslational modifications are obvious possibilities. Middle T-antigen has not yet been observed to have the same type of biochemical or physical heterogeneity found for large T-antigen, but this might only reflect technical problems in studying it. However, two apparently different molecular weight forms have been reported as phosphoacceptors in an in vitro phosphorylation reaction (Schaffhausen and Benjamin, 1981) (see below). Middle T-antigen does show extensive antigenic heterogeneity (Dilworth and Griffin, 1982),the aPy MT series of antibodies recognizing varying proportions of the total available middle T-antigen molecules in cells. The nature of this heterogeneity is unclear, although the tertiary structure of middle T-antigen is probably important in its recognition by aPy MT13 in that the form of middle T-antigen recognized by this antibody sediments significantly faster than the rest of the molecules during

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velocity centrifugation in sucrose gradients (our unpublished observations). The aPy C series of antibodies, on the other hand, recognize all the detectable metabolically labeled middle T-antigen. For both large and middle T-antigens, no primary sequence alterations have been detected among the different antigenic forms using either an approach based on analysis of tryptic peptides by a two-dimensional procedure or “Cleveland-type” limited proteolytic degradations (Cleveland et al., 1977). There is evidence, however, that different antigenic forms of middle T-antigen have different activities in an in vitro phosphorylation reaction. One of the features of the aPy MT series of monoclonal antibodies is their ability to immunoprecipitate a number of lower molecular weight polypeptides together with middle T-antigen (Fig. 6). Since the same polypeptides are recognized by a number of different monoclonal antibodies, their precipitation is unlikely to be a result of an antibody cross-reaction. (For a discussion of this phenomenon, see Lane and Koprowski, 1982.)A number of polypeptides from this “middle T-antigen family” have been analyzed by two-dimensional separation of their tryptic peptides, labeled in vivo with [35S]methionine(Fig. 8). In the resulting fingerprints, the methionine-containing spots have been tentatively assigned to peptides predicted by the amino acid sequence. In particular, the N-terminal peptide (numbered 1) which can be labeled in vitro by formyl [35S]methioninefrom the appropriate tRNA, and the peptides Met-Arg (labeled w, amino acids number 37 1 and 372, Fig. 5 ) and Ala-His-Ser-Met-Gln-Arg (labeled x, amino acid numbers 38 1 - 386) which comigrate with chemically synthesized peptides (Hunter et al. 1978; our unpublished data) have been identified. Peptide x, the C-terminus of which is only 35 amino acids from the C-terminus of the entire middle T-antigen, as well as peptide w, are present in all of the polypeptides immunoprecipitated together with middle T-antigen. This essentially rules out that the size variability observed within the middle T-antigen family could be accounted for by premature termination, or limited proteolysis at the C-terminus. Further analysis of the individual polypeptides that make up the middle T-antigen family indicates that they are all related to middle T-antigen but lack variable amounts of amino acid sequence at the N-terminus. (Tryptic peptides not normally present in middle T-antigen are also observed in individual analyses, presumably reflecting altered N-terminal sequences.) These polypeptides have apparently not been detected in precipitates obtained with antitumor sera, but since the latter often appear to be directed against the N-terminus of the T-antigens, this may not be surprising; an alternative explanation is that they are masked by proteolytic cleavage products of large T-antigen. The origin and significance of this particular series of polypeptides is unclear; as yet, they have only been identified in material from lytically infected cells

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using the aPy MT series of antibodies. In addition to polypeptides smaller than middle T-antigen, a higher molecular weight (60K) polypeptide, antigenically related to middle T-antigen, is also observed. This protein contains most of the tryptic peptides found in middle T-antigen, as well as a number of peptides not previously observed in any of the T-antigens (Ito, 1979;see Fig. 8). Possible mechanisms for generating these various polypeptides include unusual mRNA splicing during lytic infection (see Soeda et al., 1979a), aberrant initiation of protein synthesis, transcription and translation from defective viral DNA molecules which may contain some host sequence, transcription of a polyomavirus DNA molecule integrated into the host chromosome, or (for the smaller members of the family only, with distinct N-termini) limited proteolysis at defined sites. None of these mechanisms would, on its own, however, appear to account for all of the available data regarding this interesting, and possibly highly relevant, middle T-antigen family of polypeptides. WITHIN THE CELL D. LOCATION

In attempting to understand the functions of the T-antigens, a question of prime importance becomes the location of each of them within the cell. Subcellular fractionation, followed by immunoprecipitation, provided initial evidence that large T-antigen was predominantly nuclear in location, middle T-antigen was bound to membranes, and small T-antigen was present in the cytosol (It0 et al., 1977b; Silver et al., 1978; Ito, 1979;Turler, 1980). However, such studies were limited by difficulties in achieving complete purification of subcellular fractions. Moreover, they gave no FIG. 8. Two-dimensional tryptic peptide fingerprints of [3JS]methionine-labeledmiddle T-antigen (55K) and the other members of the “middle T-antigen family,” with sizes (as deduced from SDS- PAGE) indicated. The polypeptides were immunoprecipitated with the monoclonal antibody a P y MT5 and isolated from a gel similar to that shown (Fig. 6). The designation of individual “spots” on the two-dimensional fingerprint and their identification have been previously reported for the 55K species(Ito, 1979;Ito et al., 1980):Spots 1 and 2 are also found in large T-antigen and spots a and b (as well as 1 and 2) are present in small T-antigen. These therefore can be considered as being derived from the N-terminus of middle T-antigen (see Fig. I). The remainder of the peptides are unique to middle T-antigen. In the species smaller than 55K, that is, the 34K and 23K polypeptides, tryptic peptides, spots 1 and 2, that correspond to those from the N-termini of the antigens are absent, whereas those, such as x and w, which are derived from the C-terminus of middle T-antigen (see text), are present, arguing for a C-terminal relationship among the polypeptides. Major unidentified spots probably come from novel N-termini tryptic peptides; minor spots are impurities. The larger, 60K, protein has a fingerprint similar, but not identical, with that of the 55K protein, and can be seen to contain tryptic peptides from both the N- and C-termini of the middle T-antigen, as well as additional species. The origin of this protein is unclear.

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BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

FIG.9. Cellular localization of middle and large T-antigens by immunofluorescence. 3T6 mouse cells were infected with wild-type (A2 strain) polyomavirus, incubated for 42 hours at 32”C, fixed with formaldehyde, permeabilized, reacted with appropriate antibodies and antigens detected using different fluorochrome-conjugated antiimmunoglobulin sera (Hansson et al., 1983). Antibodies used in detection of large T-antigen were mixtures ofaPy LTI, 4, and 7 and of middle T-antigen were mixtures of aPy MT5 and 16 (see Fig. 6). Cells were photographed under phase contrast (a and b), epifluorescent optics specific for large T-antigen (c and d), and epifluorescent optics specific for middle T-antigen (e and f). Panels a, c, and e show the Same cellular field photographed using a X40 planapochromat objective lens, and panels b, d, and f show a similar field observed with a X63 planapochromat objective lens. Significant variation in T-antigen staining among cells is observed. Noteworthy are the bright patches of staining in the nucleus observed for large T-antigen, indicative of compartmentalization.

POLYOMAVIRUS AND CELLULAR TRANSFORMATION

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information regarding the organization and exact positioning of the T-antigens within any cellular compartment. The monoclonal antibodies now provide specific probes for studying the exact subcellular locations of large and middle T-antigen. The immunofluorescence observed for large and middle T-antigens in lytically infected cells using these reagents is shown in Fig. 9. As expected, large T-antigen is found within the nucleoplasm, with no staining of the nucleoli or the cytoplasm being observed. Within the nucleus, in many lytically infected cells, the large T-antigen was shown to be concentrated into discrete areas which may be related to viral DNA replication or virion assembly within the mucleus. Middle T-antigen fluorescence showed a very “grainy” pattern and an extranuclear distribution. Fluorescence was brightest in the immediate vicinity of the nucleus and the nuclear membrane was often observed to be stained. No staining was seen at the edges of cells, or at cell-cell contact points, although diffuse staining spread throughout the cell. It is not clear whether the higher staining in the nuclear region is a result of the greater cell thickness at this point, or to a localized concentration of the antigen. Immunoelectron microscopy data (Hansson et uf., 1983) on large and middle T-antigens in lytically infected cells are shown in Fig. 10. Large T-antigen is again seen to be concentrated in discrete areas within the nucleoplasm, with no staining of the nucleolus; the small amounts detected in the cytoplasm may result from synthesis or transport into the nucleus. From these pictures, little new information can be deduced about the precise location of the protein. However, one interesting observation is that considerable staining is observed around the nuclear pore structure of the nuclear envelope, which may implicate this protein in the processing and transport of mRNA (Webb et af.,1981). Results with middle T-antigen-specific antibodies were more surprising. These antibodies stain cytoplasmic membrane structures in both lytically infected and transformed cells. In contrast to expectation, and in apparent contradiction with studies based on membrane fractionation (Schafiausen et af.,1982),the predominant location of the protein relatively early after infection was found to be on membranes distributed throughout the cytoplasm of the cell, apparently corresponding to the rough endoplasmic reticulum. The external face of the nuclear membrane also appears to be stained, while the plasma membrane is stained only to a minor extent, but appears to show increased staining late in infection.

E. In Vitro FUNCTIONS OF THE T-ANTIGENS There are very few biochemical activities that can be precisely attributed to the T-antigens of polyomavirus. At best, the term “associated with” still

210

BEVERLY E. GRIFFIN AND STEPHEN M . DILWORTH

applies to most activities, even the better characterized ones. This is a consequence of the lack of pure proteins with which to conduct biochemical studies, The current molecular biological and immunological techniques have now reversed the problems of enzyme biochemistry. Originally, when an enzymic activity was identified, efforts were made to purify this activity away from other proteins; with present technology, however, more and more scientists are identifying proteins (and often purifying them) without any knowledge of their activities. Deducing unknown activities is obviously problematical, and requires considerable guesswork and elimination. Therefore, a knowledge of the properties of the protein being studied, its subcellular location, etc. is clearly of great value. Investigations of this type for the T-antigens of polyomavirus have the considerable advantage that defined viral mutants with altered properties exist (see Section VI). These provide both hints about function and substrates for the determination of activities. It is hoped that in the near future, it will prove possible to isolate reasonable quantities of purified proteins that will allow the enzymic activities associated with the polyomavirus T-antigens to be determined and lead to a fuller understanding of viral - host interactions. Recently, progress on the purification of middle and small T-antigens has been made (Horwich et al., 1980, 1982; Walter et al., 1982; our unpublished results). A few of the properties of the T-antigens have been tentatively defined and these are discussed. 1. Large T-Antigen

The large T-antigen of polyomavirus has frequently been compared with the large T-antigen of the closely related papovavirus, SV40. If the analogy were exact, one would predict that the hrt mutants of polyomavirus (see FIG.10. Subcellular localization of middle and large T-antigens by immunoelectron microscopy. 3T3 mouse cells were infected with wild-type (A2 strain) virus, left at 37°C and examined 20 hours postinfection. (A) Permeabilized cells examined for large T-antigen using a technique previously described (Nygren and Hansson, 1981) and the monoclonal antibody Py LTI, shown at two magnifications. Most of the staining is found to occur within discrete areas in the nucleus, and around the nuclear pores. A small amount ofcytoplasmic staining isalso observed (arrow). (B) (See page 212.) Cells examined for middle T-antigen using the monoclonal antibody Py MT16. shown at two magnifications. All of the staining occurs at cellular membranes. The bulk of the staining is in association with the rough endoplasmic reticulum, but a minority is also found at the plasma and other intracellular membranes. Similar results have been observed with infected mouse 3T6 cells and, in preliminary studies, with virally transformed rat cells. Data taken from Hansson el al. (1983). At later times postinfection, a greater proportion of middle T-antigen is found in association with the plasma membrane (H. A. Hansson, personal communication). Cells that have not been permeabilized show irregular patches of middle T-antigen at the plasma membrane.

FIG. IOA.

FIG. IOB.

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

213

Section VI) should be transforming. To date, all studies suggest the opposite. The “lytic” properties associated with both viruses appear to be similar, however. Polyomavirus large T-antigen has been shown to bind to doublestranded DNA, with a low affinity for nonspecific sequences and a high affinity for specific sequences near the viral origin of DNA replication (Paulin et al., 1975; Clertant and Blangy, 1977; Gaudray et al., 1977, 1980, 1981; Hayday et al., 1983; C. Prives, personal communication). Using a DNA protein immunoprecipitation assay, similar to that devised by McKay (198 1), and monoclonal antibodies to ensure specificprecipitation, Cowie et al. (1983) have demonstrated the existence of two sites on polyomavirus DNA molecules to which large T-antigen binds. One site is located near the region where the 5’ end of early RNA transcripts map, and another near the origin of replication (see Fig. 4). It is tempting to speculate that the two sites may account for two of the known in vivo functions of large T-antigen, namely, the initiation of viral DNA synthesis and the switch from early to late transcription. Similar results have been obtained with the adeno-SV40 large T-antigen related protein, D-2 and polyoma virus DNA (Tjian, 1980), adding some validity to speculations about one protein based on studies with another. Similar to data on SV40, an ATP phosphohydrolase activity has been detected in partly purified polyomavirus large T-antigen (Gaudray et al., 1980), but the relationship of this activity and the DNA binding activity (if any) is not clear. It is most likely that polyomavirus large T-antigen has other activities not associated with DNA binding (see Section VIII), but the determination of such activities, and the definition of those related to DNA binding, now depends on the availability of pure protein.

2. Middle T-Antigen Immunoprecipitates of the polyomavirus T-antigens contain an activity that transfers phosphate from the y-position of ATP onto either middle T-antigen (Eckhart et al., 1979; Schafiausen and Benjamin, 1979; Smith et al., 1979) or the heavy chain of rat immunoglobulin (Smith et al., 1979), in an assay similar to that devised by Collett and Erikson ( I 978). This activity appears to be associated with the viral middle T-antigen since it is missing in the hrt mutants of polyomavirus that have defects in middle and small T-antigens (Schafiausen and Benjamin, 1979), and can be shown to be altered in the mlt mutants that affect middle and large T-antigens (Smith et al., 1979; Dilworth, 1982) and in a mutant that results in premature termination of middle T-antigen (Carmichael et al., 1982). This protein kinase activity has the unusual property of transfemng phosphate onto the amino acid tyrosine (Eckhart et al., 1979) rather than onto the more common phosphate acceptors serine and threonine. A number of tyrosinespecific protein kinase activities have recently been described (for review, see Hunter and Sefton, 198I), most of them apparently having a role in either

d

rz

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

215

cellular transformation or the control of normal cell proliferation and growth. This unusual activity, associated with a number of transforming agents, has generated much interest in the proteins involved and suggeststhe possibility of a common mechanism for transformation events induced by several viruses. Obviously, a more detailed understanding of the polyomavirus middle T-antigen protein kinase activity would be useful in attempts to understand its role in cellular transformation. Previous studies of the middle T-antigen associated protein kinase activity has involved the use of antitumor sera to immunoprecipitate the T-antigens. This approach is somewhat problematic as the sera from individual tumor-bearing animals give quite different reactions in the assay (Smith et al., 1980). Recently, monoclonal antibodies have been used to investigate this phosphotransfer reaction (Dilworth, 1982). Figure 1 I shows a comparison of the polypeptides precipitated by the 10 available T-antigen monoclonal antibodies either from cells labeled in vivu with [35S]methionine,or proteins labeled in vitru with 32P (from [Y~~PIATP). With appropriate antibodies, immunoprecipitated large T-antigen has been shown to contain a small amount of phosphoserine or phosphothreonine, but no phosphotyrosine residues. The more relevant tyrosine phosphorylation is detected with middle T-antigen. However, the presence of immunoprecipitated middle T-antigen is not always sufficient for phosphate transfer to occur. This might to some extent be expected, since antibody blocking of the kinase active site, or the receptor site, could conceivably occur with certain monoclonal antibodies. It is noteworthy that despite the recognition of the great majority of the available methionine-labeled middle T-antigen in cells, aPy C immunoprecipitates do not incorporate phosphate into middle T-antigen in vitru. Further, little incorporation is seen in aPy MTlO and aPy MT 13 immunoprecipitates; these antibodies recognize only minor populations of the middle T-antigen molecules (Dilworth, 1982). In appar~

~~

FIG.1 1. A comparison of proteins immunoprecipitated from 3T6 mouse cells infected with wild-type (A2 strain) polyomavirus, labeled (A) in vivo with [3sS]methionineor (B) in vitro with [y3*P]ATP.Polypeptides were separated by gel electrophoresis(SDS - PAGE) and visualized by autoradiography. Control tracks, designated N, show immunoprecipitates using supernatant tissue culture fluid from parental myeloma cells; other tracks show immunoprecipitates using monoclonal antibodies, as indicated. Noteworthy among these data are the fact that whereas a single species (55K) is immunoprecipitated from in vivo labeled cells with aMT5 and 16, these antibodies immunoprecipitate two species, 55K and 57K, in the protein kinase assay. In the latter assay,aMT7 immunoprecipitates the single 55K protein, but with an enhanced degree of phosphorylation. Both the 55K and 57K proteins contain phosphotyrosine and are indistinguishable by tryptic fingerprint analysis (Fig. 9). The locations of large (LT), middle (MT), and small (ST) T-antigens, as well as a host 80K protein immunoprecipitated by aPy MT I3 (in A) or by aPy MT7 (in B) are indicated; the latter was found to contain a phosphotyrosine residue. The 80K host proteins do not appear to be identical. Data taken from Dilworth (1982).

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BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

ent contrast, immunoprecipitates obtained with aPy MT5 and aPy MT16 incorporate phosphate into two polypeptides. One of these is indistinguishable from the middle T-antigen isolated from cells labeled in vivo, that is, about 55K in size, while the other apparently is 2K larger. Similar data have been obtained with antitumor serum (Schaflhausen and Benjamin, 198 1). Immunoprecipitates with aPy MT7, although containing only about 50% of the middle T-antigen known to be present in cells, incorporate five times more phosphate than observed with other antibodies, but labeling appears only in the 55K protein. The phosphate-containing species have all been ’shown to be variants of middle T-antigen and the incorporated phosphate shown to occur as phosphotyrosine. Differences of phosphate incorporation observed between aPy MT7 and aPy MT5 or aPy MT I6 immunoprecipitates have been correlated with the antibody binding sites, an enhancement of the protein kinase activity probably occurring in the case of immunoprecipitates with aPy MT7. Further experiments (Dilworth, 1982) have suggested that the failure to observe phosphate incorporation in aPyC precipitates is a consequence of the absence of the antigenic site recognized by aPyC antibodies in molecules responsible for the in vitro phosphotransfer activity. We suggest, therefore, that this activity is not a general property of middle T-antigen but resides in a minor population of the total protein in lytically infected cells. A likely candidate for a species containing the activity would be one of the smaller proteins belonging to the “family” of middle T-antigens, discussed previously. The results of studies on in vitro phosphorylation indicate that there may be quite different functions associated with the different antigenic forms of the middle T-antigen, and this may reflect different subcellular localizations of the protein. It is still not clear whether the phosphotransfer reaction is an inter- or intramolecular reaction or whether any protein other than the middle T-antigen is involved in the reaction. In this regard, it is interesting that many other protein kinases are capable of intramolecular or autophosphorylation (for review, see Flockhart and Corbin, 1982). In the case of polyomavirus, an additional intriguing observation is that an 80K host protein is immunoprecipitated together with middle T-antigen in virally infected cells (Dilworth, 1982). Under conditions in which middle T-antigen is phosphorylated, the 80K protein is also phosphorylated (on tyrosine). Thus, this host protein could be a substrate for a middle T-antigen kinase activity, or, less likely, be to some extent responsible for the phosphorylation of middle T-antigen. Segawa and Ito (1982) have found that small amounts of middle T-antigen are phosphorylated in vivo; from membrane fractionation experiments, the phosphorylated antigen appears to be associated with the plasma membrane. At this juncture, it is difficult to consider these experiments as conclusive, however, since the experimental procedure also

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allows significant amounts of large T-antigen to be detected in the plasma membrane (see Section IV).

3. Small T-Antigen For polyomavirus, no function either in vitro or in vivo has been associated with this small (22K) protein which has been shown to be present in the cytoplasm of infected cells (Tiirler, 1980).Its extensive homology (see Fig. 3) with the corresponding protein of SV40 (which appears to be important in transformation), and the clustering of an unusually high number of cysteine residues, suggests that a relevant process may be being overlooked. Friedmann et al., (1978) have pointed to sequence relationships between small T-antigen and a number of hormones, such as glycoprotein hormones, thyroid-stimulating hormone, luteinizing hormone, and chorionic gonadotropin. A relationship between small T-antigen and metal binding proteins such as the metallothioneifis (Kojima et al., 1976) is also noteworthy. Another possible function (and one that correlateswell with the abundance of cysteine residues) is that it acts as an intermediary in the acetylation of host histones. Schafiausen and Benhjamin ( 1976) have shown that cellular histones (particular H3 and H4) associated with virions are highly acetylated; differencesin acetylation patterns have been observed with hrt mutants. Studies with mlt mutants suggest that polyoma small T-antigen alone is not sufficient for inducing cellular transformation and may indeed play no role in this process. The considerable amount of heterogeneity found to be associated with both large and middle T-antigens, and possibly even with small T-antigen, although not yet identified, and the differing activities possibly associated with individual species, suggests that considerable caution must be applied in attempts to purify these proteins. Conventional purification techniques could easily result in enrichment for a particular form of a T-antigen, and complicate the determination of activities associated with any one antigen. Consequently, the approach we are adopting for T-antigen purification is that of affinity chromatography using monoclonal antibodies covalently attached to an agarose support. To date, considerable enrichment for both large and middle T-antigens has been achieved by this approach. For the purification of middle T-antigen, the amount of protein synthesized in a lytic infection can be increased by using cells infected with a tsa mutant, in that shifting from the permissive to the nonpermissive temperature, subsequent to infection, has been shown to result in an overproduction of early mRNA and proteins (Cogen, 1978). This appears to be a valid approach for obtaining middle and small T-antigens, since the DNA sequence of the complete early region of the tsa-a DNA has not been found to have any detectable alterations, relative to wild-type DNA, other than the mutation

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responsible for the temperature-sensitive phenotype (M. D. Jones, S. M. Dilworth, and T. Sheedy, unpublished data). F. In ViVO FUNCTIONS OF THE T-ANTIGENS Functions for the T-antigens in vivo have been deduced from studies on the alterations of effects produced by infection of cells with viral early mutants. Unfortunately, comprehensive data are not yet available for many of the more recently constructed mutants. Most of the evidence related to function comes from earlier investigationswith the fsa mutants, which have a thermolabile large T-antigen (Oxman4 al., 1972; Paulin and Cuzin, 1975; Ito et al., 1977a), ana the hrt mutants, which have defects in the middle and small T-antigens. Some of the details of changes in cellular responses to mutant relative to wild-type viral infections are discussed subsequently. Here, we will confine ourselves mainly to considerations of the antigens themselves. 1. Large T-Antigen The hrt mutants (see Section VI) are capable of growing lytically in polyomavirus transformed mouse fibroblasts and primary mouse embryo cells (Benjamin, 1970; Feunteun and Benjamin, I982), as well as in cultured mouse epithelial cells (B. E. Griffin, unpublished). This suggests that in these cells, the expression of large T-antigen alone among the early functions is sufficient for inducing viral replication and assembly. The hrt mutants are also capable of inducing a single round of cellular DNA synthesis, and consequently are to a certain extent mitogenic (Schlegel and Benjamin, 1978). However, since wild-type virus is capable of inducing multiple rounds of DNA synthesis, presumably middle or small T-antigens are necessary for the full mitogenic effect. Cells grown in the presence of tsa mutants at the permissive temperature, then shifted to the nonpermissive temperature (or grown in the presence of cytosine arabinoside), have been found to “overproduce” early viral mRNA, relative to cells irifected with wild-type virus (Cogen, 1978). Thus, large T-antigen is probably responsible for the “early” to “late” gene switch and acts as an autorepressor. When cells infected with fsa mutants are shifted to the nonpermissive temperature, they complete an already initiated round of viral DNA replication, but do not initiate new rounds of DNA synthesis (Francke and Eckhart, 1973), suggesting that large T-antigen is crucial for the initiation of viral DNA synthesis. In some cases, large T-antigen may also be responsible for the initiation of the transformed state (Fried, 1965), but at this stage it is unclear whether this is a direct or an indirect function of the protein (see Section VII).

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It has also been reported that large T-antigen regulates the integration of polyoma DNA into host chromosomes (possibly the reason for its requirement in the initiation of transformation) and the reversible generation of polyploid cells; again the data are deduced from the temperature sensitivity of these functions in tsa infected cells (Colantuoni et al., 1982; Dailey et al., 1982). The N-terminus of large T-antigen, at least, has been suggested to be necessary for the growth of transformed cells in low serum (Rassoulzadegan et al., 1982).That is, whereas established lines of rat fibroblast cells could be transformed with a cloned fragment of polyomavirus that was competent to express only the viral middle T-antigen (Treisman et al., 1981b), the resulting cell lines lacked the ability to grow to high density when starved of serum and ultimately reverted to an apparently normal phenotype. Nor did they produce colonies in soft agar in low serum medium. Thus, although transformed by some criteria, they did not express the full range of phenotypes appropriate to transformed cells. The missing phenotypes could be shown to be restored by complementation with a cloned fragment of polyomavirus that contained the 5’-portion of the early region DNA present in the hrt mutant, NG-18. Although these data have been interpreted to mean that two viral functions are needed for the expression of the “fully transformed” state of the cells, an alternative explanation is also possible. That is that the middle T-antigen expressed by the original cloned fragment of DNA is not fully competent, but lacks some feature essential to its function in transformation that can be supplied either by the whole animal (in tumor formation, Treisman et al., 1981) or by other viral sequence (Rassoulzadeganet al., 1982).In this context, it is worth keeping in mind the experiments of Horwich et al. (1982) which show that the small T-antigen fusion protein expressed from plasmids appears to be extremely labile, at least in Escherichia coli, whereas no such lability has been observed for small T-antigen itself in vivo.Therefore, the possibility must be considered that middle T-antigen synthesized at least in part from cloned DNA is not identical with the product expressed in vivo.

2. Middle T-Antigen The tsa mutants of polyomavirus, although capable of abortive transformation at the nonpermissive temperature (Stoker and Dulbecco, 1969),are defective in the establishment of a stably transformed state (Fried, 1965). This has been interpreted as implying that although the tsa gene (now known to encode large T-antigen) may govern the integration and stable perpetuation of the viral genome, it is not necessarily responsible for the transformed phenotype. Later work has amply justified the latter conclusion, The phenotype of the hrt mutants (Benjamin, 1970) has provided

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further insight regarding a “transforming gene”; studies with these mutants has allowed this entity to be equated with either the middle or small T-antigen, or both. The hrt mutant viruses have lost the ability to induce morphological changes in the appearance of cells, disrupt the cellular actin cable network, increase nuclei and nucleoli size, increase lectin agglutinability of the cell surface, and increase uptake of glucose (Schlegel and Benjamin, 1978).They are also defective in initiating multiple rounds of cell division in confluent cultures, but are capable of integrating into the host chromosome, and are expressed, with no evidence of concomitant cellular transformation (Lania et al., 1979). Moreover, they also fail to induce endogenous cellular factors that are believed to be necessary for viral growth in established mouse fibroblast lines; this gives rise to so-called “host range” phenotype (Goldman and Benjamin, 1975). The mutants are also deficient in the ability to stimulate cells to acetylate histones H3 and H4 to the extent that they are acetylated in minichromosomes that contain wild-type viral DNA (Schaflhausen and Benjamin, 1976). What is lacking in studies with hrt mutants are definitive whole animal experiments. It could be argued that these mutants, being large T-antigen competent, might be capable of tumor induction in the appropriate species, albeit at a very slow rate. To test this hypothesis, experiments are under way using one of the better characterized hrt mutants, NG 18,and nude mice. After more than 8 months, tumors have become apparent in these animals, although the causative agent has not yet been identified (B. E. Griffin, unpublished observations). A comparison of mutants of polyomavirus that have lesions in large and middle T-antigens, the mlt mutants, with hrt mutants, suggests that the middle T-antigen is the protein that is primarily responsible for the transforming effects normally observed with polyomavirus (Griffin et al., 1980a; Ito et al., 1980). Some mlt mutants are defective in their ability to transform cells, but retain a capacity to replicate similar to that observed with wild-type viruses (Griffin and Maddock, 1979; Magnusson and Berg, 1979;Ding et al., 1982b).This suggests that they essentially have a competent large T-antigen, and in that the small T-antigen is present, protein responsible for the transformation defect is middle T-antigen. Analysis of the T-antigens produced by a number of polyomavirus transformed cell lines provides some confirmatory evidence for this notion. That is, different lines show a wide variability in the amounts of large T-antigen produced, in many cases producing no full-sized antigen, but all produce apparently normal middle and small T-antigens (Israel et al., 1980b; Ito and Spurr, 1980; Lania et al., 1980a). The hazard of drawing biological conclusions from studies on hrt and mlt mutants is that both of these are double mutants. The use of precise molecular biological techniques has enabled the above conclusions to be

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more directly confirmed. Two single site mutants of polyoma have been constructed that prematurely terminate expression of the gene coding for middle T-antigen, while producing little or no effect on the large and small T-antigens (Carmichael et al., 1982; Templeton and Eckhardt, 1982). In both cases, the resultant virus is capable of lytic growth, but not of transforming cells. Moreover, the use of a bacterial plasmid that contains a polyoma DNA fragment which is missing a sequence corresponding to the intron deleted in middle T-antigen mRNA, and consequently is capable of expressing only the middle T-antigen among the known early functions, has been used to confirm directly the role of middle T-antigen in transformation. The recombinant DNA, introduced into an established rat cell line either by transfection techniques or fusion with bacterial protoplasts, causes changes in the cell phenotype; a loss of cellular actin cable networks is observed and the cells acquire the ability to grow in soft agar and to form tumors in syngeneic animals (Treisman et al., 1981b). However, the presence of middle T-antigen alone does not appear sufficient to induce transformation of primary rat embryo cells or to permit the growth in low serum characteristic of cells transformed by wild-type virus (Rassoulzadegan et al., 1982, 1983). Thus, functions other than those expressed by middle T-antigen may be required for the elaboration of all the properties associated with transformation. For example, complementary functions from the other viral antigens may be required, or, similar to the situation for untransformed cells, extracellular growth factors may be needed for cellular proliferation (P. Kaplan and B. Ozanne, personal communication). Middle T-antigen alone, however, in certain cells is possibly fully competent for the induction of tumorigenesis, although further work is necessary to prove this. At this juncture, it may be worth recalling those tumor surface transplantation antigens (TSTA) that are capable of inducing specific cellular immunity which can lead to tumor rejection when transplanted into a syngeneic host (for review, see Allison, 1980; Law et al., 1980). In the case of polyomavirus and SV40, they are assumed to be virally coded, cell surface associated antigens, but they have not yet been identified. Some doubts concerning the validity of this assumption come from studies of Dalianis et al. (1982) which show that both hrt (middle and small T-antigen defective) and mlt (large and middle T-antigen defective) mutants can induce tumor rejection. Israel et al., (1980a) have shown that for polyomavirus, the TSTA activity is not associated with large T-antigen. A prime candidate in polyomavirus for this important biological entity would therefore seem to be one of the species that make up the middle T-antigen family, possibly the relatively minor species that has been shown by immunoelectron microscopy to be associated with the plasma membrane.

BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

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A

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0.50

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tsA mutants

26

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hrt mutants

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mlt mutants

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FIG. 12. A schematic representation of the location of “early” mutants of SV40 and polyomavirus. (A) The “early region” which lies between 0.67 and 0.14units on the physical map of SV40 (see Fig. 2) has two known classes of mutants, the dl 0.54-0.59mutants and tsA mutants; their locations, relative to the SV40 small (t) and large (T) antigens are shown. (B) The “early region” which lies between 72 and 26 units on the physical map of polyomavirus has three known classes of mutants, hrf, mb, and fsu mutants; their locations in relation to the three viral T-antigens (small-, middle-, and large-T) are shown (see Tegtmeyer, I981, and text for discussion of mutants). Adapted from Griffin ( 1982).

VI. Mutants of Polyomavirus

Until a few years ago, there were only two classes of mutants known which could be correlated with the “early genes” of polyomavirus. These were the tsa and hrt classes,’ exemplified by tsa-a (Fried, 1965) and NG-18 (Benjamin, 1970), respectively. Recently, another class of mutants, designated mft and exemplified by dl-8 and dl-23 (Griffin and Maddock, 1979; Smolar and I We defer to the convention of genetics and designate these classes by three lower case letters.

I

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Griffin, 198I), has been described. The location of these classes of mutants relative to the “early” mutants ofSV4O are shown in Fig. 12. Several discrete types of mutations within the noncoding region of the viral genome which can affect expression of early genes have also recently been isolated and examined. For purposes of discussion, we will treat the latter mutants as if they were a single class, and designate them ncr. Only a few “late mutants” of polyomavirus have yet been thoroughly characterized. All mutants will be considered mainly in terms of the effects individual mutations produce on viral gene expression. A. tsa MUTANTS

These temperature-sensitive mutants were selected followingnitrous acid treatment of the polyomavirus genome. In the case of the mutant described by Fried (1 965) ( tsa-a) and those by Di Mayorca et al. (1 969), the viral strain used was a large plaque strain. At least one of the tsa mutants, tsP155, described by Eckhart (1969), is instead derived from a small plaque isolate. All of the tsa mutants examined to date appear to be defective in viral DNA synthesis and some are also defective in the induction of cellular DNA synthesis (Francke and Eckhart, 1973; Eckhart, 1975). In general, cells transformed by these mutants at the permissive temperature (32°C) remain transformed when shifted to the restrictive temperature (39°C);the initiation of transformation is not, however, observed at the higher temperature. These results have indicated that the lesion in these mutants, shown subsequently to affect the viral large T-antigen, alters the initiation but not the maintenance of transformation. There appear to be exceptions to these general conclusions, however. For example, the early mutants ts52 and possibly ts609, which are not complemented by tsa-a but can be complemented by late mutants, appear to show only a slight temperature effect in transformation assays (Eckhart, 1975). Two other experiments are worthy of note in this connection. In one, Bourgaux and co-workers have demonstrated that mouse embryo cells can be transformed at the restrictive temperature by tsP155 (Bourgaux et al., 1978) and have established a cell line (the “Cyp” line) from these cells. In the other, we (Novak and Griffin, 1981b) and Basilico and co-workers (della Valle et al., 1981a) have shown that, although there might be a small quantitative difference, qualitatively there is no detectable distinction in cellular transformation when isolated DNA from tsa mutants (either tsa-a or ts25) is used in transfection experiments at either the permissive or restrictive temperatures. Further, Seif and Cuzin (1 977) have described two types of rat cells that were transformed by tsa mutants. One, the so-called “A”-transformant, was selected by growth in soft agar at the permissive temperature, and maintained the transformed phenotype at the restrictive temperature. The other, the “N”-transformant,

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selected by dense focus formation, did not. Data derived from studies on Aand N-transformants are discussed in detail by Hand ( 1981 ) and are considered below. The lesions in those tsa mutants that have been mapped (for review, see Ito and Griffin, 1982)have all been shown to be located within the C-terminal portion of large T-antigen (Feunteun et al., 1976; Miller and Fried, 1976). The DNA sequence changes in four tsa mutants (tsa-a, ts25, ts52, and ts48) have been determined (Deininger et af., 1981; Thomas et al., 1981) and their locations are indicated in Fig. 3; mutants ts25 and ts52 have different base changes but they are located within the same codon. DNA sequence studies further confine the lesions in these mutants to the C-terminal one-third of large T-antigen. Similar analysis has shown that in the entire early coding region, no further base change occurs for tsa-a (M. D. Jones, S. M. Dilworth, and T. Sheedy, unpublished data), whereas for ts48, a second site lesion occurs that introduces amino acid changes into both middle and large T-antigen (Deininger et al., 1981). The latter lesion lies, however, in a region of the genome that can be deleted with no apparent effect on either transformation or replication (see mutant 45, below) and therefore may essentially be “silent.” Whereas data on viral DNA synthesis are consistent among tsa mutants, data on transformation are not. These can be contrasted with the hrt mutant studies in which data on transformation in vitru are essentially consistent whereas those on virus growth are not. Some of the apparent anomalies with the tsa mutants may be a consequence of different interactions between the virus and the various host cells used in transformation studies. Many early experiments with tsa mutants were camed out on hamster cells, subsequent studies mainly with rat cells, and recent studies by Bourgaux and his group with tsPl55 have involved mouse cells. The latter, homologous system appears of particular interest. Since the stimulation of host DNA synthesis is often observed to be affected by lesions in tsa mutants, it is possible that large T-antigen (a DNA binding protein) may interact anomolously with the different chromosomal DNA of heterologous cells. Moreover, we do not yet know whether cells that can be transformed are those that are actively dividing, or the converse, or whether cell division is an important aspect of transformation. In this context, recent studies by Folk et af. (198 1) show that even “cloned” cell lines (hamster BHK-2 1 cells, in this case) are heterogeneous and differ in their response to polyomavirus. We have argued (Novak and Griffin, 1981b) that the role of polyomavirus large T-antigen in cellular transformation may be indirect. One explanation for the temperature sensitivity observed with virions, but not with “naked” DNA, could be that one of the functions of large T-antigen in the cell is to convert the DNA in virions into a form suitable for integration into the host chromosome and thus for transformation. This could, for example, involve removing cellular histones, or even possibly one of the viral capsid proteins

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from viral minichromosomes. It is perhaps worth noting in this context that removal of all the proteins in vitro from DNA isolated from virions is not easily accomplished (Frearson and Crawford, 1972). Alternatively, a dose effect may be responsible for the apparent discrepancy between the in vivo and in vitro data, since large amounts of viral DNA are introduced into cells by the in vitro calcium phosphate transformation technique. A further anomaly in studies of tsa mutants may be inherent in the sequence of the parental molecule used to generate individual mutants. As has been mentioned earlier (see Fig. 4)and discussed again in the case of ncr mutants, the DNA sequence in the region of the genome encoding the viral origin of replication, putative promoters, and possibly other “control” signals, varies from strain to strain. This is particular apparent in the case of small plaque strains (see Griffin, 1977;also, personal communications from A. J. Levine, P. Bourgaux, and H. E. Ruley). It can deduced from the work of Vogt et al. (1 976) that the parental strain for tsa-a had a different sequence from either the A2 strain of virus sequenced by us (Soeda et al., 1980b) or the strain 3 sequenced by Friedmann’s group (Deininger et al., 1980). If viral or cellular replication or the levels of viral T-antigens influence transformation as they well may, then possibly not only is the lesion responsible for the thermosensitivity important, but also the unique controlling sequences derived from the parental molecule. A further striking anomaly is observed between two of the tsa mutants. Eckhart (1 975) first showed frequencies of transformation of ts25 as at 32°C and, correspondingly, for at 39°C and 2 X (39°C) and 7 X lo4 (32”C), relative to wild-type virus. ts52 as 6 X More recently, DNA sequence analysis of ts52 and a strain of ts25, ts25E, showed that the lesions in these two mutants appeared at the same glycine residue, it being converted to cysteine in the case of ts25E and serine in the case of ts52 (Deininger et al., 1981; Thomas et al., 1981). The transformation results need to be confirmed with the precise mutant stocks used for sequence analysis, but if there are no other significant alterations in these mutants, the glycine residue, present 12 amino acids from the C-terminal end of strain 3 large T-antigen (or I7 amino acids in the A2 strain), becomes a focal point in the study of any role for this antigen in transformation.

B. hrt MUTANTS This class of mutants (host range, nontransforming) has lesions in both small and middle T-antigens but none in large T-antigen. With a few exceptions, hrt mutants have been isolated and studied mainly by Benjamin and his group (see Staneloni et al., 1977). The mutants complement tsa mutants for growth and transformation (Eckhart, 1977; Fluck et al., 1977) and to date, without exception, have been found to be nontransforming and nontumorigenic. The prototype hrt mutant, NG-18, has been shown to have a 187 base pair deletion and thus to be a frame-shift mutant; the lesion

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BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

results in premature termination of both small and middle T-antigens (Soeda and Griffin, 1978; Hattori et af., 1979). Sequence changes in other mutants are described by Carmichael and Benjamin (1 980). The study of hrt mutants has lent much support to the concept of a virally coded gene responsible for transformation and for focusing attention on its product, the middle T-antigen. Cells infected with most of the hrt mutants make remnants of proteins that appear to be related to middle T-antigen (Silver et af., 1978). Perhaps the most difficult of this class of mutants to rationalize, and therefore possibly the most interesting, is NG-59. This mutant has an in-frame lesion (Carmichael and Benjamin, 1980) and produces apparently “normal” amounts of 56K and 22K proteins, presumed to be middle and small T-antigens, respectively (Silver et al., 1978); the structural alteration in NG-59 involves the addition of an isoleucine residue, as well as a change of an aspartic acid to asparagine in the two antigens (see Fig. 4). If there were not evidence to the contrary, it would be tempting to speculate that in the light of the drastic phenotypic change observed and the apparently slight sequence change in NG-59, the mutant might contain an important second-site modification. Evidence against this suggestion lies in the data of Feunteun et af., (1976) who show that the lesion can be rescued by a 379-long oligomer derived from the HpaII-4 restriction fragment of polyomavirus DNA (see Fig. 1); this entire region in the mutant has apparently been sequenced in a search for second-site alterations (Carmichael and Benjamin, 1980). Therefore, it must be concluded that the amino acid change has occurred in a functionally important domain of the middle T-antigen, possibly within “an active site.” Alternatively, the lesion may produce a con formational change that affects a functionally significant area. A further possibility is that the sequence change in the mutant might result in an alternative splice site being used to create the messenger for middle T-antigen. As noted earlier, a large number of putative alternative splice sites exist for the mRNAs of middle and small T-antigen (Soeda et al., 1979a).The lesion in NG-59 has created a nine-residue-longA,T-tract in the DNA near the 5’-side of the intervening sequence. It is plausible that this could alter the conformation of the RNA, and were an alternative splice site used, the protein could still appear indistinguishable from middle T-antigen by gel electrophoresis, but contain more profound sequence changes than predicted by the simple DNA sequence alteration observed. Several possible explanations exist for the failure of hrt mutants to transform cells. Assuming that the mutants can infect the recipient cell, one is that a viral transforming gene has been inactivated, while another is that the mutant DNA fails t o become integrated into the host chromosome, or integrates in such a way that cell death results and no transformation is observed. In order to distinguish among these possibilities, Lania et a/.

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(1 979) isolated rat cells with apparently normal phenotypes, that had been exposed to an hrt mutant, and were able to show that mutant DNA was present in these cells both in an integrated and in a free state. These studies, assuming that they represent hrt mutants in general, emphasize the importance of the viral gene product per se in transformation. The sequence of the DNA in the hrt mutant used in these experiments has not been determined, however, nor have similar results been reported for any of the better characterized mutants. The hrt mutants were originally selected by their ability to grow in polyomavirus transformed mouse 3T3 cells but not in untransformed cells (Benjamin, 1970). This implied that a complementation factor for a growth defect existed in the transformed cell. Since an apparently normal large T-antigen is induced by hrt mutants, one of the conclusions allowed by these data is that either small or middle T-antigens are required for viral DNA replication, or packaging, in fibroblasts. More recently, it has been shown that several types of cells, not carrying polyomavirus information, also can serve as permissive hosts (Goldman and Benjamin, 1975; Goldman et al., 1979). This has been explained by suggesting that hrt mutants are “null,” and cellular “permissivity factors,” varying from cell to cell, determine their expression. We have shown that in mouse kidney cells, cultured in media that permits the growth of epithelial cells (Taub et al., 1979), NG-18 is capable of producing either viral DNA or virions in a manner indistinguishable from wild-type virus (B. E. Griffin, unpublished data). Moreover, polyoma mutants that are phenotypically and genotypically similar to hrt mutants have now been isolated from primary mouse embryo cells (Feunteun and Benjamin, 1982).It remains to be proved whether cellular permissivity factors are the explanation for growth defects in hrt mutants, or whether there is some external change in the virion which requires cellular receptors other than those normally present in established mouse fibroblast lines.

C. mlt MUTANTS Genotypically, hrt mutants are double mutants with lesions in middle and small T-antigens; mlt mutants can be considered to be complementary in some respects in that they are also double mutants, but with lesions in middle and large T-antigens. Unlike hrt mutants, which are apparently phenotypically all alike, the mlt mutants vary considerably, some behaving as if they were essentially defective in middle T-antigen, some as if defective in large T-antigen, and some being indistinguishable from wild type virus. Many mlt (middle, large T-antigens)mutants are deletion mutants (see Figs. 5, 7, and 13); to date, only two point mutants have been reported. The first of these mutants to be described, dl-8 and dl-23 (Griffin and

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BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

FIG. 13. A n analysis ofT-antigens induced in mouse cells infected with wild-type (A2 strain) polyomavirus. rnh mutants dl-1013, 1014. 1015, dl-8 and dl-23, and the hr/ mutants. NG-18. Cells were labeled. proteins immunoprecipitated using antitumor antibodies, and separated as described (Fig. 6). The location ofthe three viral antigens, large (L-T), middle (M-T). and small (S-T) T-antigens, and the capsid protein VPI, as well as their sizes, are indicated. As expected, NG- I8 induces only large T-antigen, among the early proteins. m/f Mutants induce all three antigens, with the sizes of the large T-antigens correlating with sizes expected from the sequences of the mutants (Magnusson et a/., 198 I ; Smolar and Griffin, 198 I). The sizes of the middle T-antigens do not all correlate with the sizes ofthe deletions in the mutants, but appear to reflect further structural changes. Most notable are the pair of mutants dl-8 and dl-23 whose sequence should differ by only four amino acids but whose apparent mobilities are about 50K and 43K, respectively (Ito ef a / , 1980: see text). VPI appears to accompany large T-antigen in immunoprecipitates (Dilworth and Griffin. 1982). Since this structural protein appears to form covalently bound complexes to itself, probably linked by disulfide bridges (Walter and Deppert, 1975). it is possible that it can form similar linkages with large T-antigen.

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Maddock, 1979), have proved quite useful in subsequent functional studies since their properties lie toward the extremes of properties displayed by this class of mutants. That is, dl-8 grows poorly yet transforms cells in culture, and the latter produce tumors in animals with an efficiency generally greater than that found with wild-type virus; dl-23 grows well in vitro but transforms poorly (Griffin et al., 1980a). Virus from the latter mutant produced small foci in rat cells after a long period of time, but, in our hands, using rat-1 cells as the recipient, did not produce colonies in soft agar. Some cell lines established from foci, after passage, eventually produced tumors in syngeneic rats, others did not. Seif (1 980), on the other hand, found with rat 3T3 cells that colonies in soft agar were observed 3 - 4 weeks subsequent to infection with wild-type virus and 6 - 8 weeks after infection with dl-23, or SV40. These apparently conflicting data may reflect differences in the recipient cells used in the two experiments or time differences. McCance (198 1) has studied both growth and persistence of the dl-23 and dl-8 mutants in mice and found both mutants to have a reduced capacity to replicate, relative to wild-type virus, and to persist in vivo, thus emphasizing the importance of the viral- host interaction. Mutants with phenotypes similar to that of dl-23 have been described by Magnusson and Berg ( 1979); their mutant 1015 has a lesion located adjacent to that of dl-23 (Magnusson et al., 198I ; Smolar and Griffin, 1981) and is essentially nontransforming. Bendig et al. (1980a) isolated a mutant, designated mutant 45,that has a deletion which essentially maps between those ofdl-8 and dl-23 (Smolar and Griffin, 1981). Mutant 45 is in all its studied properties indistinguishable from wild-type virus. Lesions in these and some other rnlt mutants are shown in Fig. 5. These deletion mutants are proving useful for locating regions of functional activities in the two antigens and tentatively correlating them with amino acid changes in the corresponding proteins. Additional mlt mutants have been isolated (Ding et al., 1982b; Gelinas et al., 1982; G. Magnusson, personal communication) with properties similar to one or the other of the mutants described above. From a consideration of the deletions found in all the mlt mutants reported to date and their properties, we have concluded (Ding et al., 1982b) that the polypeptide Glu-GIu-Tyr-Met-Pro-Met-Glu, located toward the center of middle T-antigen, is critical for transformation, and may be correlated with a protein kinase activity associated with middle T-ant igen. Recently, two reports of the isolation of single-site mutants have appeared (Carmichael el al., 1982; Templeton and Eckhart, 1982). In both cases, a mutation has been introduced in such a way as to create a termination codon within middle T-antigen, toward the C-terminus of the protein; this has little or no effect upon the viability of the species but, as would be

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BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

predicted from earlier studies (see below), results in a failure ofthe mutant to induce cellular transformation. The mutant P$ 1387-T, constructed by Carmichael et al. (1982), removes 37 amino acids from the C-terminus of middle T-antigen, and that of Templeton and Eckhart, MOP1033, much more (see Fig. 5). A comprehensive study of the role of the sequence at the C-terminus of middle T-antigen in transformation has been carried out (Novak and Griffin, 198la). Rat-1 cells were transfected with cloned polyoma DNA fragments containing increasingly large deletions from the portion of the DNA that would encode the hydrophobic region at the C-terminus of middle T-antigen, presumably its cellular membrane attachment site (Novak and Griffin, 1981b; Carmichael et al., 1982)and transformation was studied. Any alteration of the C-terminus of middle T-antigen was accompanied by a dramatic decrease in transformation efficiency; up to 36 nucleotides, corresponding to the terminal 12 amino acids, however, could be removed without totally abolishing transformation. No transformants were obtained when sequences corresponding to a further two amino acids were removed. Data on the physical properties of the three classes of mutants of polyoma virus, tsa, hrt, and mlt, which appear to have a direct or indirect effect upon transformation, have been tabulated by Ito and Griffin (1982). D. ncr MUTANTS About 9% of the polyoma virus genome appears to be noncoding (Soeda

et al., 1980b). This region contains the designate viral origin of replication (with sequence that is to a large extent homologous to those seen in other papovaviruses around their origins of replication, Soeda et al., 1979b), as well as the putative “promoters” for early and late transcription, and possibly other functions not yet identified (see Fig. 4). Except for the sequence thought to specify the origin of replication, the rest of the region bears little homology to corresponding sequences in the other papovaviruses and may well reflect host cell adaptation. Because the whole noncoding region has not yet been well defined in terms of functions, we have combined all the mutants from this area into a single class which, for the purposes of this article, we designate ncr (noncoding region) mutants. The first such mutants to be described had deletions between the viral origin of replication and the coding region for the T-antigens (Griffin and Maddock, 1979; Magnusson and Berg, 1979; Wells et al., 1979; Bendig and Folk, 1979). None of the mutants differed significantly from wild-type virus in their properties. All grew without helper virus and all could transform cells in culture. One mutant (mutant 75) subsequently described by Bendig et al. ( 1980b) had a fourfold reduction in the amount of DNA synthesized relative to that observed with wild-type virus; this was the largest effect

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23 1

observed among the mutants with respect to replication. When the sequence of the large plaque A2 and (strain-3) strains of polyomavirus became available (Soeda et al., 1980b; Deininger et al., 1980, respectively), it was easy to understand how many of the mutants arose, since the Hue11 enzyme used in their generation, previously thought to have a single recognition site in polyomavirus, was shown to have two contiguous sites, cleavage of which removed 11 base pairs from the viral genome; the viral strain used by Folk and collaborators (Bendig et al., 1980b)seems, however, to contain only one Hue11 site. The conclusion allowed from the above studies is that polyomavirus can tolerate fairly large deletions within parts of this region of the genome with relatively minor concomitant effects upon either growth or transformation. It is noteworthy that, in many of the mutants, sequences that have been postulated to represent ribosome, RNA polymerase, and large T-antigen binding sites have been deleted (see Fig. 4) and therefore are clearly not essential, at least qualitatively. A mutant recently described by Ding et al. (1982a) has 49 base pairs of this portion of the genome deleted, and in their stead has acquired 95 base pairs with limited homology to the deleted sequence and apparently of mouse origin. This mutant (din-21) has properties similar to those observed with mutant 75. Similar deletion - insertion mutants have also been isolated by W. R. Folk and shown to contain mouse DNA (personal communication). A mutant, designated dl- 1023, recently isolated by Magnusson and co-workers (Luthman et al., 1982) removes nine base pairs of a region designated as part of the origin of replication (Griffin et al., 1974). Not surprisingly, it is nonviable but it retains a “normal” ability to transform cells, suggestingthat replication may not be a prerequisite for transformation. Polyomavirus contains within its noncoding region two very similar inverted repeat structures, separated by about 50 base pairs, that resemble known origins of replication (Day and Blake, 1982) (see Fig. 14). It has always been assumed, but not rigorously proved, that the sequence at the HpaII 3,5-junction, which shows remarkable homology with the corresponding sequences of SV40 and BKV (Soeda et al., 1979b), serves as the viral origin of replication. Because the two inverted repeats are structurally so similar, it is tempting to speculate that they both might be involved in viral DNA replication. A number of mutants have been isolated with deletions on the “late side” of the putative viral origin of replication, in the region included ifi Fig. 14 (Tyndall et al., 1981; Luthman et al., 1982). Most of the mutants described in the latter instance appear to transform normally but vary in their growth properties. One of the mutants (dl-1025) should allow the hypothesis of a “second site” origin of DNA replication to be tested directly, since it lacks the A,T-rich sequence around nucleotide 5150 and much of the hairpin loop. A total of 27 base pairs have been deleted, relative to wild-type DNA. The mutant transforms with an effi-

c-9

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24

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FIG. 14. A model for the “modulation” of DNA replication. The sequence of the “noncoding” region of polyomavirus allows for two very similar hairpin (or cruciform structures) to be formed, both of which are preceded by very A,T-rich tracts and followed by purine (or purine-rich) tracts. The sequence shown in this figure includes these structures, which correspond to two boxes labeled “HP’ (Fig. 4),and all the sequence between them; from studies with deletion mutants (see text), at least one “HP’-region appears to be ofprimary importance in viral DNA replication. All current data suggest that the 185-nucleotide-long sequence shown here defines the maximum sequence involved in the initiation of DNA replication. Determination of primary DNA sequences on a number of viral strains, variants. and mutants would suggest that whereas limited sequence heterogeneity occurs within this region, particularly in the areas which specify the cruciform structures, the postulated overall secondary structure, and possibly a tertiary structure, ispreserved(Friedmann eral., 1978: Soedaet al.. 1977. 1979, 1980b;Deiningereral.. 1979;Herbomelera/., 198I:Tyndallefal., 1981;alsoA. J. Levin, W. R. Folk. and M. Yaniv, personal communication). Interestingly, one of the differences between the A2 strain of virus and strain 3 (Soeda et a/., 1980b Deininger er a/., 1980)lies in the number of base pairs that could exist in the “pseudo-origin,” the A2 strain having one less. This variation lends support for the DNA conformation in that the double mutation at positions 5 172 and 5 186 has only a small effect upon the energy ofthe structure. (In this figure, the A2 strain sequence has been used to produce the structure designated “putative origin” and the strain 3 sequenceto produce the“pseud0origin,” since this maximizes the similarities between the structures.) As discussed (text), the two cruciform structures could compete for protein or cellular sites important in replication, and thereby act as modulators of this event. The eight base pair repeat (boxed) could alter the distance between the two structures if the “early strand’ sequence of one repeat moved along to pair with the “late strand” sequence of the second repeat, as shown (bottom). Were two proteins involved in DNA binding, a change in distance could result in the displacement of one or the other, producing concomitant alteration in a regulatory event. Alternatively. one of the sites might be used to bind to nuclear membranes, in an event preceding G transitions, as replication (see Buchler-White ef al., 1980).Many cellular “host range” mutants (see ncr mutants, text) have been found to have A indicated, as well as duplications and rearrangements within this region, which appear to release a block to replication found for wild-type virus in undifferentiated cells. Moreover, many strains of polyomavirus, such as the Toronto small plaque strain, also vary in sequence over part of this region (H. E. Ruley, personal communication). Sequences either side of that shown here would appear to regulate transcription (Kamen ef al., 1980). In the light of discussion about a functional role for large T-antigen in recombination events, it should be noted that the local DNA structure around a cruciform junction is indistinguishable from recombinational intermediates postulated by Holliday ( 1964) (see also Potter and Dressler, 1976; Mizuuchi ef a/., 1982). As support for a model that allows cruciform conformations to exist in the DNA, possibly upon interaction with proteins, it has recently been shown (Lilly, 1980; Panayotatos and Wells, 1981) that even quite short double-stranded inverted repeat DNA sequences can exist in equilibrium with such structures, as judged by sensitivity to SI single-strand-specific nuclease.

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BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

ciency similar to the latter, but grows less well. Assuming large T-antigen is expressed in dl-1025, this would appear to negate the postulate, except for the fact that the deletion creates a new inverted repeat, rather similar to that deleted, which has the same CCAG sequence at its apex. In the growth defective mutants described by Tyndall et al. ( 198I), viral DNAs with fairly large deleted segments have been cloned via linker sequences into plasmids, and their expression studied. A number of mutants with deletions that encompass the entire “second-site origin,” and polyomavirus-transformed mouse cells (COP cells) that express the large T-antigen but lack the sequence that has been designated as the origin of replication, are complementary for growth. This hypothesis of a second-site origin that might act as an inhibitor or modulator of replication, possibly by mimicking the “real” origin in its interaction with an initiation factor(s) thereby removing the latter from its normal functional site, or by physically interacting (at the DNA level) with the origin sequence, remains to be evaluated. To us, the complex tertiary structure potentially allowed by the inverted repeats within the noncoding region suggests a regulatory role for the DNA (Soeda et al., 1979b). Some of the ideas apparent in Fig. 14 can be tested once appropriate point mutants become available. It has long been known that whereas primary cultures or established lines of mouse cells are permissive to polyomavirus, no gene expression occurs in undifferentiated, or embryonal, mouse cells even though the virus can penetrate these cells. Viral mutants have now been isolated in which the block to expression has been removed. The simplest of these mutants (Py F441, Fujimura et al., 1981) appears to have a single A G base change within the 5 5 residues that span the two hairpin loops (Fig. 14); this small change allows productive infection of some mouse embryonal carcinoma (EC) cells. If tertiary DNA structure of biological significance occurs within this region, the change could involve a conformational alteration. It is also noteworthy that the single transition mutation removes a termination codon. For wild-type polyomavirus, no coding function has been attributed to this region. For SV40 a “corresponding” position on the genome, previously called the “agno gene” region, is known to have a short open reading frame and recently a small protein (6 1 amino acids) has been isolated which appears to arise from the expression of this sequence; it is interesting that it seems to have DNA binding activity (Jay et al., 1981). Mutants similar to Py F441 (Py FlO1,andPyFl 1 1,Fujimuraetal.. 1981;FuJimuraandLinney, 1982;and PyhrN-2 and N-5, from the Toronto small plaque strain of virus, Sekikawa and Levine, 1981) have duplications of the region in which this A G base change occurs. Three further mutants of this class have been G described (Vasseur et al., 1982);although none appears to have the A base change, two of them (Py EC F9-HM and F9-1) have an alteration (T A) that also removes the putative termination codon. All mutants that

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-

-

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grow in F9 cells appear to be related to the strain 3 virus, at least with respect to the region designated “pseudo-origin” in Fig. 14. The same holds true of mutants described by Tanaka et al. (1983). Although it a priori seems unlikely that a mutation has “created” a protein related to the “agno gene” product, it is, nevertheless, of obvious interest to determine whether synthesis of a novel protein accompanies viral infection in undifferentiated mouse cells treated with these mutants. Wild-type polyomavirus can also infect mouse EC cells, but no viral functions appear to be expressed. Blangy, Yaniv, and their co-workers (Vasseur et al., 1980; Katinka et al., 1980, 1981) have described a number of polyomavirus mutants that have the capacity to grow in these cells. Relative to wild-type virus, the mutants have alterations which appear complex. The simplest explanation for many of the sequence alterations observed is that they have arisen by a duplication of viral sequence within the origin region (possibly in a manner similar to that which generates defective molecules, Griffin and Fried, 1979, followed by subsequent deletions (H. E. Ruley, personal communication). In this connection, it should be emphasized that many strains of polyomavirus, notably the Toronto and P16 small plaque strains, have duplications in the noncoding region, similar to those found in SV40, whereas the A2, A3, or strain-3 large plaque strains do not. Some, but not all, of the mutants which grow in EC cells have the same A G transition observed in the mutants described above and, in addition, contain sequence changes which would marginally alter the the second-site inverted repeat structure or the distance between the viral origin of replication and this region (Fig. 14). (Some of the mutants described by Luthman et al., 1982, have deletions which lie within this region, but they do not appear to have been examined for growth in undifferentiated cells.) Tanaka et al. (1982) have also described similar mutants. It appears to be clear that the sequence covered in Fig. 14 is of primary importance in allowing the host range change observed with these mutants and that they provide valuable information with respect to replication. In that the region to the left ofthe sequence illustrated in this figure is of considerable importance in transcription (Kamen et al., 1980), the properties of some of the mutants probably also reflect changes in the levels of messenger RNAs.

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E. LATEMUTANTS Relatively little attention has been paid over the past few years to the late region of polyomavirus, and the role(s) of the various capsid proteins in packaging. This neglect is to some extent now being rectified (see Section VIII), but most studies on packaging to date have not yet reached the state where they involve late region mutants. Properties of some of the late mutants have been previously described (Eckhart, 1974).

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A small number of mutants exist that appear to have lesions in the minor capsid proteins, VP2 and VP3. Recently, it has been found that one of the early tsa mutants, ts48, in addition to its “early” lesions (Bendig et al., 1979) has an alteration in the late region that abolishes the normal termination codon for the VP2 and VP3 proteins, allowing an additional 2 1 amino acids to be added to the C-termini of these proteins (Eckhart et al., 198 1). Virion assembly, interestingly, does not seem to be sensitive to this change. Another late mutant, ts59, has been shown to have multiple lesions, one of which renders the VP2/VP3 proteins thermolabile (Gibson et al., 1977). Yet another mutant, ts3, which maps in the region of the genome coding for VP2 (or VP2/VP3) has an effect that can be bypassed when DNA rather than virions are used for infection (Eckhart and Dulbecco, 1974). Blackburn and Hare (198 1) have described an interesting mutant, from strain 3049 of polyomavirus, which appears to overproduce late mRNAs and capsid proteins. The sequence ofthe lesion in this mutant has now been mapped in the region of genome that codes jointly for VP3 and VP2 (Thomas and Hare, 1982). Some of these mutants, when better characterized, should prove useful in defining the roles for the minor capsid proteins. Llopis and Stark (198 1) have carried out studies on SV40 VP2/VP3 mutants which suggest that these proteins have regulatory roles which modulate late transcription. Similar studies do not yet appear to have been carried out with mutants of polyomavirus. Due to the critical role(s) of VPI in infection and encapsidation, it is perhaps surprising that so little effort has gone into finding mutants that allow this protein to be studied in detail. Bendig et al. (1979) have selected mutants that lack a Hind11 site in polyomavirus, which lies near the end of VPl. They describe three mutants which code for proteins smaller than wild-type VP1 and, with all three, find no detectable change in the properties of the mutants relative to wild-type virus. We have shown that changes around the BamHI site (a three base pair insertion which results in loss of the restriction enzyme recognition sequence), and a similar lesion at the Sstl site within the VPl gene, have no detectable effect upon encapsidation (M. D. Jones and B. E. Griffin, unpublished). Preliminary studies with monoclonal antibodies (Dilworth and Griffin, 1982) have suggested that a strong interaction may exist between VPI and large T-antigen, the nature of which is not clear. The whole question of encapsidation, signals for packing, roles of late genes in regulation, and the relationship between early and late proteins would appear to be a fruitful area for research. The need for more, well-characterized, late mutants would appear obvious. VII. Cellular Transformation

The term “transformation” is used to describe a wide range of cellular alterations assayed in v i m in tissue culture cells. These include the ability of

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cells to grow in semisolid medium or in suspension, the loss of contact inhibition of growth and movement, the ability to form tumors in syngeneic animals, the loss of a defined cytoplasmic actin cable network, an increased surface lectin agglutinability, an altered cell morphology, an increase in nutrient uptake, a relative independence of exogenous serum, and the secretion of increased quantities of proteases or protease activators. The term “transformed cell,” therefore, does not describe a unique state, but represents a series of cellular changes. It has previously been suggested that cellular transformation may be a two-step process (Vogt and Dulbecco, 1963), the steps being divided roughly into growth alteration (reflected by an ability to grow in low serum) and change in the membrane properties of the cell. We have attempted to rationalize these events in terms of the known viral gene products (Griffin ef al., 1980b). Each of these events has a finite possibility of occumng spontaneously within a cell growing in vitro, but the probability of both occumng in the same cell is small; but with low frequency established cell lines do undergo spontaneous transformation. When one step is provided by the virus, the probability of transformation occurring becomes much greater. The involvement of a two- or even multistep process may explain some of the ambiguities discussed in the previous section with regard to the role of polyomavirus in inducing such a state in cells. Moreover, it suggests that some of the ambiguities may be of cellular rather than viral origin. Data presently available from studies with polyomavirus would seem to suggest that it is a completely competent transforming virus, encompassing all the functions necessary to produce the required cellular alterations. On the other hand, SV40 appears to be less competent to induce all aspects of transformation and is more reliant for a part of the process on the cell itself. This postulate is obviously simplistic and may require modifying, but it appears to account for a large amount of data on both viruses. To note only a few examples, the work of Risser and Pollack (1974) and Pollack el al. (1975) showed that it is possible to isolate minimally and totally transformed cell lines both of which express the SV40 large T-antigen. PontCn ( 1971) pointed out that SV40 and polyoma transformed cell lines have distinct morphological differences. Seif (1 980) observed that the dl-23 mutant of polyomavirus which is probably defective in middle T-antigen activity, but functional for large T-antigen (Griffin et al., 1980a), transformed rat 3T3 cells with a time course similar to that observed with SV40. In the case of both SV40 and polyomavirus, the expression of a large T-antigen function(s) might allow for a decreased dependence on serum and induce the first of the transformation steps in cells in culture, although the mechanism by which this is accomplished is not clear. It is interesting in this context to note that with polyomavirus, large T-antigen is found concentrated around the nuclear membrane pores (see Fig. 1I), and could possibly alter the flow of molecules entering or leaving the nucleus. In neither case does it appear,

MEMBRANE

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POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

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FIG.15B. FIG. 15. Two cellular processes that might be altered by the action of polyomavirus middle T-antigen and lead to expression of the phenotype associated with cellular transformation. (A) A postulated role for the membrane protein vinculin (see Hynes, 1982), actingasa protein link between the actin cable network of the cytoskeleton, and the intrinsic membrane proteins, particularly those involved in cellular adhesion to the substratum (through adhesion plaques) (see Pastan and Willingham, 1981). Middle T-antigen could break this interaction by phosphorylating vinculin in a manner suggested for the p p 6 W gene product (Sefton ef a/., 1982). (B) A postulated, and simplified, arrangement of the cellular synthesis, processing, selection, and recycling of membrane components in eukaryotic cells (Tartakoff, 1980). These events control the functioning of all cellular membranes by ensuring that the correct components are present in the membranes for performing their respective functions. Precise details of how these events occur have not been determined. In such a cycle, however, disruption at any component would be expected to influence not only that site but many others, so no defined point of action can even be suggested. In this model, plasma membrane function could be severely altered by events occumng well away from the membrane itself. Although very preliminary, data presented elsewhere (Fig. 10) would suggest that this process might be of relevance in cellular transformation by polyomavirus. GERL, Golgi endoplasmic reticulum lysosome.

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BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

however, that the expression of large T-antigen alone is adequate to induce a fully transformed state in cells. Middle T-antigen, for which there is no counterpart in SV40, may invoke the next step in the transformation process by an alteration of membranes and, under certain conditions, may provide the only viral function necessary to induce cellular transformation. The wide range of membrane changes that accompanies the transformation process, and their possible induction by a single protein would suggest a large-scale disturbance in the normal balance of cellular control. It would seem extremely unlikely that middle T-antigen can cause all of the cellular changes directly; it is more realistic to assume that it can influence one (or a small number of) vital control reaction, which then leads, possibly by a series of events, to the expression of the rest of the properties of transformed cells. If this proves to be the case, it will undoubtedly be difficult to establish which is the initial event in the expression of the transformed phenotype, and which are the effects that follow on from it. The concept of middle T-antigen as a protein phosphorylating enzyme is obviously attractive because of the wide range of biological consequences this could invoke. A. MODELSFOR TRANSFORMATION

One of the prevalent theories of cellular transformation proposes as an important event the loss of the cellular actin cable network which is thought to be involved in the maintenance of the morphological and surface adherence properties of normal cells. This network may be attached to integral membrane proteins through an intermediary “protein bridge.” The protein, vinculin, has been suggested as providing this bridge (for review, see Hynes, 1982) (see Fig. 15A). The subcellular location of a number of transforming gene products, notably the well-characterized Rous sarcoma virus (RSV) protein, pp60 src, on the internal face of the plasma membrane, and the detection of a protein kinase activity expressed by this protein (for review, see Collett and Erikson, 1978; Sefton et al., 1982) have resulted in the suggestion that phosphorylation of vinculin might be a primary event in transformation. Evidence for this suggestion comes from the fact that among the cytoskeletal proteins in RSV transformed cells, increased levels of phosphotyrosine could be detected only in vinculin and, to a lesser extent, filamin and vimentin (Sefton ez ul., 1980).That the disruption ofthe bridge between membrane proteins and the actin cable network could cause dramatic changes in cell morphology, and possibly disrupt the transmembrane signals that control the cell’s response to its environment, is an attractive model but there is no evidence for this being the primary cause of cellular transformation, even for RSV. In fact, the subcellular location of pol yomavirus middle T-antigen and the pp60 src of transformation competent mutants of RSV (Garber et af., 1982) would seem to question the

POLYOMAVIRUS A N D CELLULAR TRANSFORMATION

24 1

generalization of the above model. In the case of both viruses, although some of the protein is found associated with the plasma membrane, most of it is associated with internal cellular membranes (see Fig. 1 1B). It cannot be discounted that the minority of plasma membrane associated protein is responsible for transformation, but it seems more than likely that the bulk of the protein also plays some important role. Therefore, an alternative model for transformation needs to be considered. The surfaces of transformed cells have been shown to have an increased lectin agglutinabilitycompared with normal cells (for review, see Nicholson, 1974), although the cause of this is not clear. The increased agglutinabilityof polyomavirus transformed cells, unlike cell morphology changes, appears to be inhibited by inhibitors of cellular DNA synthesis (Schlegel and Benjamin, 1978). In other cases, it has been argued that the enhanced agglutinability is preceded by a change in cytoskeletal components, and it is perhaps noteworthy that increased lectin agglutinability can be created even in normal cells by mild proteolytic treatment (Burger, 1973). Thus, alteration of a membrane component could explain this as well as other changes (Nicholson, 1974). Cellular membranes are fluid, active entities (Moore et al., 1979; Holzman, 1981; Geisow, 1982), and, although the overall flow processes are little understood, they must obviously be well controlled and coordinated since they determine the functions performed by individual membranes and interactions between membranes. It can therefore be envisaged that any alteration in the correct functioning of any part of this membrane flow is likely to have a wide range of effects on the properties of the cell. The control of these processes, as well as those previously discussed, could well be mediated by the action of protein kinases, as are many other processes within the cell (for review, see Cohen, 1982; Flockhart and Corbin, 1982), and could be a site of action for transforming gene products (see Fig. 15B). This model, like the previous one, has the advantage that the action of a single protein can account for a wide range of cellular alterations. It has the practical disadvantage that a single point of action cannot be specified. There are no strong data available to support any particular detailed model for virally induced transformation. An accompanying problem is that the normal functioning of the processes involved is far from being defined, at least at the molecular level. Ironically, the disruption encountered in transformed cells may well lead to a fuller understanding of the normal functioning of cell components. It cannot be discounted that cellular transformation may be induced by a number of alternative routes, and both of the above models (or others), in concert or individually, could be involved in the relevant changes. The tight control encountered in any cellular reaction suggeststhat any disruption in a control pathway, although initiated at different sites, could ultimately lead to a similar result (Pastan

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BEVERLY E. GRIFFIN A N D STEPHEN M. DILWORTH

Membrane Binding

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FIG. 16. Comparison of the transforming proteins of Rous sarcoma virus (RSV) and polyomavirus. Both proteins have associated protein kinase activities and both have been isolated from the membranes of cells that have been infected with virus. It has been shown that changes in the amino-terminal of pp60'" leads to decreased membrane association and tumorigenicity (Kreuger et al., I982), whereas changes in the carboxy-terminal sequence of middle T-antigen have similar effects (Novak and Griffin, I98 I b; Carmichael e/ a/., 1982). These data suggest that membrane association may play a critical role in both cases in promoting in vivo tumorigenicity. Another property that correlates with tumorigenicity in each case is the protein kinase activity associated with these proteins, as measured in an in vilro assay (Collet and Erikson, 1978; see text). This activity is postulated to phosphorylate the tyrosine residue of a variety of protein substrates, as well as result in autophosphorylation in vivo. pp60'" has been shown to be phosphorylated at the tyrosine residue in a sequence, Leu-lle-Glu-Asp-Asn-Gln-Tyr (Smart el a/., I98 I ; Patschinsky et a/., 1982; Pike el a/., 1982), whereas middle T-antigenisthought to be phosphorylated at a tyrosine residue in a similar highly polar sequence, Glu-Glu-Glu-Glu-Glu-Glu-Tyr (Schamausen, 198 I ; Smolar and Griffin, 198 I ) (see Fig. 5). These similarities are strikingenough to allow the speculation that the two viruses might follow comparable routes for inducing cellular transformation. The cautionary note in such speculation is the observation that whereas the overall levels of phosphotyrosine in cellular proteins are increased following infection with RSV, the same appears not to be true in the case of polyomavirus (SeRon ef a/., 1980).

and Willingham, 1981; Rothman, 1981; Tartakoff, 1982). Throughout the future work, the separation of cause and effect is going to be a major problem. It would be optimistic to expect all modes of transformation to have a common cause or proceed by a common route. However, the apparent similarity between polyomavirus and retrovirus "transforming genes" (see Fig. 16) suggests that the mechanisms may be limited in number, and similar in nature. We will review briefly the evidence that points to middle T-antigen as the protein primarily responsible for transformation by polyomavirus, and some of the data that suggest subsidiary roles, probably related to replication and/or cellular growth, by one or both of the other viral antigens.

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B. INTEGRATION OF VIRALGENES INTO HOSTCHROMOSOMES It appears to be uniformly accepted that cellular transformation by polyomavirus involvesintegration of the viral genome (in part, in total, or in tandem copies) into the host chromosome with concomitant expression of some of the viral genes. To our knowledge, there has been no report of a transformation event in which viral DNA was not detected in the chromosomal DNA of the resulting cell. Much of the literature over the past 5 years on transformation by polyomavirus has therefore involved study in some detail of the arrangement of this DNA and its expression in transformed cells. Unfortunately, these studies are fraught with contradictions which have not yet been satisfactorily resolved. For example, if one considers one of the current hypothesesthat viral integration into host cells can give rise to chromosomesin which host genes fall under the control of viral promoters, and thus become subject to abnormal expression,then the prediction would follow that integration should show some specificity. The evidence against any specificity (either viral or host) in SV40-transformedcells is well known (Botchan et af.,1976). Similar evidence against specificityhas also been put forward for polyomavirus (Basilicoet al., 1980;Kamen et al., 1980;Lania et af., 1980b; Della Valle et af., 1981b; Hayday et af., 1982b; Ruley et af., 1982). Some preliminary evidence in favor of specificity appears, however, in two recent publications,.where, although only essentially one transformant was investigatedin each case, the viral integration site was very similar in the two instances and was located in the distal part of the late region (Chartrand et al., 1981; Mendelsohn et af., 1982). This apparent discrepancy, if real, may have a simple explanation. Hiscott et af.(1 98 1) showed for SV40 that viral -cellular joins found in newly transformed cells differ from those found in cells after passage for a number of generations in culture. Basilico and co-wokers have published results from a number of varied types of studies which point to the instability of integrated DNA (Prasad et af., 1976; Zouzias et al., 1977; Basilico et af., 1979, 1980; Colantuoni et af., 1980). We would not be surprised if further investigations showed that within any particular host and cell type, there will prove to be preferred sites of integration, determined by DNA sequences, but for expression of viral genes, absolute specificity will not be required, as long as the event allows adequate amounts of certain gene products to be produced and the cell to be protected from large quantities of other gene products. We discuss the identity of these gene products below. C. FREEVIRALDNA Many transformed cells, in addition to possessing integrated viral genornes, have also been found to contain extrachromosomal forms of viral

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BEVERLY E. GRIFFIN AND STEPHEN M. DILWORTH

DNA. Questions about the origin of these free copies of viral DNA in transformed cells, and whether, by allowing integration into host chromosomes to occur subsequent to transformation, they can account for the apparent lack of sequence specificity in this process, or whether they survive when cells are injected into animals to produce tumors, remain largely unresolved. Most of the evidence suggests that free viral genomes do not represent autonomously replicating, that is, plasmid, forms of the virus, such as those found in cells transformed by papilloma (Law et al., 1981; Moar et al., 1981) or Epstein-Barr viruses (Lindahl et al., 1976). Rather, they result directly from excision of polyomavirus DNA from integrated forms of the viral genome. Many transformed cells have been shown to contain head-to-tail tandem repeats of viral DNA within the cellular DNA (Birg et al., 1979; Basilico et al., 1979; Colantuoni et al., 1980; Lania et al., 1980b;Novak et al., 1980;Chartrand et al., 1981; Mendelsohn et al., 1982). Whether tandem copies of the genome originated prior to, or following, the integration event (or both) is still largely a matter of speculation. Colantuoni et al. (1980) demonstrated that amplified viral sequences can arise subsequent to integration in the presence of a competent large T-antigen, but the data do not exclude the possibility that they also could arise before integration from DNA derived, for example, from a rolling circle mode of replication. However, in most Cyp cells (see Section VI,A), the integrated viral sequences consist of tandem copies of complete and incomplete genomes (Chartrand et al., 1981). This is not readily explained by a simple integration of a rolling circle intermediate. Whatever the pathway may be for the generation of integrated tandem copies of the viral genome, it seems fairly clear that they are one source, at least, of the “free” DNA. In this regard, a number of individual clones of rat cells transformed by two closely related rnlt deletion mutants, dl-22 and dl-27 (Ding et al., 1982b; and unpublished data) were investigated at early passage and in no case were free copies of viral DNA observed; both of these mutants, although competent for growth, produce truncated large T-antigens with an internal deletion (see Fig. 5 ) . Since “tandem integration” is the general rule, these cell lines appear to be an exception, and strengthen the argument for some function in large T-antigen being required for generating integrated species that then act as precursors of nonintegrated DNA. The mode by which free viral DNA is released from integrated forms seems even more problematical. For example, it would appear from many studies that excision may involve replication and require a functional large T-antigen (Basilico el al., 1979; Gattoni el al., 1980; Sylla et al., 1980). The absolute requirement of the latter is questioned, however, in a recent study that shows the presence of free polyomavirus DNA in transformed cells which, although containing the viral origin of replication, cannot code for a full-sized large T-antigen. In order to observe the excision event, transformed rat cells were fused with mouse cells and excision is argued then to

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have arisen as a result of homologous recombination, presumably depending on a mouse-encoded recombination activity (Lania et al., 1982).If this is the case, it is not clear why mouse cells that have been transformed with polyomavirus, and contain tandem copies of the viral genome, fail to produce free copies of the DNA. In this regard, the Cyp cells isolated by Bourgaux and co-workers ( 1978) (transformed at the nonpermissive temperature (39°C) with a tsa mutant, tsP155, of polyomavirus) contain tandemly integrated copies of viral DNA, but appear to release detectable free copies only when shifted to 32"C, the temperature at which the large T-antigen would be functional (Bourgaux et al., 1978; Sylla et al., 1980). Moreover, superinfection of Cyp cells with wild-type polyomavirus does not result in the accumulation of free copies of the resident genome (Delbecchi et al., 1981). Thus, neither replication nor recombination per se seems to explain adequately the presence of free copies of the viral genome. From results with a different type ofexperiment, it has been suggested that in vivo selection occurs against cells that contain free copies of the viral genome (Lania et al., 1981). That is, in experiments in which transformed rat cells, containing free viral DNA, were used to produce tumors in syngeneic animals, cell lines established from these tumors were found to have no free DNA (Lania et al., 1981). Other experiments, also using rats, have shown that the same animals are competent to cope with free copies of the viral genome (Chowdhury et a!., 1982). Since both of these sets of experiments were carried out in a similar manner, it is at present difficult to understand why in one case a change between cells propagated in vitro and in vivo appears to occur, whereas no change was observed in the other case. There seems little doubt that the viral large T-antigen is involved in some, or all, of the processes discussed above, but to what extent this involvement reflects a role in replication, or a recombination function, is not clear. Moreover, the extent to which a cellular function can compensate for a defect in this protein remains obscure. Since transformation is accompanied by specific chromosomal translocations in the case of at least one other DNA tumor virus, notably the Epstein - Barr virus, the data discussed above suggest that some of the earlier experiments in this general area, described by Eddy ( 1969), should be reinvestigated using the better defined cell lines now available and more modern technology. IN TRANSFORMATION D. ROLESOF T-ANTIGENS

Another controversial issue concerns the relative roles of the different viral T-antigens in transformation. Except for one conflicting report, there now exists an overwhelming body of evidence in support of an important positive role for the viral middle T-antigen in cellular transformation. This evidence has been discussed previously and, by and large, shows that almost any perturbation of this protein results in some concomitant effect upon

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transformation. Some minor apparent exceptions to this statement exist in data derived from studies on the mlt mutants, but the major exception comes from experiments described by Trejo-Avila et al. ( 1981). Their data suggest that type-N rat cells that lack the phenotype associated with transformation are phenotypically stable even though they express middle T-antigen. These observations are discussed subsequently. Assuming that in the large majority of cells transformed by polyomavirus, the role of middle T-antigen, whatever it may be, is crucial to the event, the question remains whether any other viral protein is involved. Since the viral capsid proteins are apparently not expressed in transformed cells (Fenton and Basilico, 198I), this leaves the viral small and large T-antigens to be considered. There are no small T-antigen-specific mutants and the only data to be had on this protein come indirectly from studies of mlt mutant viruses. It has been shown in some cases that in rat cells, at least, the expression of small T-antigen is not sufficientto produce a fully transformed cellular phenotype (Griffin et al., 1980a).Moreover, these studies show that small T-antigen is not responsible for several of the phenotypic markers associated with transformation, such as disruption of cytoskeletal actincables and increased levels of plasminogen activator. The small size (22K) and unusual structure (extremely rich in cysteine)of this protein make it attractive to consider a hormone-like role for it (Friedmann et al., 1978). In an attempt to include small T-antigen in events contingent upon the expression of viral genes, we have previously proposed a two-step progression model for transformation, with small T-antigen possibly playing a mitogenic role (Griffin et al., 1980b). However, there is at present no direct evidence that it is involved at all in transformation. Horwich et al. (1980) have recently made proteins related to polyomavirus small T-antigen in Escherichia coli. Hopefully, one of these may ultimately prove useful in the search for a function of this small viral antigen. The protein which evokes considerable contention vis-A-vis transformation is the viral large T-antigen. Our personal bias (Novak and Griffin, 1981b) is that the large T-antigen plays no direct role in transformation. Moreover, properties of the hrt mutants support this notion, since these nontransforming mutants can induce a full-sized large T-antigen (see Section VI); rat cells infected with an hrt mutant have been found to contain both free and integrated copies of the viral DNA, supportingthe notion that the large T-antigen is functional in these cells (Lania et al., 1979). Using wild-type viral DNA, Israel et al. (1 979) showed that not only was DNA that had been cleaved in such a way that full-sized large T-antigen could not be expressed able to induce tumors in animals, but the frequency of tumor induction was considerably higher than when genomic DNA was used. They further demonstrated (Israel et al., 1980a)that this enhancement effect was not due to any change in the levels of TSTA, thus dissociatingthis

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latter function from the large T-antigen. These data, which suggest that the presence of large T-antigen might even have an inhibitory effect upon transformation, were confirmed and extended in a series of publications which proved that fragments of DNA capable of coding for less than half of the large T-antigen could induce cellular transformation in culture, or tumors in animals, at least as efficiently as genomic DNA (Chowdhury et al., 1980; Novak et al., 1980; Moore et al., 1980; Hassell et al., 1980; Bastin et al., 1980).Note that in all the transformation studies discussed above, small T-antigen may be expressed. Novak and Griffin ( 198la,b) produced evidence to show that the minimum DNA sequence essential for efficient transformation was that which allowed expression of the full-sized “normal” middle T-antigen. The latter experiments were carried out using viral DNA in which variable amounts of sequence coding for the region around the C-terminus of middle T-antigen had been removed, the resulting DNA cloned in plasmids, and subsequently used to transfect rat cells (see Fig. 5). One puzzling piece of evidence arose from these studies that still has not been resolved. That is, DNA (p42, Fig. 5 ) that could code for all of middle T-antigen, plus eight additional plasmid-detransformed only rived amino acids (Gly-Thr-Pro-Ile-Cys-Leu-Phe-Phe), 490as efficientlyas other clones which allowed a normal protein termination codon to be read. These data suggest an inhibitory effect by the plasmid polypeptide on the function(s)of middle T-antigen, or decreased stability in vivo of the fusion protein. An alternative intriguing possibility is that a functional form of the viral protein may be posttranslationally modified at its C-terminus (see Section V,F,2). Using a similar approach, Katinka and Yaniv (1982) have studied the effect of alterations in the DNA encoding the N-terminus of middle (and, of necessity, small and large) T-antigen(s) on transformation. Their results essentially parallel those of Novak and Griffin (1 98 1a,b). That is, the removal of any of the sequence coding for middle T-antigen resulted in a marked decrease in the efficiency of transformation. It is worth noting that there were some anomalies in this interesting study. Further evidence against a direct role for large T-antigen in transformation lies in several extensive analyses of a number of virally transformed cells, which show that viral sequence coding for the C-terminal portion of large T-antigen is frequently absent (It0 and Spurr, 1980;Israel et al., 1980b; Lania et al., 1980a). Analysis of the viral proteins demonstrated that although some of these cells contained polypeptides considerably smaller than, but related to, large T-antigen, similar proteins were not found in all cells (It0 and Spurr, 1980). These polypeptides were identified as being derived from the N-terminus of large T-antigen. Moreover, continuous passage of cells in culture appeared to result in a loss of T-antigen, or some part of it, from the cells. These data are reminiscent of one of the in vivo studies, mentioned above, where T-antigen-positive cells appeared to lose

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the ability to express this antigen on being reisolated from animal tumors. Ito and Spurr (1980) detected a 39K protein in transformed cells, whose tryptic fingerprint suggested that it might even have the amino terminal portion of large T-antigen and the carboxy terminal of middle T-antigen (see Fig. 1). This arrangement would generate a defective “middle T-antigen” deprived of the cluster of cysteine residues normally found in this protein (see Fig. 5). One final, apparently conclusive, piece of evidence against a primary role for large T-antigen in transformation of certain types of cells, at least, comes from studies with a recombinant piece of DNA in which, by correctly “splicing” the viral DNA, only the expression of middle T-antigen is allowed (Treisman et al., 1981). This DNA transforms rat cells and produces tumors in syngeneic animals, although with an efficiency only about half that of a recombinant clone that contains coding sequences for all three of the viral T-antigens. The above data can be put on the “negative” side of the scale, that is, suggesting little direct involvement of the viral large T-antigen in transformation. They do not preclude the possibility that it is important in the mediation of virion DNA integration (establishment) or in regulating the synthesis of middle (or small) T-antigen (maintenance), at least in fibroblast cells. Moreover, on the “positive” side can be placed many of the data derived from studies with a number of the temperature-sensitive, tsa, mutants (reviewed by Eckhart, 1974; see also Lania et al., 1980b). These results support the concept that large T-antigen is important in initiating the transformed state of cells, although it is not required for its maintenance, a concept widely quoted. Less attention has been paid to those tsa mutants, notably ts52, tsPl55, and possibly ts609, which appear to transform at both the permissive and nonpermissive temperatures (Eckhart, 1975; Bourgaux et af.,1978).Studies with the latter still emphasize the role of large T-antigen in replication, but provide questions about its role iq initiating transformation. (At this juncture we will postpone further consideration of this discrepancy since it weakens the case for large T-antigen.) Further evidence in favor of a role(s) for large T-antigen in transformation comes from studies by Cuzin and his collaborators. In their type-A rat-1 cells, the data are consistent with studies on other tsa mutants which suggest a role in the initiation of transformation; data derived from studies of their type-N cells, however, imply a need for the large T-antigen not only in the initiation, but also in the maintenance, of transformation (Seif and Cuzin, 1977; Perbal and Rassoulzadegan, 1980; Perbal, 1980; reviewed, Rassoulzadegan et al., 1980) and thus appear to be in conflict with other data derived from studies on tsa mutants. That is, N-type cells, originally transformed with a tsa mutant, appear to revert to a nontransformed phenotype when shifted to the nonpermissive temperature for the large T-antigen, implying a maintenance

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function for this protein. In an attempt to reconcile these data with the large body of accumulated evidence against a role for large T-antigen in the maintenance of transformation, or possibly even in its initiation, such as those discussed above, Rassoulzadegan et al., ( 1 98 1) have demonstrated that recombinant DNA containing a viral sequence which codes only for the N-terminal region of large T-antigen can complement N-type cells for the maintenance of transformation at 39°C. Thus, they argue the need for two functions in transformation, one camed by the middle T-antigen and another by the N-terminal portion of large T-antigen. Since the interpretation of these data gives results different from all other studies with tsa mutants, we assume that type-N rat cells represent unique types of cellular mutants. These cells may hold important clues about the nature of the viral - host interactions, it is regrettable that they have not been better characterized or studied in other laboratories, or that similar type-N cells have not been isolated using different tsa mutants. Before discussing all these discrepancies,it is perhaps worth recalling that “growth” control is very complex and involves the interaction at different levels of many cellular functions. Therefore, the presence and expression of viral genetic information is doubtless only one of many factors contributing to the phenotype of the “transformed cell” (Eckhart, 1975). Most of the studies on processes leading to transformation, and resultant transformed cells, have been carried out on what can be considered heterobgous cells (rat and hamster) for polyomavirus, and only a few studies on homologous mouse cells (for examples of the latter, see Bourgaux et al., 1978, 1982; Ito and Spurr, 1980; Chartrand et al., 1981, and references therein). Although the reason for this is obvious (see Section VIII), this means that comparisons between results involving different host cells must be treated cautiously. With this in mind, it would appear that some of the apparent discrepancies might be resolved if, as appears likely, host cell DNA synthesis is an important contributing factor to transformation. Many of the tsa mutants, while failing to synthesize viral DNA at the nonpermissive temperature, nontheless initiate synthesis of mouse cellular DNA at this temperature (Eckhart, 1975). It is not clear that the same degree of initiation of cellular DNA synthesis occurs with rat and hamster cells. Different cells might therefore exhibit varying sensitivities to tsa mutants in a way that is only indirectly related to viral gene expression. Another factor that may contribute to these discrepancies concerns the level of viral gene expression. Cogen (1 978) has shown that the accumulation of polyomavirus-specific early mRNA is under the control of large T-antigen (see also Section V,D). Cells treated with tsa mutants at the nonpermissivetemperature might be expected to be exposed to less ofall the viral T-antigens than cells treated at the permissive temperature, since large

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T-antigen controls not only viral replication but appears to affect the expression of the other antigens as well. Because transformation is usually studied for a finite time, it is possible that the doses of functional middle T-antigen in tsa infected cells rarely reach those required for transformation. There are no experiments to our knowledge that preclude this argument as an explanation for the failure of some tsa mutants to initiate transformation at the nonpermissive temperature, whereas others transform in a way that appears to be independent of the lesion in large T-antigen. In the latter case it seems unlikely that the explanation lies in mutant “leakiness,” at least in studies with ts52 (Eckhart, 1975) and tsP155 (Bourgaux et al., 1978). It would be interesting to compare the levels of the T-antigens induced by these mutants with those induced by other tsa mutants, notably the frequently studied tsa-a and ts25. The prediction is that they might prove to be higher, particularly in the tsP155 case, since this mutant was derived from a small plaque strain (Toronto) of polyomavirus which was shown in early experiments by Stoker (1 960) to be unusually pathogenic in hamsters; with this virus, 4 days after treatment, abnormal cells were already observed in the kidneys of infected animals. The results of two other, different, kinds of studies may also be related to the level of viral gene expression. In one, rat cells were transfected with DNA derived from tsa mutants of polyomavirus. Contrary to general expectations, it was found that cells could be transformed at either the permissive or nonpermissive temperature; both rate and quantitative differences in transformation were observed, however (Novak and Griffin, 1980b; Della Valle et al., 198la). Our experiments (Novak and Griffin, 1980b) were carried out with both tsa-a and ts25 to prove that the observations were not mutant dependent. In another approach, Basilico and co-workers showed that in the presence of an active large T-antigen, loss of some of the integrated viral DNA can occur at a fairly high rate, with some cells even reverting to a normal phenotype (Basilico et al., 1979; Gattoni et al., 1980). “Cured” revertants appear to make mRNAs containing viral information that differs from that in normal early mRNAs, in that they use either a second-site polyadenylation signal present in the polyomavirus genome at 99 map units (see Soeda et al., 1980b)or a signal within the host. They encode information for the expression of middle and small, but not large, T-antigens. It is argued that these cured cells fail to express a transformed phenotype because the viral antigen levels are too low (Fenton and Basilico, 1981). Reinfection with virus particles results in retransformation of these cells. There remain to be resolved the numerous data on studies of N-type transformed cells (see above). The simplest explanation is that these are very special cell mutants, either selected from among a normal cell population or generated by the action of large T-antigen (Seif and Cuzin, 1977). Several studies from other laboratories contain data possibly relevant to this subject.

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For example, there is much evidence to suggest that viral integration is unstable in the presence of a functional large T-antigen (Dailey et al., 1982). Moreover, Bourgaux et al. ( 1982)showed that at the temperature permissive for expression of a thermolabile large T-antigen, DNA consisting of an entire viral genome with about lo6daltons of mouse DNA covalently linked to it, is excised from Cyp mouse cells. Such an excision event obviously places the viral information remaining in these cells in a different environment from that present before the excision occurred. The resultant cells in this case, designated R cells, unlike the N-tyRe cells, remain transformed (Herring et al., 1980). In another recent study, Neer et al. (1983) have mapped flanking viral-host sequences in a transformed rat cell line, and compared them with the corresponding sequence in normal rat fibroblasts. Their data suggest that viral integration has resulted in the deletion of sequence about 3 kb in size from the host chromosome. From these data on the instability of host and viral sequences in the presence of a functional large T-antigen it would be surprising if on occasion the virus did not effect cellular mutations. This would certainly allow the behavior of N-type cells to be explained. Moreover, in such a fluid environment, viral gene expression probably differs markedly from one transformed cell line to another. The fact that T-antigen-negativecells appear to have a selectiveadvantage in populations of polyomavirus transformed cells (It0 and Spurr, 1980; Israel et al., 1980b; Lania et al., 1980a; Dailey et al., 1982)would seem to reflect a strong drive toward chromosomal stability. Recent investigations implicate the large T-antigen in recombinational events that result in the overall chromosomal instability observed in transformed cells (Colantuoni et al., 1982; P. Bourgaux, personal communication). These studies, and the implications allowed by numerous apparently conflictingdata considered above, suggest that in addition to stimulating both viral and host cell DNA replication, large T-antigen may have a function related to recombination (see Fig. 14). Most of this discussion has centered around the role of the viral large T-antigen in transformation. This is a consequence of the fact that most transformation studies to date have been carried out on cells transformed with tsa mutants. Extrapolating from data derived on studies with an mlt mutant, dl-8, which in our hands transforms and produces tumors more efficiently than wild-type virus, we have argued that replication and transformation may actually act as “antagonistic” events (Griffin et al., 1980a; Smolar and Griffin, 1981). There is as yet no direct evidence for this with polyomavirus, but there is evidence with SV40 (Lomax et af., 1978) and with adenovirus (Ginsberg et al., 1975). It is interesting to note a recent paper on SV40 (Small et al., 1982) which shows that origin defective mutants of this virus have greater transforming potential than the corresponding wild-type virus. Earlier, Lomax et al. (1978) showed that the

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efficiencyof transformation of (semipermissive) human cells by SV40 was greater (at both the permissive and nonpermissive temperatures) for growth-deficient mutants than for wild-type virus. They proposed that a reduction in the potential of the viral genome for cell killing could reflect additional opportunities for the expression of transformation. Thus, from the previous discussion, it can be deduced that a failure to replicate would not only prevent cell lysis, but increase the stability of the transformed phenotype. When all the available data are considered, they appear to us to suggest that middle T-antigen plays a direct role in cellular transformation, probably by altering events taking place at cell membranes. The role of large T-antigen, on the other hand, is more indirect, though it appears to correlate with accessory functions such as cellular DNA replication, or DNA recombination and expression of the viral genes, the latter two functions being undoubtedly important in transformation. In the face of some of the conflictingdata on the role of large T-antigen in transformation and the fact that DNA, which can express only middle T-antigen, transforms cells in culture less efficientlythan wild-type viral DNA and appears to be incapable of transforming primary rat embryo cells (Rassoulzadegan et al., 1982), questions about a “helper” for middle T-antigen, be it another T-antigen, a modification of the protein itself, or a host cell function, must remain paramount among those that need solving in order to understand transformation by polyomavirus. Preliminary success on the purification of the T-antigens, and the creation of genetic species with the ability to express a single T-antigen in cells, provides hope that a fuller understanding of cellular transformation induced by this virus is not too far away. VIII. The Lytic Cycle

Much more effort over the past few years has gone into defining the viral genome and its expression with regard to transformation than at looking into the events that follow on from cell penetration and viral expression in the permissive cell. Yet the two events appear to be so closely related that it is difficult to see how either can be understood except by studies in parallel. This statement perhaps explains why we have come to favor experiments carried out in the natural host (mouse) of polyomavirus, as being more relevant to the ultimate understanding of the unique relationship that has been established between the virus and its host and the processes that are altered in the destruction of this relationship, than corresponding studies in other (heterologous) hosts. It also argues the need for cell mutants and improved in vitro systems for investigating individual viral - cellular interactions. Characteristic features of the DNA sequence of the “late region” have recently been discussed (Arrand et a/., 1980; Soeda et al., 1980c; Srivatsan et al., 1981).

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The structure of the polyomavirus particle and the events leading to lytic expression have been carefully and thoroughly reviewed by Consigli and Center (1978); Acheson (198 1); Challberg and Kelly (1982); and DePamphilis and Wassarman (1 982). As these authors stress, very little is known about the early events of attachment, penetration, and uncoating of the virus, in either permissive or nonpermissive host cells, and even less is known about events leading to the assembly of polyomavirus. If one takes this further, although it is well documented that host cell replication is stimulated by the virus, very little is known about the cellular changes that appear as a consequence. The process of polyomavirus adsorption to mouse cells (3T6) has recently been reinvestigated and receptors containing the sialo-oligosaccharide sequence, N-acetylneuraminicacid (NeuAc) a(2,3) Galpl, 3GalNAc, identified as being competent to allow for both viral infection and virus-mediated hemagglutination (Cahan and Paulson, 1980; Fried et al., 1981). Bolen and Consigli (1979, 1980) have shown that these two activities are nonetheless separable and have correlated them with different regions of the major capsid protein, one (infection)being associated with a 14K polypeptide and the other (hemagglutination)with a 16K polypeptide, generated from VPl upon treatment with EGTA and DTT. As mentioned earlier, Bolen et al. ( 1981) have shown by isoelectric focusing that “intact’ VP 1 is in itself a “family of proteins.” It therefore becomes a very interesting problem to define the relationship between the 14K and 16K polypeptides and the parental species. Of further interest in this context is the fact that SV40 virus lacks the ability to agglutinate guinea pig erythrocytes; it therefore seems reasonable to predict that the latter function might reside in a part of the VPl protein where there is little sequence homology between the two viruses (see Fig. 3). Once penetration has occurred, little information exists on the uncoating of virions. The early data of Frost and Bourgaux (1975)suggested that some initial uncoating could occur in the cytoplasm, but subsequent studies (MacKay and Consigli, 1976; Chlumecka et al., 1979) indicate that it takes place exclusively in the nuclei of the cell. The electron micrographs of MacKay and Consigli (1976) are particularly enlightening in that they allow the virus particle to be visualized during its passage into and through the cell, although they are not very informative with respect to entry into the nucleus. Also, little or nothing is definitively known about the biochemical events that initiate the uncoating process, although it has been speculated that removal of calcium ions and reduction of disulfide bonds may be involved (Brady el al., 1977, 1978). In an interesting paper, Winston et al. (1980) describe a procedure which allows them to isolate and analyze “uncoating intermediates” of polyomavirus from the nuclei of infected primary mouse kidney cells. From this study, they propose a model which involves removal of viral capsid proteins in the nucleus to generate a 48 S

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“uncoating intermediate,” which then becomes attached to the nuclear membrane structure in a manner that allows the viral genome to interact with the host enzymes it requires for replication and transcription. When nuclei were sonicated, a 190 S “uncoating intermediate” was isolated from which one protein analogous to the cellular HI histone and other proteins, postulated to be involved in expression of the viral genome, were obtained. Presumably the 48 S uncoating intermediate is related to the minichromosomes of polyomavirus described elsewhere (Herbomel et al., 198 1). Like their SV40 counterparts (Saragosti ef al., 1980), polyomavirus minichromosomes have been shown to have two DNase I hypersensitive regions, over an area of about 260 base pairs, near the viral origin of replication (see Fig. 4); it has been suggested that hypersensitivity reflects the absence of nucleosomes in this region (Herbomel et al., 1980). Of equal interest is the observation of a DNase I resistant site that lies between the sensitive sites. It was suggested that this site might be in some manner protected against the nuclease action, possibly by the presence of a protein which could be, but probably is not, large T-antigen, since the major binding sites of this protein appear to lie elsewhere (Tjian, 1980; Cowie et al., 1982; M. Ginsburg, personal communication; C. Prives, personal communication; see Fig. 4). In this context, with lytically infected 3T6 mouse cells, a significant amount (up to 25%) of polyomavirus DNA appears to be tightly associated with the nuclear matrix (Buckler-White ef al., 1980). It is therefore possible that a matrix protein can become bound to the DNA, protecting it from degradation by DNase I; it remains to be seen whether there is any specificity in this binding. As an alternative to these suggestions, some protection against DNase I may be afforded by tertiary structures that could exist within the DNA. In Fig. 14, the protected region appears as a cruciform structure, distinct from, but similar to, that designated as the viral origin of replication. Some evidence for unusual structure in this region comes from the observation that a HinfIIBglI subfragment of virus DNA, containing the region shown in Fig. 14, migrates anomalously slowly on polyacrylamide gels, behaving as if it contained about 20 more base pairs than expected from the DNA sequence (M. Ginsburg, unpublished). The unusual electrophoretic behavior of particular fragments of DNA is not unique and has recently been discussed (Ross and Landy, 1982; Lyamichev et al., 1982). Of relevance here is the observation that a 436-bp fragment from the lambda origin of replication, which is not strongly biased in its base composition, has an apparent mobility corresponding to 504 bp on a polyacrylamide gel (Moore ef al., 1979). Indeed, it seems possible that this phenomenon, which reflects an unusual DNA structure not yet clarified, may even be useful for predicting locations of origins of replication, for example, in human papilloma virus DNA whose sequence is known but origin@)of replication unknown (Danos et al., 1982).

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Following the pioneering work of Reichard and his group (reviewed by Acheson, 1981;Challbergand Kelly, 1982), a series of experiments has been initiated by Gourlie et al. (198la,b) and Krauss and Benbow (1 98 1) aimed at identifying the enzyme activities associated with minichromosomes and their roles in the replication of polyomavirus DNA. In their assay, replication of minichromosomes in nuclear extracts could be demonstrated, but no Form I (supercoiled) DNA was produced. Presumably a factor@)required for the ultimate maturation process was lost during the extract preparation, or was possibly not of nuclear origin. Mastromei et al. (1 98 1) have further shown that isolated nucleoprotein complexes are not fully competent since addition of heterologous Drosophila enzymes will stimulate replication. Data from Benbow, Pigiet, and their collaborators (Buckler-White et al., 1980, 1982; Krauss and Benbow, 1981) point to the asynchrony of the replication process (this might explain the origin of species such as D50, the “multiorigin” defective molecule frequently encountered in preparations of polyomavirus DNA, Griffin and Fried, 1975) and to the presence of associated cellular DNA polymerase a and p (but not y), topoisomerase I, and RNase H activity with polyomavirus minichromosomes. Their system, like that of isolated nuclei (Eliasson et al., 1981), may prove useful in understanding the roles of the individual enzymes involved, although it presently appears still to be of greatest value in analyzing elongation rather than initiation or termination of replication. The absence of conditional lethal cell mutants for analyzing DNA replication in mammalian systems means that to date very little headway has been made in pinpointing individual steps in the replication process. Reichard and collaborators suggest this problem may be overcome by the use of monoclonal antibodies to individual enzymes, when these become available (Eliasson et al., 1981). Replication of polyomavirus DNA may be further complicated, because, although it appears that for most of the lytic cycle, replication proceeds via a bidirectional mode from a fixed origin site, earlier thought to be by a discontinuous symmetric process (see Narkhammar-Meuth et al., 198la,b) and now reported to be asynchronous (Buckler-White et al., 1982), there is also evidence for a second, asymmetric “rolling circle” mode of replication (Bjursell et al., 1979). Possibly, a switch from one replication mode to the other occurs half-way through the infectious cycle, as is the case for bacteriophage lambda. It would perhaps be of value to study replication at different times postinfection. The rolling circle mode of replication offers one explanation for the presence of tandemly repeated DNA sequence in most of the transformed cell lines analyzed to date (see above). Further complicationsvis-5-visreplication arise from the fact that it is not yet known to what extent integration of viral DNA into host chromosomes occurs during the lytic cycle (Turler, 1977). The isolation of viable mutants containinginserts of cellular DNA located near the viral origin of replication (Ding et al., 1982a; W. R. Folk, personal communication) supports the

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notion that integration may occur during the lytic cycle. If so, whether this is a significant or an essentially minor event, whether it is common or rare, and to what extent cell cycle asynchrony figures in viral replication are open questions. In this regard, it has been demonstrated that the semipermissiveness of hamster cells to polyomavirus is a consequence of cell heterogeneity (Folk et al., 1981). As emphasized above, most of the in vitro studies on replication have provided little information about the initiation event. In an attempt to study this process, Clertant and Cuzin ( 1980)used nuclei from tsa mutant infected cells and showed in vitro that at the nonpermissive temperature, viral DNA synthesiswas inhibited but could be stimulated by addition of extracts from wild-type virus-infected cells, further emphasizing the dependence of polyomavirus on the large T-antigen for DNA replication. Of all the events involved in DNA replication, we would predict that progress over the past few years will soon allow detailed information on initiation to become available; the recent success in specific initiation of E. coli DNA replication in vitro will act as a stimulus in this regard (Kaguni et af., 1982). The monoclonal antibodies (Dilworth and Griffin, 1982) will allow large T-antigen@) to be purified, replication-defective transformed mouse cells provide the means of assaying for replication (Tyndall et af., 1981; Binetruy et af., 1982) and data from a variety of mutants (discussed above), together with the known DNA sequence, allow predictions to be made about additional mutants that can be constructed to provide insight into initiation. The sequence that appears, from a consideration of all available data on polyomavirus, to representing the limits of the viral origin of replication encompasses about 200 base pairs (see Fig. 14) and has many features in common with the E. cofi K-12 replication origin, extensively studied by Oka et af. (1980). We propose a model to show how replication might be modulated by the structure(s) inherent in the DNA sequence. Thus, the primary sequence allows, on each strand of the DNA, for the formation of two 22-base-long “hairpin loops” (or four-stranded structures, Soeda et af., 1979b), flanked by similar regions, which could possibly compete with each other for a membrane attachment site or an initiation pratein(s). Moreover, the latter might be shifted from one site to the other (or even onto a third site elsewhere in the viral DNA or in the host chromosome) by an alteration in steric location of the relevant structures with respect to each other. We postulate that this fluid relationship might be allowed, as illustrated, by the eight base pair repeat sequence located between the two hairpin loops. It has already been noted (see Section VI) that one of the features that allows a host-range change of polyomavirus is an A G transition within one of the two repeated sequences, as shown in Fig. 14. In the model presented here, this would undoubtedly have an effect on the tertiary structure of this region. Additional point mutants within the

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repeat sequence or at hydrogen-bonded sites within a “loop,” as well as small deletions within the region between the two loops would, we believe, be very instructive in defining features important in replication. [The driving force for generation of such secondary structures for polyomavirus, and concomitant tertiary structures, could come from a protein interaction(s), or, as suggested by Mizuuchi et af.(1982), from the supercoiling of the DNA.] As a step in this direction, Magnusson and Nilsson (1982) have investigated the properties of an evolutionary variant of polyomavirus that appears to have an additional 250 base pairs of sequence relative to wildtype DNA and contains two origins of replication. This variant, designated 1001, replicates about twice as fast as wild-type DNA. Since the lesion also seems to alter transcription of “early” mRNAs, it is, however, not ideal for studying replication alone. It has been known for some time that viral infection results in the stimulation of host cell DNA replication (Franke and Eckhart, 1974). Khandjian et af.(1980) studied this stimulation in more detail and showed that it extended, not unexpectedly,to cellular RNA and protein synthesis. Is this a general or speciJicstimulation?To our knowledge, there appears to be only one recent publication on this very interesting subject. Using methotrexate-resistant 3T6 cells, Kellems et af.(1979) showed that polyomavirus resulted in a four- to fivefold increase in dihydrofolate reductase synthesis, and they were able to show that the enzyme expression was controlled by at least two regulatory pathways. It is regrettable that there are no other studies of this nature, and it seems clear that approachessimilar to this are necessary for elucidating the cellular events that occur during transformation. The work from Schimke’s group is important in that it points to the possibility of the occurrence of specific rather than general, or random, mechanisms of positive control of polyomavirus infection. As indicated earlier, once viral DNA replication is terminated, very little is known about packaging and the events leading to cell lysis. A recent reinvestigation of structure by X-ray diffraction (Rayment et af., 1982) led to the conclusion that the viral capsid is composed of 360 VP1 units comprised of pentameric capsomeres. Consigli and collaborators, by employing a reducing agent and a Ca ion chelator, disassembled virions into capsomeres and a 48 S DNA-protein complex (Brady et af., 1978). They have recently been able to reassemble “virions” that retain some of their hemagglutination and infectivity properties with these components (Yuen and Consigli, 1982). This appears to be an important step forward in the study of polyomavirus assembly. All studies to date point to the importance of VPl in polyomavirus structure, assembly, and infectivity.No role for the minor proteins, VP2 and VP3, which constitute less than 20% of the virion proteins, has been implicated in any of these processes, although it has been suggested that the

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corresponding SV40 proteins might have regulatory roles (Llopis and Stark, 1981). Studies with antitumor antisera and monoclonal antibodies raised against the large T-antigen have indicated that a strong association exists between this protein and VPl (Dilworth and Griffin, 1982). This has subsequently been confirmed with a monoclonal antibody to VPI (A. J. Street, unpublished results). Since large T-antigen is also a DNA binding protein, we question whether it may not also be involved in an early nucleation step leading to formation of minichromosomes and capsomere formation. If so, is the specific binding of large T-antigen to DNA near the origin of replication a reflection of a replication control process, or of a packaging event, or both? IX. Conclusions

From the data discussed, it is obvious that many of the “giant steps” taken in molecular biology in the 1970s have been instrumental in allowing the elucidation of the intricate arrangement of the polyomavirus genome. Much now is known about the structural organization of the virus, and many predictions about gene expression and expected cellular responses to viral products can be made. Unfortunately, the impressive accumulation of interpretable data has not really extended across the cellular barrier. Relatively little more is known about the biochemical changes in the cell that accompany viral infection than was known when the reviews of Eddy ( 1969) and Pontkn (197 1) were written. The major achievement of the molecular biological studies has been to provide overwhelming evidence in support of a virally coded “transforming gene” and thus keep polyomavirus viable as a valuable model system for studying oncogenesis. Considerably more headway will undoubtedly be made once reasonable quantities of viral proteins become available. Many of the reagents needed for this work are already at hand. Thus, the next few years should see many questions about the roles of viral gene products being answered in a direct way, rather than deduced from indirect assays. The problem already looks, however, more complex than suggested by the first figure of this article. Several of the proteins, notably middle and large T-antigens, as well as the viral capsid species VP 1, appear to consist of closely related “families” of proteins, rather than unique and discrete molecules. The possibility must be faced that individual members of a particular family might have different functions. One of the more interesting of the relationships among the viral proteins is that between the middle and small T-antigens. DNA sequence analysis predicts that with the exception of the C-terminal tetrapeptide, all of the amino acids found in small T-antigen are also present in middle T-antigen. Since overlapping reading frames are fairly extensively used in the case of polyomavirus for maximizing coding capacity, it seems unlikely

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that small T-antigen is merely an anachronism, duplicating some of the functions of middle T-antigen, but this possibility must be considered. It might be easier to answer this question were the problem of the potential host origin of the C-terminal part of middle T-antigen resolved. Considering all the current interest in the isolation and study of “oncogenes,” it is somewhat surprising that the possibility of using the polyomavirus genome as a probe for mouse “onc” genes is not being more actively pursued, particularly since mouse 3T3 cells have played such an important role in establishing the existence of such genes. It seems to be accepted that polyomavirus could be a potentially useful vehicle for cloning genes and amplifying their expression in mammalian cells (Fried and Ruley, 1982; Hassell et al., 1982). It is less obvious that the present “wild-type” polyomaviruses are the best vectors. In fact, preliminary evidence would suggest that they are inferior to SV40 in this regard. However, our knowledge about the functional domains of the polyomavirus genome are rapidly becoming well enough understood to allow a virus to be tailor-made for these purposes. Finally, it would appear that the early optimism regarding the use of viruses to understand many important cellular events has not been misplaced. As more “control” signals, such as those leading to DNA replication, become defined in lower organisms in terms of structural elements within the genome, and similar elements are identified in viruses such as polyoma, it would be surprising if they did not prove faithful mimes of the corresponding events in mammalian cells. Thus, the concept of polyomavirus as an excellent tool for understanding its host should gain further support within the next few years. ACKNOWLEDGMENTS We wish to thank our many colleagues who read this manuscript and made critical and valuable suggestions. We are particularly indebted to Drs. Pierre Bourgaux and Tomas Lindahl in this regard. We also acknowledge with gratitude manuscripts or information received before publication from Drs. C. Basilico, T. L. Benjamin, P. Bourgaux, F. Cuzin, W. Eckhart, A. Hayday, Y. Ito, G. Magnusson, H. Manor, C. Prives, M. Yaniv, and G. Walter. We are grateful to Drs. M. D. Jones, H. E. Ruley and W. R. Folk, Miss M. Ginsburg, and Mr. A. J. Street for permission to cite unpublished work. Finally, we thank Mrs. S. Somani for much help with the manuscript.

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THE PATHOGENESIS OF ONCOGENIC AVIAN RETROVIRUSES

Paula J. Enrietto and John A. Wyke Tumour Virology Labocatory. Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, England

I. Introduction .....................................................................

11. Possible Mechanisms of Retrovirus Oncogenesis. ................................ A. Neoplasia Directly Induced by Virus Infection ...............................

B. Neoplasia as an Indirect Result of Virus Infection.. .......................... Ill. Diseases Caused by Avian Retroviruses,. ........................................ IV. Pathogenesis of Transforming Retroviruses: The Role of v-onc Genes ........... A. The Diversity of v-onc Genes and Their Structure.. ... ................... B. The Functions of v-onc Genes.. .............................................. V. Pathogenesis of Nontransforming Retroviruses: The Possible Role of c-onc Genes ................................................................... A. Structure and Function of the Long Terminal Repeat,. .>.................... B. Evidence for Insertional Mutagenesis in Avian Leukosis ..................... C. The Relationship between Viral and Cell Oncogenes.. ....................... VI. The Host Response .............................................................. VII. Conclusions and Prospects.. ..................................................... A. The Nature of Oncogenes .................................................... B. The Consequences of onc Activity in the Cell ................................ C. The Optimist’s View ......................................................... References .......................................................................

269 270 270 273 274 278 278 283 292 292 295 30 I 305 306 306 306 308 309

I. Introduction

Retroviruses are of interest first as pathogens in a number of vertebrate species important to man and second as a major tool of the experimental oncologist. The speed and reliability with which these small viruses cause tumors in animals and analogous changes in tissue culture suggested that an understanding of retroviral functions would provide basic information on the primary mechanisms of cancer causation. This belief is beingjustified by results of recent years. Molecular biologists have identified a number of genes, originally in retroviruses and subsequently in uninfected vertebrate cells, whose activity is needed to induce neoplasia of various types. The functions of these genes and their role in cell metabolism are now being sought and it is here that complexities arise that presage the problems of understanding fully the effects of retrovirus infection on the whole organism. Pathologists have long realized that retroviruses cause a complex of neoplastic and nonneoplastic diseases in any particular species. The disease 269 ADVANCES IN CANCER RESEARCH, VOL. 39

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form IeseNed. ISBN-O-12-0066394

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patterns were simplified when it was realized that field isolates of the viruses were mixtures of agents (Gross, 1970),but even a single homogeneous strain could show pathogenic heterogeneity. Some of this complexity reflects host factors such as the age of the animal, its genetic, immunological, hormonal, and nutritional status, the effect of concurrent disease, the route of virus infection, the dose of infecting virus, and modulation or mutation of the virus postinfection. However, even within an individual animal it is clear that a single virus strain can cause pathogenic changes in several tissues, yet leave others apparently unaffected. The challenge that now faces retrovirologistsis to integrate the knowledge of pathologists with that of the cell and molecular biologists into a full and coherent explanation of retrovirus pathogenesis. The gap between the disciplines, although narrowing, still exists. It is the aim of this article to examine the information that impinges on this gap and the sort of approaches that might attempt to bridge it. Being eclectic in concept, the article will also be selective in content. Because of limitations in our space and our knowledge we will concentrate on examples provided by the avian retroviruses, but, where a particular point is well illustrated by retroviruses of other species, these will be noted. Further information about any particular point, and fuller citation of original work, will probably be found in the recent compendium edited by Weiss et al. (1982). II. Possible Mechanisms of Retrovirus Oncogenesis

The means by which infection with retroviruses, or any other oncogenic viruses, might lead to neoplasia can be envisaged even without a detailed knowledge of the viruses. These means are listed in Table I and this article considers the extent to which these theoretical mechanisms have been shown to operate in reality.

INDUCEDBY VIRUSINFECTION A. NEOPLASIA DIRECTLY 1. The Virus Persists in the Tumor

Most strategies of viral oncogenesis involve infection of the tumor cell lineage and hence depend on the virus being noncytocidal. Moreover, the role of the virus has been most readily investigated in those tumors in which continued neoplasia relies on the persistence of all or part of the viral genome. The retrovirus life cycle makes such mechanisms particularly plausible. The single-stranded genomic RNA is converted by the virion RNA-dependent DNA polymerase into a double-stranded DNA provirus, which is then inserted stably into the host chromosome and persists in

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27 1

TABLE I THEMEANSBY WHICH RETROVIRUSES MIGHT INDUCE TUMORS A. Direct: Either the tumor cells or their ancestors have been infected by virus I. Virus present: All or part of the viral genome persists in the tumor cells a. Viral oncogenes: The provirus cames a gene whose product initiates and/or maintains neoplasia b. Insertional mutagenesis: Proviral integration disrupts normal regulation of gene expression 2. Virus absent: Transient infection causes a stable heritable neoplastic change in the tumor cell lineage B. Indirect: Neither the tumor cells nor their ancestors need to have been infected by virus. Tumors arise among proliferating cells, either of two ways I . Regeneration of damaged tissue or 2. Mitogenesis of immune competent cells

descendants of the original infected cell (Bishop, 1978). This integration event permits two subdivisions of this mode of neoplasia (Table I). Until recently attention focused on the possibility that retroviruses carried genes that encoded a protein whose function induced and maintained neoplasia (Vogt, 1977). A number of such viral oncogenes (v-onc genes) were described and it was found that homologous sequences (c-oncgenes) existed in uninfected eukaryote cells suggesting that, during evolution, retroviruses acquired these genes from their hosts and now reintroduce them under the control of viral transcriptional promoters (Bishop, 1981). This results in their aberrant expression and ensuing neoplasia, as we shall discuss in Section IV. In acquiring onc genes the viruses almost always lost genes needed for their own replication, with the result that most agents with v-onc genes cannot replicate without a complementing nondefective “helper” retrovirus. However, many oncogenic retroviruses have retained the three genes needed for their replication (see legend, Fig. 1)and lack detectable onc genes. The modes by which such agents induce neoplasia have recently been studied as reviewed in Sections V,A and B. In a few cases evidence suggests that they act as insertional mutagens, proviral integration causing stable inherited changes in the host genome (Varmus, 1982). These changes might include enhanced cell gene expression mediated by viral regulatory elements (“Promoter insertion”; Nee1 et al., 198l), disruption of coding or cell regulatory sequences by the simple presence of the provirus, and genomic deletions or rearrangements resulting from the insertion process. Proviral integration is apparently a simple insertion without massive deletions or transpositions (Varmus, 1982) and the only examples of retroviral inser-

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Agag wbA orb6 pP75 p68

Aenv

on 9(19

APol

myb

7

. Agag

E 28

myb

Aenv

pP135

FIG. I . The structure of representative avian Sarcoma and defective leukemia provirus genomes and their v-onc products. The viruses are identified on the left of the figure. For each, the upper line represents the proviral genome, with solid boxes denoting LTR regions; the lower open boxes represent the protein products of the v-onc sequences. The orientation of viral genes is shown on the genomes: the replicative genes, present in their entirety in RSV at the top of the figure, are gag (encoding a polyprotein cleaved to virion structural components), pol (encoding the viral RNA dependent D N A polymerase), and env (specifying virion membrane glycoproteins). The symbol A denotes a deletion in the respective gene. The nomenclature of v-onc proteins follows Coffin el al. (198 I): approximate molecular weight (X lo+) is preceded by p (for protein) or P (for fusion protein). An additional p indicates a phosphorylated molecule, while g denotes glycosylation. ? indicates a protein that is thought to exist but has not yet been detected.

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tional mutagenesis analyzed so far have caused stimulation of host genes in the vicinity of the proviral integration sites. An important finding, that the genes stimulated include c-oncgenes, stresses the significance to neoplasia of c-onc activity, as discussed in Section V,C.

2. The Virus Is Absentfvom the Tumor Some oncogenic viruses may act transiently, perhaps by inducing a mutation in a single cell, and are then lost from the tumor lineage. In practice such an oncogenic mechanism is hard to distinguish from one in which virus persistence is essential and we know of no authenticated case of a tumor induced by a “hit-and-run” virus. The problem is obvious when one considers the phenomenon of tumor progression, the stepwise acquisition of a fully neoplastic phenotype. A virus that may be essential to the induction, and indeed the maintenance, of a preneoplastic or neoplastic phenotype, can become dispensable once the tumor lineage has undergone further progression. Since its initial interaction with the cell was not transient, the virus has not operated by a “hit-and-run” mechanism, but this distinction is not demonstrable in the light of the subsequent disappearance of the virus. AS AN INDIRECT RESULTOF VIRUSINFECTION B. NEOPLASIA

It is also conceivable that a tumor might arise as a consequence of virus infection of other cells in the organism. Two schemes by which this could occur can be envisaged, both postulating that virus infection stimulates proliferation of a specific cell population, these dividing cells being more prone to neoplastic change than the corresponding quiescent population in an uninfected host. Cell proliferation might be a regenerative response to a cytocidal virus infection [it has been suggested that the induction of hepatocellular carcinoma in individuals with chronic viral type B hepatitis may in part be due to such an effect (Blumberg and London, 1980)l. Most retroviruses, however, are not very cytotoxic and even with those that are, such as the reticuloendotheliosis virus complex, there is no evidence that tumor production results from a proliferative response to this effect. There is, however, evidence that retrovirus infection can exacerbate immune deficiency, influencing the host reaction to preexisting tumor cells (Section VI), and possibly favoring tumor development. A more specific cell proliferation can occur among immunologically competent cells responding to the antigenic stimulus of virus infection. Murine leukemia virus-induced thymic lymphomas may arise because viral glycoproteins recognize receptors on T lymphocytes and act as T cell mitogens (McGrath and Weissman, 1979). In this instance infected T cells

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PAULA J . ENRIETTO AND JOHN A. WYKE

provide an autocrine stimulus and the proposed pathogenic mechanism is not indirect, but it is also possible that uninfected cells respond to lymphokine mitogens produced as part of an immune response to chronic virus infection (reviewed by Ihle and Lee, 1982). These proposals are still tentative and there is no evidence as yet for such a phenomenon in the diseases induced by avian retroviruses, although host immune responses clearly influence the course of diseases (Section VI). Even if such indirect mechanisms were postulated it might be difficult to validate them. On the one hand the tumor cells could be fortuitously infected with virus, particularly since such proliferating cells might be a better “soil” for virus infection. On the other hand, lack of detectable virus infection in any cell population does not prove that its ancestors were uninfected, as pointed out in Section II,A,2.

111. Diseases Caused by Avian Retroviruses

The discoveries of virus-induced avian neoplasms and studies on their pathology have been reviewed extensively (for example, Gross, 1970; Purchase and Burmester, 1978; Beard, 1980). We will extract from these surveys only such information as is needed to appreciate the work on pathogenesis discussed in later sections. Even after due allowance for the virus and host variation outlined in Section I, the pervasive impression from work on retroviral neoplasia is that ofa pleomorphic host response. The full extent ofthis is difficult to judge, for studies have been performed over several decades during which concepts in histology and standards of histochemical investigations have varied, as have experimental protocols. Moreover, many tumors are metaplastic or anaplastic and if they also invade or metastasize then their tissue or origin may be difficult to determine. Nonetheless, avian retroviruses as a whole can definitely induce hemopoietic, other connective tissue, and epithelial tumors in tissues derived from all three embryonic layers. In addition, a single virus can be neoplastic in multiple cell types and, even in a single tissue, can induce tumors showing varying degrees of pleiomorphism. Studies in vitro have helped to clarify this situation, largely by culturing specific cell types and analyzing the affect on them of genetically homogeneous retroviruses. These findings, combined with in vivo observations, have led to a classification of avian retroviruses that was based initially on their pathogenic potential but is fully consistent with subsequent knowledge of their genetics and biochemistry (Table 11). It thus forms a useful framework in which to consider mechanisms of pathogenesis. Five main groups of avian retroviruses are shown in Table 11. The first group comprises viruses originally obtained either as helper viruses for the replication-defectivemembers of other groups or as the causal agents from a

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275

variety of tumors, but mainly from lymphoid leukosis (hence the viruses are known generically as lymphoid or avian leukosis viruses: LLV or ALV). Most of these viruses take several months to induce clinical neoplasia and they cannot transform cells in vitro; a few produce tumors such as osteopetrosis with a latency as short as 3 weeks and have some effect on appropriate cells in culture (Section V,B,l). However, their major common feature is that the only genes they are known to possess are those needed for virus replication. The second category, viruses isolated from sarcomas of various sorts, include Rous sarcoma virus (RSV) and a group called defective sarcoma viruses (because they cannot replicate without ALV helpers). In susceptible hosts they produce tumors of connective tissues of both mesodermal and ectodermal origin within a few weeks. In vitro they transform fibroblasts, other mesenchymal cells, and a variety of epithelia. They all carry v-onc genes, distinct from viral replicative genes, that initiate or maintain neoplasia. These genes are genetically different in different viruses, but they show functional similarities (Sections IV,A and B). Members of the third group of viruses were originally isolated from a variety of tumors of the hemopoietic system and since their replication requires a helper ALV they were named defective leukemia viruses (DLV). Their pathogenic spectrum, however, includes not only hemopoietic tumors but also sarcomas of other tissues and carcinomas, all of which are induced with a latency of a few weeks (hence the group’s other title, acute leukemia viruses). Like the sarcoma viruses the DLV all carry v-onc genes that are vital to neoplasia. The nature of these genes, together with the types of cells transformed in vitro and diseases caused in vivo, define three subdivisions of DLV (Table 11, and Graf and Stehelin, 1982). (1) Avian erythroblastosis virus (AEV) carries a gene called v-erb and transforms erythroblast-like cells and fibroblasts in vitro. (2) Myelocytomatosis virus (MC29) and related agents bear a sequence denoted v-myc and transform macrophage-like cells and fibroblasts in vitro. (3) Avian myeloblastosis virus (AMV) and E26 virus have v-myb sequences and transform myeloblast-like cells. These three groups comprise the avian sarcoma/leukosis virus (ASLV) complex that, on the basis of viral replicative genes, is distinct from the other avian retrovirus categories, the reticuloendotheliosis group viruses (REV). REV, like ASLV, are subdivided on pathobiological criteria (Table 11)into a group of several nontransforming, nondefective helper viruses (resembling ALV) and the rapidly pathogenic, transforming, replication-defective REV-T (resembling DLV). Unlike most ALV, the nondefective REV viruses can cause disease fairly rapidly (Witter and Crittenden, 1979), a property that may or may not be associated with their cytotoxicity in vitro (Keshet and Temin, 1979).

TABLE I1 PATHOLOGICAL AND BIOLOGICAL PROPERTIES OF AVIANRETROVIRUSES Virus group" Avian Sarcomaleukosis complex

Pathogenic groupb

Virus typeb

Representative strains

Viral oncogene

Cells transformed in vitro

Nondefective Avian leukosislike virus

Endogenous viruses Exogenous avian leukosisviruses

evz lo. I1. l2 RPL 12, RAV- 1, MAV-2(0)

None None

None None; some are cytopathic

Sarcoma viruses

Rous sarcoma virus

Bryan, Prague, SchmidtRuppin strains

src

Fujinami-type sarcoma viruses Y73-type sarcoma virus

FSV, PRCII, URI Y73. Esh sarcoma virus UR2 AEV strains ES4 and R

fps

Fibroblasts, myoblasts, chondroblasts, various epithelia Fibroblasts

Yes

Fibroblasts

ros erb

Fibroblasts Erythroblasts, fibroblasts

N I .

m

Defective leukemia viruses

UR2 sarcoma virus Erythroblastosis viruses

I

Disease spectrum Nonpathogenic B-cell leukosis (frequent), osteopetrosis, nephroblastoma (frequent with some strains), various sarcomas and carcinomas, anemia, nephritis Various sarcomas of mesodermal origin, plus gliomas (ectodermal)

Various sarcomas of mesodermal origin

Erythroblastosis, sarcomas, carcinomas

Reticuloendotheliosisvirus complex

Nondefective viruses

Defective leukemia virus

MC29-type viruses

MC29, CMII, MH2, OK10

myc

Macrophages, fibroblasts

Myeloblastosis viruses

AMV, E26

myb

Myeloblasts

Chick syncytial virus, duck infectious anemia virus, spleen necrosis virus REV-T

None

None, but cytopathic

re1

Fibroblasts

Various carcinomas, sarcomas of mesenchymal and hemopoietic tissue, myelocytomas Myeloblastosis, erythroblastosis, rarely carcinomas and sarcomas W e l l leukosis, lymphoproliferative lesions, reticuloendotheliosis, anemia Reticuloendotheliosis

Classification based on genetic criteria. The virus responsible for the lymphoproliferative disease of turkeys forms yet another group, but since little is known about it it is not considered here. Weiss e? al. (1982) consider that the endogenous viruses of quails and pheasant should form a distinct group, but the reasons for this are not clear. Classifications based on a combination of pathological, biological, and genetic criteria.

27 8

PAULA J . ENRIETTO AND JOHN A. WYKE

IV. Pathogenesis of Transforming Retroviruses: The Role of v-onc Genes

The concept that tumor viruses contain genes responsible for converting a normal to a malignant cell was the force that drove much early work on the molecular biology of these agents. It transpires that those retroviruses capable of transforming appropriate cells in tissue culture and rapidly inducing tumors in animals do indeed bear such viral oncogenes and these are distinct from the genes required for virus replication. A. THEDIVERSITY OF v-onc GENESAND THEIRSTRUCTURE

The first convincingly defined v-onc was the v-src gene of Rous sarcoma virus (RSV). Unlike other transforming retroviruses, RSV is nondefective and can replicate without the help of a nontransforming virus. In fact, RSV contains more genes than are needed for replication; it segregates mutants with smaller RNA genomes that can still replicate but can no longer transform cells or cause sarcomas (transformation-defectiveor td mutants). RSV mutants that replicate but bear conditional (temperature-sensitive, ts) defects in transformation have also been isolated. The lesions in ts mutants are all located in that part of the RSV genome that is missing from tdviruses, thus defining a region, v-src, presumed to encode a protein-mediating cell transformation (reviewed by Vogt, 1977). After a lag of several years the src gene product was identified in transformed cells, and in the products of in vitro RSV RNA translation, by precipitation with antisera from mammals bearing RSV-induced tumors (Brugge and Enkson, 1977; Purchio et af., 1978). It is a protein, unrelated to any virion polypeptides, whose function is discussed in Section IV,B, 1. The replication defects of other transforming retroviruses hampered the isolation of td and ts mutants and the consequent genetic definition of their transforming genes. This problem was circumvented by direct biochemical examination of the genomes of these viruses and the proteins they encode, thus identifying presumptive v-onc genes whose nature is now being validated by the isolation of naturally occurring or genetically manipulated mutants. The genomes of these transforming viruses are now studied after molecular cloning, but they were originally investigated by making nucleic acid probes specific for sequences that were absent from their helpers and were thus presumed to be unrelated to replicative genes. Such probes not only identified putative v-onc genes in the viruses but also detected homologous c-onc sequences in normal host cells. The existence of a cellular counterpart for every retroviral v-onc gene studied suggested that the viruses acquired these genes from their hosts and thus gained control of their expression. This idea not only turned the spotlight on c-onc genes and their

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role in the cell (Section V,C) but it raised questions about the way in which the viruses captured such genes and the effect this would have on onc expression and activity (see Graf and Stehelin, 1982). The known transforming retroviruses seem to have acquired c-onc genes by “illegitimate” nonhomologous recombination events, resulting in a number of different patterns of onc insertion among the residual viral replicative genes (Fig. 1). One invariant feature is the retention by the proviruses of long terminal repeat (LTR) regions that contain regulators essential for virus gene expression and replication (Section V,A and Varmus, 1982). These controlling elements maintain v-onc expression at a high level, largely independent of cell control, but the form in which the v-onc product is expressed (Fig. 1 and Table 111) is determined by the position of the onc gene with respect to viral coding regions and viral RNA splicing signals. In many cases the onc insert is near the 5’ end of the virus and it is expressed, together with sequences of part of the viral gag gene, as a gag- onc fusion protein. Such proteins are readily recognized products of transforming retroviruses because they contain antigenic determinants of virion structural genes and hence can be precipitated by antivirion sera. Fusion proteins are the only known gene products of the avian defective sarcoma viruses: Fujinami sarcoma virus (FSV) and the sarcoma viruses PRCII, PRCIV, UR1 and 16L all contain onc sequences derived from the cellularfis genes. They encode gag-fps fusion proteins of the various sizes shown in Fig. 1 and Table 111. The fusion proteins of Y73 and Esh sarcoma viruses are coded in part by yes inserts, while UR2 carries a distinguishable insert, v-ros (Table 111; for review see Neil, 1982). Many avian defective leukemia viruses also encode fusion proteins (Fig. 1 and Table 111; for review see Hayman, 1981). The pP1 lWg-mJ’c protein of MC29 is important in neoplasia because several virus mutants that encode proteins lacking some myc-specific peptides have a reduced tumorigenicity and can no longer transform macrophages (although they still transform fibroblasts) (Ramsay et al., 1980; Ramsay and Hayman, 1982). However, some DLV show different transformation strategies. Linial and colleagues, studying the myc-containing DLV MH2, have used hybrid selection with a cloned myc gene to obtain from transformed cells a subgenomic MH2 messenger RNA that, upon translation, yields a protein of 57,000 molecular weight. It is likely that this p57 molecule is the only protein needed for transformation and tumorigenicity, since a virus mutant that encodes p57, but not the MH2-specific pP1008ng-mJ’c fusion protein, is able to transform macrophages and fibroblasts in vitro and is highly oncogenic in vivo (M. Linial, personal communication). The situation with OK 10 virus may be comparable, for there is no evidence that the pP20@‘~-~0~-mJ’c fusion protein is involved in tumorigenicity. Moreover, OK 10-transformed nonproducer

TABLE 111 RETROVIRUS ONCOGENES~

w

DO

0

Oncogene

Probable animal origin

re1 src

Turkey Chicken, quail

fps

Chicken

Chicken 10s

ski mYb

Chicken Chicken Chicken Chicken

Protein product9

Representative viruses* ?

Avian reticuloendotheliosisvirus-T Rous and B77 sarcoma viruses, recovered avian sarcoma viruses

PP~O

Fujinami sarcoma virus PRCII sarcoma virus URI sarcoma virus 16L sarcoma virus Y73 sarcoma virus Esh sarcoma virus UR2 sarcoma virus SKV viruses Avian myeloblastosis virus Avian leukemia virus E26 Avian myelocytomatosisvirus MC29 Avian myelocytomatosisvirus CMII MH2 virus OK 10 virus

(and variants) pP140 pP I05 pP150 pP 142 pP90 pP80 pP68 ? P110, PI25 ? pP135 pP110 pP90 pPl00 p57 pP200 ?

Protein kinase activity

+

+ +

mos ras

Chicken Chicken Mouse Rat

abl fos fese

Mouse Mouse, cat Mouse Cat

erb-A e r bB

fms sis

pP75 P68 PP37 PP2 1 pP29 PP2 1 pP90-pP160d PP55 pP85 pP110 gP 180 P28

Table modified from Coffin ef al. (1981) and Weiss et al. (1982) (which contain appropriate references). The list of viruses bearing any particular onc gene is not complete, but serves to illustrate the variety of isolates obtained. Protein nomenclature follows Coffin et al. (198 1) as described in the legend to Fig. 1. For clarity, superscripts defining the genes have been omitted. * Size of fusion protein varies with virus strain. Thefes gene is closely related to thefps gene, but at present is considered as distinct. a

c

Cat Woolly monkey, cat

Avian erythroblastosis virus Avian erythroblastosis virus Moloney murine sarcoma viruses Kirsten and Harvey murine sarcoma viruses Rasheed rat sarcoma virus BALB murine sarcoma virus Abelson murine leukemia virus FBJ murine osteosarcoma virus Synder-Theilen feline sarcoma virus Gardner- Amstein feline sarcoma virus McDonough feline sarcoma virus Simian sarcoma virus

282

PAULA J. ENRIETTO AND JOHN A. WYKE

cells contain a subgenomic virus-specific mRNA of a size sufficient to encode an as yet unidentified protein of about 50,000 daltons (Chiswell et al., 1981). Avian erythroblastosis virus exhibits yet a different pattern (Fig. 1). The erb insert comprises two domains that are expressed separately: erb-A as a pP75gag-erbAfusion protein and erb-B as a protein of 68,000 daltons that is encoded on a subgenomic mRNA as a precursor of 62,000 molecular weight (Privalskyand Bishop, 1982; Hayman et al., 1983b).Mutagenesis studies on the erb inserts show that deletion of erb-A sequences reduces, but does not abolish, transformation. On the other hand, a complete erb-B is needed for erythroblast transformation and partial erb-B functions are required for fibroblasttransformation. Thus, a “host range” mutant of AEV which codes for truncated erb-A and erb-B products will transform fibroblasts but not erythroid cells (Royer-Pokora et al., 1981; Frykberg et al., 1983).In addition a distinct, recently isolated erythroblastosis virus (AEV-H), which has erb-B but no erb-A sequences, is able to transform fibroblasts in vitro (S. Kawai and K. Toyoshima, personal communication). Various ts mutants of AEV have also been isolated but their lesions have not been mapped biochemically (see Graf and Stehelin, 1982). At this time I7 v-onc genes have been reported in vertebrate retroviruses, 10 of them in birds (Table 111). There are many examples in which different virus isolates have independently acquired the same onc sequence, so, although new onc genes continue to be discovered, the total number of cellular genes capable of subversion to v-oncactivity may not be much more than 20. Nonetheless, this suffices for a great deal of variation in pathogenicity and the potential complexity is increased by the fact that related onc inserts may encode fusion proteins of differing size in different virus strains (for example, the various myc andfps containing viruses in Table 111).We do not yet know how different proportions of virion and onc gene products in a fusion protein bear on differences in the pathogenicity of various isolates that contain related onc regions, but it seems probable that such heterogeneity has phenotypic consequences. For example, both AMV and E26 virus contain myb inserts, but whereas the former causes only myeloblastosis in chickens, the latter induces in addition erythroid tumors (Stehelin et al., 1980; Graf and Stehelin, 1982). It is possible that the basis for this difference lies in the form in which myb is expressed: E26 encodes a gag- myb fusion protein whereas AMV probably produces on a subgenomic mRNA an independent, but as yet unidentified, myb product (Bister et al., 1982; Beug et al., 1982) (Fig. I and Table 111). It is possible, however, that the heterogeneity among onc inserts themselves, as indicated by nucleic acid hybridization, conceals some underlying similarities. For instance, the v-yes gene of Y73 sarcoma virus appears

AVIAN RETROVIRUS PATHOGENESIS

283

unrelated to v-src by nucleic acid hybridization, but the nucleotide sequences of the two genes are about 70% homologous and, since many differences are third base changes, the two amino acid sequences are even more similar (Kitamura et al., 1982; see Griffin, 1982, for a review). Since v-yes and v-src share with v-fps of Fujinami sarcoma virus and several other oncogenes an associated protein kinase activity that phosphorylates tyrosine (Section IV,B; Table 111) it is possible that this whole group may be related and this may bear on their similar pathogenic capabilities. The amino acid sequences that flank potentially phosphorylated tyrosine residues in the v-onc products of this group are very similar, suggesting that the proteins possess related active sites (Neil et al., 1981; Patchinsky et al., 1982; Neil, 1982) and this resemblance may extend to other parts of the proteins. Moreover, the v-fps genes of avian-defective sarcoma viruses are related to the v-fesgenes of feline sarcoma viruses (Shibuya et al., 1980), and comparable relationships (more apparent in protein products than in the genes that encode them) are seen among the various ras sequences acquired from rat and mouse cells by murine sarcoma viruses (Table 111; see Weiss et al., 1982). These resemblances suggest either a remarkable convergent evolution of v-onc functions during their sojourn in viruses or, more probably, their descent from c-onc homologs that are parts of gene families whose functions have been conserved even more strongly than their sequences. OF v-onc GENES B. THEFUNCTIONS

To understand how retroviruses induce tumors, observations made at the cellular and organismic level must be correlated with those made at the molecular level. There are several stages in accomplishing such correlations: ( 1 ) defining and characterizing oncogene products and their biochemical activity, (2) identifying the targets for oncogene activity and elucidating the pleiotropic effects within a cell that lead to neoplasia, and (3) explaining the different consequences of oncogene activity in different cells within an individual host and between individuals. The first stage is becoming well understood in the case of some oncogenes; there is a great deal of information about the second stage, although as yet little insight; the third stage is still largely unexplored. 1 . The Nature and Function of v-onc Products a. Avian Sarcoma Viruses. The RSV src gene product has been studied extensively. It is a 60,000-dalton protein, pp6WC,phosphorylated at two major sites: one in the amino-terminal portion at serine, the other in the carboxy-terminal portion on tyrosine. The serine phosphorylation is by a CAMP-dependent protein kinase, while the tyrosine phosphorylation is

284

PAULA J. ENRIETTO A N D JOHN A. WYKE

cAMP independent (Collett el al., 1979; Hunter and Sefton, 1980).The area around this tyrosine site resembles that around phosphotyrosine residues in defective sarcoma virus proteins (Section IV,A) in that arg appears seven residues from the phosphotyrosine, there is a predominance of acidic amino acids, and glu recurs in the configuration glu-x-x-glu-tyr. The phosphorylation at tyrosine may be autocatalytic and this points to the enzymatic activity associated with pp60"", that of a protein kinase specific for tyrosine residues (Collett and Erikson, 1978; Levinson et al., 1978; 1980; Erikson et al., 1979; 1980; Collett et a!., 1980; Sefton et a/., 1980b; Purchio, 1982). This unusual activity was first demonstrated in immune complexes of pp60"" and immunoglobulin, where the heavy chain of the immunoglobulin became phosphorylated in the presence of [y-32P]ATP.Because the pp60"" associated kinase activity was thermolabile in cells infected with viruses ts for transformation, it seemed to be a property of pp60"" itself. Definitive proof has come using molecular clones of src DNA inserted into Escherichia coli which subsequently synthesized pp60"" that has a protein kinase activity (Gilmer and Erikson, 1981; McGrath and Levinson, 1982). Using purified pp60"" its enzymatic activity has been studied (Erikson et al., 1979; Levinson et al., 1980). ATP, CTP, and other nucleoside triphosphates can serve as phosphate donors and the activity, which is cAMP independent, phosphorylates several exogenous substrates in vitro on tyrosine, including some that are not tyrosine phosphoproteins in

vivo. The active site for phosphotransfer seems to reside in the carboxy-terminal half of pp60"". Mutants which lack up to 15,000daltons of protein from the amino-terminal portion of the molecule retain activity and a 30,000dalton fragment from the carboxy-terminal half of the protein contains the phosphotyrosine residue and is more active as a protein kinase than the native pp60"". Moreover, site-directed mutagenesis in the 3' portion of src affects protein kinase activity (J. T. Parsons, personal communication) and some ts mutants that map in the 3' 40%of the gene (Fincham et al., 1982) are known to have thermolabile kinase activity (Sefton et al., 1980a). All the avian-defective sarcoma virus gag- onc fusion proteins listed in Table I11 are phosphorylated in vivo on both serine and tyrosine and they have associated protein kinase activities that seem to be directed toward the fusion proteins themselves as well as toward immunoglobulin and cell acceptor proteins. Studies on ts transformation mutants of FSV showed that the tyrosine phosphorylation of pP 140gng-fps was thermolabile, thus implicating the kinase activity associated with this protein in cell transformation. ts mutants if PRCII have also been isolated (for review see Neil, 1982). b. Defective Leukemia Viruses.The DLV fusion proteins are phosphory-

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285

lated, but this is not known to be the case for those proteins encoded entirely by onc inserts (Table 111). Moreover, unlike the avian sarcoma viruses and some mammalian retroviruses (Table 111), the putative transforming proteins have not been associated with a protein kinase activity and, at present, the biochemical activities of these proteins are unknown. 2. The Targetsfor Oncogene Activity Neoplastic cells, by definition, are growing abnormally. However, since they succeed in this growth over many cell generationstheir deviation from normal regulation as a consequence of oncogene action is probably very subtle. The complexities of normal cell growth regulation are poorly understood and the scant knowledge that we have has not so far helped to elucidate the behavior of oncogenes (indeed the converse is likely to be the case, and when we learn more about the perturbing effect of oncogene activity it may illuminate the normal situation). With this nebulous background, work on the cellular effects of oncogene products is inevitably at a descriptive stage with no clear conclusions yet apparent. Most studies fall into one of two categories: localization of oncogene products (an approach that can be applied to all proteins for which appropriate probes are available) and identification of endogenous proteins that show transformationdependent changes in tyrosine phosphorylation (obviously only applicable to onc proteins associated with a protein kinase activity). a. Localization of Oncogene Products. Normal cell growth presumably occurs in response to environmental signals, received at the cell surface and appropriately processed to direct cell division. Oncogene products might disrupt these events at a number of sites. For example, they could be secreted and then act as external growth-stimulatingsignals, they could be located in the cell membrane or cytoplasm, perturbing signal reception and processing, or they could be of nuclear location, affecting the transcription of genes related to growth regulation or influencing directly DNA synthesis. Important clues to their action might thus come from determining their subcellular location. However, all such studies to date, and indeed work on target molecules for oncogene activity (Section IV,B,2,b), are subject to a major caveat. We do not know in any particular cell the minimal dose of an oncogenic product that is needed to induce transformation. For instance, mammalian cells that are transformed by RSV may contain far less pp6OSrc than transformed chicken cells (Bishop et al., 1980). If both cell types are equally susceptible to the action of pp60“ then chicken cells presumably contain a large excess of the protein and any studies in chickens that localize the bulk of pp60”” may fail to reveal the site of a crucial minority of the molecules. Similar reasoning applies in cases in which a transformed cell (or tumor) contains only slightly more transforming gene product than an

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PAULA J . ENRIETTO AND JOHN A. WYKE

equivalent normal cell; observations on the bulk of the protein may obscure significant changes in a small proportion of it. Nonetheless, with this proviso, studies attempting to localize onc gene products (by cell fractionation, immunofluorescence, and electron microscopy using immunohistochemistry) have been very useful. Results of studies to locate pp60”” within the infected cell have varied, describing localization at the nuclear envelope around centrioles or in focal adhesion plaques, while the bulk of evidence supports its association with the plasma membrane (Rohrschneider, 1979; 1980; Willingham et al., 1979; Shriver and Rohrschneider, 198I ) . The membrane association seems to occur at the cytoplasmic face and it has been postulated that the aminoterminal 8000 daltons of pp60”” may serve as a structural hydrophobic domain through which binding occurs (Courtneidge et al., 1980; Goldberg et al., 1980; Krueger et al., 1980a,b; Krzyzek et al., 1980; Levinson et al., 1981). The importance of the amino-terminal domain of pp60” in binding to the plasma membrane has been demonstrated using variants of RSV recovered from transformed mammalian cells (Garber et al., 1982). When chick cells infected with rescued virus were studied by subcellular fraction or immunofluorescence it was found that the location of pp6OSrchad changed, being associated with the nuclear envelope and nuclear reticular membranes. Investigation of the structure of pp60” encoded by these viruses revealed changes in the amino-terminal half of the protein. Therefore, it appeared that an intact amino-terminal domain was required for plasma membrane association. However, the cells were still transformed, implying that pp60”” can act at diverse sites within the cells and perhaps on multiple targets-an idea backed by the pleiotropic effects which the virus has on a host cell (see below and Hanafusa, 1977). This observation has been extended by Krueger et al. (1982) using two virus isolates from a tumor induced by mutants of RSV that had partially deleted src genes. The recovered ASV (rASV) were shown to have alterations in the amino-terminal portion of the src proteins they encoded and altered membrane association. Again fibroblasts infected with these viruses became transformed, but when injected into chickens they showed reduced tumorigenicity, inducing differentiated sarcomas which regressed. These data support the role of the amino-terminus in the association of pp60”” with the plasma membrane and may implicate this region in the process of tumorigenesis: by directing pp60”” to the plasma membrane, the amino-terminus may be placing it in proximity to the target@)whose phosphorylation determines the tumorigenicity of the infected cells. It would be of interest to study the localization and structure of pp6PCencoded by some partial transformation mutants of RSV such as CU2 (Kahn et al., 1982)which have been shown to induce only

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some parameters of transformation in vitro and to be poorly tumorigenic in vivo [interestingly, in CU2 infected cells one of the potential targets for the protein kinase activity of pp6@", a 36,000-dalton protein (see below), is poorly phosphorylated]. It would also be worth investigating pp60"" localization in cells transformed by virus mutants that induce fusiform rather than round cell morphological transformation, since several such mutations are known to be located in the 5' half of the src gene, thus affecting the amino-terminus of its product (Fujita et al., 198I; Fincham et al., 1982). Localization of the proteins encoded by the defective sarcoma viruses is just beginning and may reveal common features with the RSV system since both appear to have associated protein kinase activities and similar pathogenicity. Among the defective leukemia viruses careful localization studies have been done only with those containing myc information. The pPllOgag-mYcproduct of MC29 is mainly a nuclear protein with DNA binding activity (Abrams et al., 1982; Donner et al., 1982). CM11 and OK10 have not been studied, but MH2 appears to be different in that pPIOOgag-mJ"is not nuclear (because no myc-specific sera are yet available p57"J" cannot be located). Recent results suggest that the AEV p6SerhB protein is membrane associated (Hayman et al., 1983b) whereas pP75gw-erbAis predominantly cytoplasmic (Abrams et al., 1982). b. Cell Proteins That Interact with pp60"'. There is now no reasonable doubt that the RSV src gene is essential for transformation and tumorigenesis and that the product of this gene, pp60"", has a tyrosine-specific protein kinase activity. However, it is not yet convincingly proved that the protein kinase activity is necessary and sufficient for transformation. Most studies with virus mutants link defects in transformation with defects in protein kinase activity, but there are a few anomalous observations. For instance, RSV-transformed vole cells may revert to a normal morphology without detectable reduction in the amounts of pp60"" or its kinase activity (Lau et al., 1980). Clearly, if pp60"" kinase mediates transformation, then its effect in these revertants has been blocked, either because of a cell mutation in the functions involved in morphological transformation, or because viral mutations prevent its access to appropriate cell targets. The amino-terminus of pp60"" may play a role in determining its cell location (Section IV,B,2,a) and a mutation in this region could well diminish transforming ability without affecting kinase activity. Such a mutant has, in fact, been described (Bryant and Parsons, in press). Moreover, a number of ts src mutations map in the amino-terminal region (Fincham et al., 1982), although the thermolability of their kinase has yet to be assessed. The mode of action of the src gene product must also be reconciled with the pleiotropic nature of cell transformation. RSV-transformed cells differ from normal cells in many respects, a number of which are thermolabile in

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cells transformed by ts src mutants and thus seem to be directly dependent on src gene activity (reviewed by Hanafusa, 1977; Vogt, 1977). There are three ways in which pp6W action might have multiple consequences; (1) the protein may have activities other than tyrosine phosphorylation; (2) the protein may have a single activity that has multiple substrates; (3) pp60”” phosphorylation of a single substrate may initiate a cascade of events with multiple effects. Studies on virus mutants that dissociate parameters of transformation address these possibilities. Not only do ts src mutants show residual transforming activity at restrictive temperature (Calothy and Pessac, 1976; Bissell et al., 1979; Calothy et al., 1980; Parry et al., 1980), but there are nonconditional td mutants that elicit some characteristics of transformation but not others (Yoshida and Ikawa 1977; Anderson et al., 1981). In some cases a partially transformed phenotype might be due to “leakiness” of the mutant function, but Weber and Friis ( 1979)studying partially transformation defective ts mutants isolated by Becker el af.( I 977) showed that there was no fixed hierarchy for the disappearance of transformation parameters when cultures of cells infected by different mutants were shifted to restrictive temperature. Indeed, some mutants were thermosensitive for cellular parameters of transformation (such as fibrinolytic activity) but cold sensitive for growth parameters, a dichotomy that cannot be explained by mutant leakiness. These findings appear inconsistent with a pleiotropism of transformation due to a cascade of events, but they suggest the existence of multiple targets for pp60”“ phosphorylation (presumably the mutants affect differentially the affinity of pp6OSrcfor various substrates). However, the possibility of a single molecular substrate for phosphorylation could still be entertained if the molecule is located at a number of sites in the cell, the consequences of phosphorylation varying with location. In this case the src gene products of the partial td mutants are envisaged as differentially defective in locating themselves within the cells, thus being comparable to the amino-terminal src variants described above (Section IV,B,2,a). The mutants in several partial td viruses isolated by Becker et al. (1977) and Anderson et al. ( 1 98 1) have been mapped to the middle region of the src gene (V. J. Fincham and J. A. Wyke, unpublished). Unfortunately, this location, remote from both the amino-terminus and the phosphotyrosine of pp6OSrc,forces us to equivocate over the nature of the mutant lesions. The questions posed above would be resolved by a full description of the identities and functions of the cell targets for pp6OSrckinase activity. In theory these targets should be readily identified. Tyrosine phosphorylation is an unusual activity and several investigators have found that the level of tyrosine phosphorylation of total cell protein is 6- to 10-fold higher in transformed cells. Moreover, in cells transformed by ts src mutants much of

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this phosphorylation is reversible upon temperature shift, suggesting that it depends on pp60"" activity. However, when specific P-tyr containing proteins have been identified a minimum of 30 have been found in transformed cells compared with 2 in cells infected with tdRSV mutants (Martinez et al., 1982). So far only a few of these have been investigated further including proteins of 130K, 51K, 34-39K, 46K, and 28K, most of which have unphosphorylated or poorly phosphorylated precursors in normal cells (reviewed by Hynes, 1980;Hunter, 1981). In addition, filamin and vimentin have low amounts of phosphotyrosine in transformed cells, the significance of which is unclear. Of this group, the two proteins best studied and considered likely important targets for pp60"" are the 34-39K protein (referred to here as 36K) and the 130K protein. The 36K protein, comprising 0.25-0.590 of the total cell protein, is present in normal cells but less than 1% is phosphorylated while 5 - 1OYo is phosphorylated in transformed cells. In addition nonphosphorylated 36K can be phosphorylated in vitro by partially purified preparations of pp60"" at the same site as the major site of tyrosine phosphorylation in vivo. Cells transformed by FSV, PRCl 1, and Y73 all show similar tyrosine phosphorylation of the 36K protein (Radke and Martin, 1979; Erikson and Erikson, 1980; Radke et al., 1980). In contrast to 36K, whose function and precise cellular location are unknown, the 130,000-dalton protein has been identified as vinculin, which is located in part in adhesion plaques through which cells attach to the substratum. Vinculin is found close to the plasma membrane and is thought to link actin cables to proteins in the membrane. When isolated from normal cells, it contains low levels of phosphotyrosine. However, in RSV transformed cells the level of phosphotyrosine increases 10-fold (Geiger, 1979; Burridge and Feramisco, 1980; Sefton et al., 1981). Vinculin is also modified in Y73 transformed cells, with increased tyrosine phosphorylation, but not in PRCll transformed cells. Despite the lack of vinculin phosphorylation some of the PRCl 1 pP105g0g-fpsis associated with cytoskeletal extracts which show increased tyrosine phosphorylation (Neil, 1982). In the search for targets of pp60" activity is was thought that the affinity of substrates for enzymes might aid their identification. Therefore, it was of interest that two proteins of 89K and 50K were routinely precipitated from RSV-infected cells along with pp60"". It has been shown that 89K, a portion of pp60"", and 50K exist as a physical complex. The 89K protein is a phosphoprotein, one of a set of proteins that is induced by heat shock or anaerobiosis. The 50K protein in the complex contains phosphotyrosine and phosphoserine, suggesting that it might be a substrate for pp60"" enzymatic activity. The phosphorylation of 50K at the tyrosine residue is dependent on the presence of pp60"" but is not temperature sensitive in cells

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infected with ts src mutants of RSV, in contrast to other potential substrates (Hunter and Sefton, 1980; Brugge et al., 1981; Oppermann et al., 1981a,b). Gilmore et al. (1982) and Brugge and Darrow (1982) postulate that 50K may be a high-affinity substrate for pp6OSrC, and evidence now exists suggesting that the 89K-pp60"-50K complex is a transport complex in which pp60"" phosphorylation at tyrosine is controlled while the protein is moved to the cell membrane (Courtneidge and Bishop, 1983). Preliminary evidence indicates that the oncogene products of the defective sarcoma viruses also exist as a complex with 89K and 50K. Protein phosphorylation regulates many enzymatic pathways (reviewed by Weller, 1979) and it is attractive to postulate that the pp60"1 kinase mediates the diverse consequences of transformation. The potential substrates studied so far seem to be phosphorylated at the same sites in normal as in transformed cells, but at much lower levels, suggesting that transformation may be due to unscheduled "over"-phosphorylation rather than completely aberrant activity. However, the plethora of potential targets for pp60"" phosphorylation makes it difficult to assess these events: we do not know which substrates are important, nor do we even know which are directly phosphorylated by pp60"". Two ways around this impasse have been attempted. One approach correlates protein phosphorylation with transformation parameters measured in cells infected by partial td mutants. For example, in the mutant CU2 a low level of 36K phosphorylation is associated with reduced tumorigenicity (Nakamura and Weber, 1982). The other gambit investigatesphosphorylation of proteins whose nature suggests that they play a role in growth control. The studies on vinculin (reviewed by Hynes, 1982)are an example. Since virus transformation affects cell shape it affects, directly or indirectly, the cytoskeleton. Both pp6OSrC and vinculin are located in part in focal adhesion plaques, points where the cell is anchored to the substrate and where vinculin may play a role by linking actin microfilaments to the cell membrane. Vinculin function may be inhibited by phosphorylation, even though there are only 0.01 phosphotyrosines per molecule, leading to loss of adhesion and cell rounding (it has been suggestedthat the lack of vinculin phosphorylation by PRCll may explain why cells transformed by this virus are fusiform rather than round). We do not know whether this change in shape is the stimulus that leads to neoplasia, but it would be interesting to test this possibility using partial tdvirus mutants and variant viruses of fusiform morphology. 3. The Cell Specificity of Oncogene Action The next question that a molecular explanation of pathogenicity must tackle is that of target cell specificity. The first facet of this problem is the capacity of some viruses to transform differing cell types. Thus RSV can

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transform fibroblasts and epithelial cells, while MC29 transforms macrophages and fibroblasts and can induce carcinomas in vivo (Section 111). The target cells for a v-oncgene may share a common substrate molecule but the possibilities discussed in Section IV,B,2,b are also relevant: the v-onc genes may have multiple functions or they may act on multiple target molecules or at various subcellular locations. We have only a few clues so far. Mutations in the erb-A region of AEV have only a quantitative effect on fibroblast and erythroblast transformation, whereas appropriate mutations in erb-B can destroy transforming ability (Section IV,A), thus indicating functional differences between the two domains of the erb insert (Frykberg et af.,1983). MC29 mutations that affect macrophage but not fibroblast transformation also dissociate these two events (Ramsay et af.,1980). On the other hand, they do not show whether this dissociation is due to two onc functions or to two targets with different affinities for the mutant onc product. The converse question is that of restriction of onc activity. Why does a given virus cause a limited range of tumors in vivo and correspondingly transform a limited range of cells in vitro while other viruses have a different pathogenic spectrum (Section III)? In addition why do some viruses cause different tumors in different (but related) hosts (C. Moscovici, quoted in Weiss et af.,1982)?The simple answer, that viruses are limited in the range of cells that they can infect, might apply in a few instances (Gazzolo et af., 1974),but in many other cases a transforming virus can apparently infect a cell and express its onc product without that cell becoming transformed (Graf et af., 1980; Durban and Boettiger, 1981). At present, reasons for the differential cell response to an oncogene product are conjectural but presumably reflect the availability of appropriate targets in the host. More detailed theories have been proposed within this framework, but although data favoring them can be obtained with some viruses, no model seems universally applicable. For instance, a popular concept is that the cells of hemopoietic tumors are neoplastic because they have been arrested during differentiation. Viral onc genes that “freeze” differentiation may do so because their c-onc counterpart functions in the cell lineage at that stage of maturation (Graf and Beug, 1978). The v-onc product might prevent further differentiation in one of two ways: (1) it is functionally similar to the c-onc protein and thus provides an excess of this product or (2) it is subtly different and blocks c-onc activity, for instance by competing for substrates. Both mechanisms predict that c-oncs are expressed specificallyin the target cells for the homologous v-onc genes. In the case ofc-erb this may be so, but c-myc and c-myb are apparently expressed in hemopoietic tissues in addition to the targets for the corresponding v-onc genes (Chen et af.,1980; Saule et af.,quoted in Graf and Stehelin, 1982). However, as emphasized in Section IV,B,2, the overall level of an onc

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product may be misleading: it is its location and access to substrate molecules that are crucial and a susceptible cell is defined by its expression of these interacting molecules. Clearly, an explanation of the cell and species specificity of v-onc action requires a full knowledge of the targets for onc functions (Section IV,B,2) as well as an understanding of the relationships between v-onc and c-onc genes (Section V,C). V. Pathogenesis of Nontransforrning Retroviruses: The Possible Role of c-onc Genes

A number of retroviruses induce tumors with a long latency (Section 111) and they cannot transform cells in tissue culture. It is not known how most of these viruses cause neoplasia, but since they lack v-onc sequences their mode of pathogenicity must be either indirect or by a direct effect that disrupts the expression of cell genes (Section I1 and Table I). In a few instances there is ample evidence for action by the latter mode, and to appreciate the means by which retroviruses might affect host gene expression one must consider various structural features of the virus (a more comprehensive review of this complex topic is given in Varmus, 1982).

A. STRUCTURE AND FUNCTION OF THE LONGTERMINAL REPEAT The retrovirus genome is a single-stranded positive sense RNA in which the coding regions are flanked by sequences essential for virus replication and expression. These include short direct repeats (R) at each end of the RNA internal to which are nucleotide tracts unique to either the 5' end (U5) or the 3' end (U3) of the genome (Fig. 2A). During transcription of this RNA into a double-stranded DNA provirus U3 sequences are duplicated at the 5' end of the molecule and U5 sequences at the 3' end, so that the coding regions are flanked by two identical long terminal repeats (LTR) in the order 5'-U3-R-U5-3'. Integrated proviruses seemingly always have the structure shown in Fig. 2C, for integration is an event with no known specificity for host sequences but high precision for the provirus. However, subsequent deletion of proviral elements can occur (Section V,B). The LTR is the key to many potential and known attributes of retroviruses. At the end of the LTR are short inverted repeat sequences, and LTR integration at any of many sites in the host generates short direct repeats in adjacent host DNA (Fig. 2B and C). Integrated proviruses are thus anatomically similar to the transposable elements of bacteria and lower eukaryotes. They also show some functional resemblances to these entities, notably in their recombination with host genes, such as c-onc genes (Section IV,A) and in their ability to excise from the genome by homologous recombination

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RNA IR R

R IR

B

B@

4 4 4

4 4 4

IR R IR

Integrated Provirus: Normal transcription

* D a u3

pi

m

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IR R IR

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-

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c - myc

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G

Insertional Mutageneeis : Viral promoters downstream of enhanced c-myc

Insertional Mutagenesis: Viral promoters in opposite orientation to enhanced c - myc

FIG.2. Retroviral regulatory elements and their effect on viral and cell gene expression. Single straight lines represent viral or cell RNA molecules; double straight lines are proviral DNA molecules; double wavy lines are cell DNA. Open boxes show positions of U3 and US sequences; solid wavy boxes denote exons of c-myc gene. R, Direct repeat sequence at termini of viral RNA; IR, inverted repeat sequences at end of U3 and U5; CR, short direct repeat sequences generated in host DNA by proviral integration; P, position of U 3 promoter region that directs viral RNA transcription.

within the LTR (Varmus et al., 1981), an event that may explain the occurrence of isolated LTRs in host DNA (Hughes et al., 1981; E. Keshet, personal communication). It has recently been stressed that genetic changes inherent in neoplasia are more readily explained by shifis in gene organization, such as translocations, than by point mutations (Cairns, 1981; Klein, 1981). The retroviruses combine oncogenic potential with the structural features needed to mediate gene transposition,and it is thus tempting to link these two attributes. There is, in fact, no evidence that retrovirus genomes

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can relocate themselves in the manner of bacterial transposons and hence no reason to suppose that neoplasia results from such transpositions within an individual animal. However, we should not entirely neglect this possibility for, during evolution, retroviruses have transposed may onc genes between host animals and the same transfer might rarely occur within a single organism. A further similarity between retroviruses and transposable elements is in the ability to act as insertion mutagens (Sections I1 and V,B). The mere physical presence of an integrated provirus could have a number of consequences for the host genome. If inserted within a coding region it could affect either transcription or the product of any transcript. Integration in intervening sequences may either reduce or enhance gene expression. Reduced gene espression is seen, for example, when a murine leukemia provirus, inserted within a previously integrated RSV provirus, ablates RSV src gene expression, apparently as a result of extending the “intron” between the 5’ leader of the src mRNA and its coding portion (Varmus ef al., 1981 ; Varmus, 1982). Gene enhancement may occur by a similar physical separation of an exon from linked cell elements that normally reduce its expression. Additional nuances are provided by LTR functions that affect neighboring host genes. Nucleotide tracts in the U3 region resemble two consensus sequences that determine the initiation and polyadenylation of RNA transcripts, and their positions in the LTR relative to the start and termination of transcription suggest that these are their functions. Moreover, the U3 regions from some viruses have been shown by in vitro reconstructions to contain “activator” elements that determine the level of activity of nearby genes (Levinson et al., 1982). Machinery both to promote and to regulate gene transcription thus resides in the LTR and, by duplicating the U3 region at 5’ end of the provirus, transcription of complete genomic RNA under the control of the 5’ promoter is ensured (Fig. 2C). Transcription normally terminates, as shown in Fig. 2C, by direction of the 3’ U3 region. Should termination fail for any reason (for example, if the 3’ LTR is deleted), transcription may continue into host sequences to the right of the provirus (Fig. 2D). Such events have been invoked to explain the first stage of recombination between retroviruses and c-oncgenes (R. Swanstrom, quoted in Varmus, 1982), but more commonly the 3’ LTR, which contains its own U3 promoter, directly mediates transcription of the 3’ R, U5, and flanking host DNA (Fig. 2E). This latter possibility was first reported by Quintrell el al. ( 1980)who observed host RNA sequences in avian sarcoma virus-transformed mammalian cells that hybridized with a probe for the viral U5 region. However, its significance became fully apparent in studies on avian leukosis.

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B. EVIDENCE FOR INSERTIONAL MUTAGENESIS IN AVIANLEUKOSIS Avian leukosis viruses cause mainly B cell lymphomas of long clinical latency in chickens, but some virus strains also frequently induce anemia, osteopetrosis, or kidney tumors (disorders which may appear more rapidly then leukosis), as well as a low incidence of other tumors (Section 111). Since ALV carry no oncogenes, the mechanisms for their tumorigenicity were long unknown, but recent results give an indication of the molecular basis of their pathogenicity, although our understanding is still incomplete. The first asymptomatic signs of ALV-induced neoplastic disease are seen quite rapidly, about 5 weeks after inoculation of virus into hatchling chicks. Multiple follicles of transformed B lymphoblasts appear in the bursa of Fabricius, most of which disappear with the natural involution of the bursa at 5 -6 months. However, a few follicles progress to slow-growing nodules that develop into rapidly growing metastatic lymphomas at 6 - 8 months (Neiman et al., 1980). Despite extensive dissemination of the virus during this gradual development of neoplasia many tumors examined contained only one or a few proviruses, which made them amenable to restriction enzyme analysis. These studies showed that the tumors (and a few premalignant nodules that were examined) contained proviruses at identifiable sites (Fung et al., 1981; Nee1 et al., 1981;Payne et al., 1981). Since provirus integration in the host as a whole can occur at many sites, this implied that the tumors were monoclonal. All the tumors contained some retroviral sequences, but intact proviruses and normal viral gene expression were not needed to maintain the tumor, and may, indeed, have been selected against (Section VI), for the tumor proviruses frequently exhibited deletions. Different tumors, such as nephroblastomas, induced in the same host originated independently from lymphomas but may also be clonal. There was evidence in many lymphomas for virus integration in specific regions of the host DNA, this region being identified later as the vicinity of c-myc, the cell homolog of the transforming gene of MC29 type DLVs. Moreover, most such tumors contained elevated levels of RNA species that hybridized with a probe for c-myc and also with a probe for the U5 region of the virus, but not with probes for other viral regions (Hayward et al., 1981). These RNAs were thus thought to be “downstream” transcripts from a viral promoter, through R, U5, and into c-myc sequences on the 3’ side of the provirus (Fig. 2E). Thus, most avian leukosis was associated with specific promotion, by viral regulatory elements, of a cellular oncogene, and it was concluded that c-myc expression is causally related to lymphomagenesis. This interpretation was strengthened by the demonstration that lymphomas induced by an unrelated avian retrovirus of the reticuloendotheliosis group, chick syncytial

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virus (CSV), were also associated with provirus integration near c-myc. A peculiarity of some CSV-induced tumors was evidence in them for amplification of the chromosomal region containing the provirus and c-myc sequences (Noori-Daloii et al., 1981; see Section V,C,l). The initial studies by Hayward and co-workers consistently found that the activated c-myc sequences were downstream of a viral promoter. However, other workers found a significant minority of lymphomas in which the provirus was integrated near c-myc, but in an arrangement preventing its action as a simple promoter for c-myc transcription: either the provirus was downstream of c-myc sequences (Fig. 2F) or it was upstream but in the opposite transcriptional orientation (Fig. 2G) (Payne et al., 1982). Nonetheless, these lymphomas show enhanced c-myc expression and Payne et al. ( 1982) conclude that the provirus influences c-myc transcription either by disrupting cis-acting cell control mechanisms or by introducing viral activator elements (Levinson et al., 1982; P. Luciw and M. Capecchi, quoted in Payne et al., 1982; Section V,A) that operate in a nonpolar fashion. The concept of neoplasia by c-onc activation was further boosted by studies on ALV-induced erythroblastosis in line 15, chickens. These ALVassociated erythroid tumors are monoclonal (unlike erythroblastosis due to the acutely oncogenic AEV), and they contain proviruses integrated near c-erb, the cell homolog of the AEV oncogene (Fung et al., 1982b). Thus, the phenotypes of the tumors associated with c-onc activation reflect the particular onc sequence involved.

Questions Which Emerge These convincing findings stimulated many attempts to link oncogenesis by nontransforming retroviruses of other species to c-onc enhancement. However, a clear-cut explanation of the pathogenesis of these nonavian viruses has yet to emerge. Indeed, even in the well-documented example of avian leukosis there are outstanding questions. The next few sections pose these questions and mention a few clues provided by recent work the answers, when they come, may be fascinating. a. Why Is c-myc Expression Associated with Bursa1 Lymphomas, While v-myc Activity in DL VsInduces a Diferent Set of Tumors (Section III)? The diverse oncogenic consequences of myc expression in different contexts is, in fact, not only a problem in comparing c-myc with v-myc activity. Even among virus strains carrying v-myc sequences pathogenicity can vary, the most notable example being the contrasting effects on quail of MC29 (which causes few tumors) and MH2 (which is strongly oncogenic) (Linial, 1982). Such heterogeneity could result from differencesbetween the myc sequences involved, from differences in behavior of gag- myc fusion proteins as

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compared to proteins specified only by myc, or from differences between gag- myc fusion proteins that contain different proportions ofgag and myc encoded sequences (see also Section IV,A). One approach to comparing c-myc and v-myc activity has used MC29 mutants that have lost myc-specific sequences (Ramsay and Hayman, 1982; Enrietto and Hayman, 1982) and have a reduced tumorigenicity (Enrietto et al., 1983). A back mutant has regained myc sequences, apparently by recombination with c-myc (Ramsay et al., 1982) but, unlike parental MC29 (which in our chicken strains rapidly induces histiocytic sarcomas, resembling the endotheliomas of other authorities), it causes a high incidence of lymphomas (Hayman et al., 1983a). This suggests that newly acquired c-myc sequences, even when expressed as a gag- myc protein, retain the capacity to induce lymphomas and it implies that the oncogenic spectrum of v-myc-containing DLVs reflects subsequentevolution of myc within the viral genomes. However, the back mutant differs from parental MC29 in its content of gag-specific sequences and we cannot yet exclude the possibility that different recombinations between gag and c-myc lead to differences in tumorigenicity. b. Why Are about 10% of Viral Lymphomas Not Associated with c-myc Activation?We have no clear idea, but there are several possibilities. First, a subset of lymphomas might differ from the majority in some as yet undiscovered fashion, and their genesis may be associated with the activity of a gene other than c-myc. Second the work of Payne et al. (1982) draws attention to the nonpolar enhancing effect of ALV LTRs on cell genes. The discovery of c-myc activation by Hayward et al. (1981) was serendipitous, v-mycbeing one of the small number of availableonc probes that the authors tested. It is thus possible that their readily interpreted findings obscured the enhancement of a nearby gene whose function is crucial to lymphoma development and which, in a proportion of cases, is activated when c-myc is not. The idea that it is not c-myc but some other gene that is causally associated with lymphoid leukosis would fit with the findings of Cooper and Neiman (to be mentioned shortly), but it is not consistent with the findings of Hayman et al. quoted above. A third possibility maintains that c-myc activation is always required to induce lymphoid leukosis and invokes the phenomenon of tumor progression to explain those cases in which c-myc activity is no longer detectable. The work of Cooper and Neiman ( 1980, 1981) supports such ideas. They identified in c-myc-expressinglymphomas, but not in normal tissues, DNA that transforms morphologically recipient NIH/3T3 mouse cells. This transforming DNA contained neither viral LTR nor c-myc sequences, promoting the concept that, although c-mycactivation is needed to induce a preneoplastic or neoplastic change in the bursa, it is subsequent unspecified events that result in the expression of a cell gene capable of transforming

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NIH/3T3 cells (see also Wyke, 1981; Cooper, 1982). These interesting results raise yet more questions, quite apart from the obvious one of the nature of the transforming DNA. For instance, although NIH/3T3 cells seem to be the most sensitive recipients known for transforming DNA from a number of tumors, they still respond to material only from a fraction of tumors tested and it is not clear why viral LTRs and activated c-myc should fail to transform them. In addition, if the DNA that transforms NIH/3T3 cells is also sufficient to transform chicken B lymphocytes, it is surprising that so few chicken lymphomas lack c-myc expression or, indeed, evidence of ALV infection. Nonetheless, the concept of a sequential activation of host genes is congruent with the step-by-step development of lymphoid leukosis from hyperplastic bursa1 follicle through progressively growing nodule to metastatic lymphoma. The lymphoid leukosis DNA that transforms NIH/3T3 cells has now been cloned and it will be informative to compare its expression with that of c-myc during the development of the disease. c. If Viral Activation ofa Host Gene Is Only the First of a Number of Gene Mutations or Promotions That Are Neededfor Neoplasia, Can This Explain the Dificulty in Demonstrating Promoter Insertion or Insertional Mutagenesis by Other Viruses That Lack v-onc Genes? Mouse mammary tumor virus induces clonal carcinomas, some of which show evidence for viral integration in a restricted portion of the host genome (Nusse and Varmus, 1982; C. Dickson and G. Peters, personal communication) and a similar situation may pertain with murine leukemia virus-induced thymomas (unpublished data of several laboratories). Enzootic bovine leukosis, like avian lymphoid leukosis, is an apparently clonal B lymphoma in which bovine leukemia proviruses are invariably found, frequently with deletions in their 5’ portions. However, there is no evidence that they are located in the same region of the host genome in different tumors nor is there detectable transcription of viral-coding sequences, LTRs, or 3’ flanking host sequences (Kettmann et al., 1982). Feline leukemia provides even more difficulties: although some cat lymphomasarcomas are clonal, as judged by the pattern of integrated proviruses, it is not clear that they all are (Casey et al., 1981). Moreover, about one-third of tumors lack detectable exogenous proviral DNA or virus expression (Casey et al., I98 1) despite epidemiological evidence of exposure to virus (Hardy et al., 1980). Feline leukemia virus may cause tumors by an indirect mechanism (Section I1,B) but it is also conceivable with these viruses of mice, cattle, and cats that provirus integration in the vicinity of a host gene leads to its expression (by enhancement rather than direct promotion) setting in train neoplastic events that may become independent of the continued presence of virus. However, it is puzzling that the virus cell interactions in these tumors should be so much more complex than in avian leukosis and, until we have more data, any mechanisms of pathogenesis are possible and any explanations approach sophistry.

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d. Why Are Some Retroviruses Nononcogenic? The DNA of chickens contains a number of endogenous ALV-like retroviruses located at the cellular ev loci. These can be expressed in the bird, notably the virus RAV-0 encoded by the ev-2 locus (Robinson, 1978),and some chicken strains show a RAV-0 viremia. However, RAV-0 is apparently quite innocuous and since, like other retroviruses, it integrates and bears viral promoters in LTRs, this benevolence demands explanation. A contributory factor, but perhaps not the whole answer, lies in the nature of the U3 promoter, which is thought to be much weaker in the nononcogenic viruses. The steady-state levels of mRNA expressed by an endogenous provirus are only one-tenth those of exogenous proviruses (Hayward et al., 1980) and, although it is dangerous to attribute this entirely to differences in promoters, this conclusion is supported by the observation that exogenous viruses grow to a higher titer (Tsichlis and Coffin, 1980). The most telling evidence is provided by a recombinant virus, NTRE-7, in which most of the genome is derived from the nonpathogenic RAV-0 but the U3 region is that of an exogenous RSV. Unlike RAV-0, NTRE-7 replicates to high titer and causes a 16% incidence of tumors in chickens, indicating that the nature of U3 is vital for both growth rate and oncogenicity. It is, however, unlikely that the difference in oncogenicity between RAV-0 and NTRE-7 simply reflects differences in growth rate, and our comprehension of NTRE-7 pathogenicity is still incomplete (Robinson et al., 1982). Moreover, the pattern of tumors induced by NTRE-7 differs from that seen with standard ALVs and these findings address another question which is discussed next. e. Why Do ALV Cause Mainly B Cell Lymphomas? Since ALV can integrate at many sites and we know that there are many c-onc genes, a simple insertional mutagenesis model of neoplasia suggests that ALV should induce many tumors at comparable frequencies. Although other tumors do occur, some at high frequency with different ALV and host combinations, the overall predominance of B cell lymphoma requires clarification. One possibility is that c-myc is the only c-onc whose expression at high level can lead to tumors. This could be either because other c-onc genes are inherently harmless at any level of expression (a point we discuss in Section V,C) or because some constraint is imposed by the postulated series of events between c-onc activation and clinical neoplasia. However, a currently more attractive explanation is that ALV interaction with the host is not as random as is suggested by the lack of demonstrable specificity of proviral integration. The recombinant virus NTRE-7 causes a spectrum of carcinomas, sarcomas, and lymphoid tumors (Robinson et al., 1982): in other words it has the oncogenic potential that one might expect of a virus that randomly enhances c-onc expression. A study of provirus integration in these tumors would be very interesting, for the sarcomas, osteopetrosis, and kidney carcinomas may result from enhancement of novel cell oncogenes.

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Robinson et al. (1982) have compared the structure and pathogenicity of NTRE-7 with other recombinants between endogenous and exogenous viruses and with pathogenic ALVs. They speculate that sequences in ALVs (that are not located in U3 or the env gene) “target” the viruses to induce particular tumors at high frequency in certain host strains. Two targeting mechanisms, that are not mutually exclusive, are hypothesized. (1) Sequences thought to lie near the boundary of the gag and pol genes direct integration preferentially to certain sites in cell DNA: for most ALV one of these sites would be near c-myc. (2) Sequences adjacent to the 3’ LTR respond to tissue-specific transcriptional controls and modulate the action of the U3 promoter in some tissues but not in others. f: Targeting Might Explain, at Least in Part, Two Other Questions. One is the variation between chicken strains in response to a single virus, examples being ( 1) the production by RAV- 1 of both erythroblastosis and lymphomas in line 15, chickens, but only lymphomas in K28 chickens (Crittenden et af., 1979; Robinson et af., 1980); (2) the induction by one strain of avian myeloblastosis-associated virus (MAV-2) of nephroblastomas in one flock (Ogura et al., 1974) but osteopetrosis in another (R. E. Smith, quoted in Graf and Beug, 1978); and (3) the high resistance of line 63 chickens to ALV-induced neoplasia despite ready replication of virus in this strain (Fung et al., 1982a). Such variation could be attributed to differences in the availability of preferred integration sites: in the case of line 63it could be postulated that there are no such sites in the vicinity of any c-onc genes. The second question concerns the mechanism of induction by certain ALVs of high incidences of osteopetrosis (Smith and Moscovici, 1969; Smith et af.,1976)and nephroblastoma (Fourcade et al., 1974; Ogura et af., 1974). Schmidt et af. (1982) studied the oligonucleotides produced by RNase T1 digestion of the RNA of virus strains inducing both high and low incidences of osteopetrosis and they found the two groups differed in oligonucleotides at the 3’ ends of their genomes. It is not clear whether the differences in osteopetrosis-inducing strains are in U3 or adjacent regions, but it is possible that in some way they enable these viruses to target on osteoblasts. Indeed, the incentive to postulate targeting in the pathogenesis of osteopetrosis is higher than in lymphoma: not only can osteopetrosis develop very rapidly, with latency ofless than 3 weeks (Franklin and Martin, 1980), but the virus MAV-2 (0)can induce osteocyte proliferation in vitro (Schmidt and Smith, 1981), a property not exhibited by any other avian retrovirus that lacks v-onc genes. It will be of great interest to elucidate the features at the 3’ ends of osteopetrosis viruses that determine their pathogenicity and to discover whether the tumors they induce are monoclonal. Relevant changes are likely to be subtle, for Smith and Morgan ( 1982) have recently isolated from MAV-2 (0) subclones of virus that differ markedly in

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pathogenicity but show no differences in growth rates or RNA oligonucleotide patterns.

C. THERELATIONSHIP BETWEEN VIRALAND CELLONCOGENES Previous sections of this article have reviewed the known mechanisms by which avian retroviruses cause cancer. In all examples for which we have adequate information neoplasia requires the activity of an onc gene under the influence of incompletely understood factors that regulate virus gene expression. There are many cases in which a v-onc is expressed and a few in which the active gene is the c-onc homolog. The coding sequences of those v-onc and c-onc genes that are known seem closely related and, in the cases of src, ras, andfis, their products seem functionally similar. This has led to speculation that ( 1) c-onc functions are inherently able to lead to neoplasia if the genes are expressed at the wrong level, in the wrong cell, or at the wrong time, and (2) such untoward c-onc expression might be implicated in tumor induction by nonviral oncogenic agents. The most likely means of enhancing c-onc expression is by increasing the gene’s transcription and it follows that oncogenic agents would act by changing the chromosomal environment of c-onc genes (for example by translocations or mutations in regulatory sequences or by effects on chromatin structure) rather than by inducing mutations in the coding regions of these genes. In this section we will survey information pertaining to this important concept -both evidence that inappropriately high c-onc gene expression can per se lead to neoplasia and some reasons why we might at present treat this interpretation with caution. There are four main sets of relevant data. 1. c-onc Activation by Avian Leukosis Viruses (Section V,B) ALV oncogenesis is convincingly associated with c-rnyc (and, less frequently, c-erb) expression. However, the long interval between first signs of bursa1 hyperplasia and eventual clinical neoplasia does provide opportunities for mutation in the relevant c-onc gene and until their functions in normal cells are understood this doubt remains. It is interesting that there is apparent c-myc amplification in some CSV-induced lymphomas (NooriDaloii et al., 1981). Gene amplification not only provides a means of increasing the levels of transcripts without necessarily increasing the transcription rate of any single gene, but it also may place genes in novel chromosomal environments and this may lead to augmented expression [the latter effect could also occur without amplification, an example being the translocations in some human B cell lymphomas that reportedly involve the c-rnyc locus (P. Leder, quoted in Logan and Cairns, 1982)l. It is also possible that amplification increases the chances of a mutation occurring in

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or around one of the c-myc gene copies. However, the significance of amplification is hard to assess since all the individuals of some rodent species show amplification of ras genes (Chattopadhyay et al., 1982) and these species are not known to have a high incidence of neoplasia. 2. The Properties of c-mos and c-ras c-mos, the cell homolog of the v-mos gene of Moloney murine sarcoma virus (MSV), possesses no introns and was readily obtained as a genomic clone in prokaryotes (Oskarsson et al., 1980). When used in transfection assays, c-mos DNA cannot cause morphological transformation, but if a MSV LTR is linked 5' to it the gene transforms efficiently (Blair et al., 1981): if the LTR is 3' to c-mos a low level of transformation is still observed. Similar experiments with the ras gene have shown that both rodent and human c-Ha-ras I sequences (that contain several introns) are able to transform NIH/3T3 cells after linkage to a viral LTR (Chang et al., 1982). Since there is little chance of c-onc mutation in these in vitro experiments they provide the best evidence so far that high expression of a c-om can cause neoplasia. However, these c-oncgenes may be exceptional: others may fail to transform either because they are inherently nontransforming or because of limitations in the response of available recipient cells. 3. Recovered Avian Sarcoma Virus When transformation-defective (td) RSV derivatives lacking a part, but not all, of the src gene are injected into chickens they induce a low incidence of sarcomas. From these tumors transforming viruses can be obtained that possess a complete and fully active src gene (Hanafusa et al., 1977; Halpern et al., 1979; Vigne et al., 1980). Hanafusa and colleagues have produced considerable evidence that these recovered avian sarcoma viruses (rASV) arose by recombination between the input td virus and c-src (Karess et al., 1979, 1980; Wang et al., 1978, 1979; Karess and Hanafusa, 1981; Takeya and Hanafusa, 1982; Takeya et al., 1982), although these conclusions have been disputed (Lee et al., 1981). Thus sarcoma-inducing genes can be obtained, a large part of whose sequence is apparently derived from c-src, but this does not prove the oncogenic potential of c-src. In particular, nucleotide and amino acid sequencing studies have shown that p ~ 6 0 " -and *~~ ~ ~ 6 0have " ~ different ~ " phosphotyrosine-containing peptides (Smart et al., 1981 ;Takeya et al., 1981). These subtle differences in the region thought to be the active site of the protein (Sections IV,A and B) may mean that pp60c-src has a different substrate specificity or responds differently to cellular controls. The mutants used by Hanafusa et al. retained the region encoding the phosphorylated tyrosine of pp60v-src, and phosphopeptide analysis of rASV pp60 showed that they possessed the tyrosine-containing phosphopeptide characteristic of p ~ 6 0 " - and ~ ~ " not that of pp60c-src(Karess and

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Hanafusa, 1981). The behavior of rASV src could thus resemble that of the parental v-src rather than c-src. However, td RSV whose deletions remove the region encoding the phosphorylated tyrosine (Fincham et al., 1982) can also induce a low incidence of chicken sarcomas from which virus possessing a fully active src gene can be recovered (Enrietto, Payne, and Wyke, 1983). Phosphopeptide analysis of the pp60"' encoded by such rASV indicates that the phosphotyrosinecontaining peptide differs from that in RSV, supporting the concept that this region of c-src can donate oncogenic activity to a recombinant src gene. However, this still does not prove that native c-src is oncogenic. The src genes in these rASV may contain short sequences from the 3' end of the v-src coding region and their src-related proteins are also all truncated in their amino terminal half. Thus it is possible that pp6OC-" differs from the rASV product in terminal amino acids. 4. Oncogenes in Naturally Occurring Tumors

The possibility that oncogenic retroviruses have revealed members of a set of host genes whose activity is crucial to neoplasia has stimulated the search for such gene activity in human and animal tumors. Two main approaches have been used: (1 ) the detection, by DNA transfer, of tumor genes that will morphologically transform recipient NIH/3T3 cells, and (2) the search in tumors for c-onc transcripts, using the battery of known v-onc genes as probes. a. Transforming DNA. Several groups have shown that tumor cells contain transforming DNA. However, such DNA has been detected in only a fraction of tumors tested, perhaps because of limitations in the response of the NIH/3T3 cells used as recipients (it would be extremely useful to have other recipient cells to expand the detection of transforming genes). DNA from normal cells transforms very inefficiently, if at all, but Cooper et al. ( 1980) reported that transformation by normal cell DNA was enhanced by shearing it to a small size, thus possibily removing cis-acting cell regulatory elements. By sequential DNA transfer the transforming DNAs have been identified and some have been cloned. It has now been found by several groups that the transforming DNA from some human bladder carcinoma cell lines contains sequences homologous to c-Ha-ras 1, whose viral counterpart is the v-onc of Harvey MSV (Der et al., 1982; Parada et al., 1982; Santos et al., 1982). Moreover, the oncogenes of several other distinct tumors, including human lung carcinomas, appear homologous to v-Ki-ras, the oncogene of Kirsten MSV (Der et al., 1982; Pulciani et al., 1983).These findings are the first suggestions that the onc genes acquired during retrovirus evolution might also be important in the genesis of naturally occurring nonviral tumors. Why do tumors contain transforming DNA whereas normal cells (which

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presumably carry homologous sequences) do not? The difference must lie either in the sequences encoding the transforming product, in regulatory flanking or intervening sequences or both. In the case of the c-Hams I gene of a human bladder carcinoma cell line a mutation in the coding region of the protein, converting the twelfth amino acid from glycine to valine, accounts for its acquisition of transforming ability (Reddy et al., 1982; Tabin et al., 1982). However, in other tumors regulatory sequences may be changed, and if this is the case then either these regulatory sequences are transferred with the coding DNA to maintain its active state or, in the original tumor, they effected an alteration in the chromatin structure of the transforming gene that survived its transfer to recipient cells. A reduction in cytosine methylation (Razin and Riggs, 1980) could be one such alteration. At present there are insufficient data to distinguish these alternatives. The c-mos gene cannot transform without a viral LTR even after cloning in bacteria (and hence loss of all its methylated cytosine) (Blair et al., 198 1). However, c-mos transformation has apparently been tested only in the presence of flanking host DNA and Cooper et al. (1980) suggest that such flanking sequences might inhibit expression. An interesting aside to these findings is that H a m s is associated with erythroleukemias and fibrosarcomas in mice but is linked to bladder carcinogenesisin man. Does this reflect pleiotropism in the c-Hams I function in both man and rat (the apparent species of origin of Ha MSVras), species differences in c-Hams I function, or differences between viral and cell oncogenes? We cannot yet say, but studies on a transforming derivative of a td MC29 mutant provide an informative parallel (Section V,B,la). The MC29 retransformant, HBI, has apparently acquired chick c-myc sequences and induces mainly lymphomas. Its pathogenicity thus differs from that of wild-type MC29 but resembles that of c-myc activated by ALV, suggesting that MC29 v-myc has either undergone mutation since its capture by the virus, or was derived from a species other than chicken. However, neither of these possibilities yet explains why all four MC29 group DLVs show a related pathogenic spectrum, quite distinct from that of ALV-induced

c-myc. b. c-onc Transcripts in tumors. v-onc probes have been used to examine c-onctranscripts in human solid (Eva et al., 1982) and hemopoietic (Westin et al., 1982) tumor lines. As with many studies on normal cells, the expression of some c-onc genes is more widespread than that of others, but there is no general or specific correlation between neoplasia and the levels of any particular onc transcripts. We can thus draw no conclusions, but must stress again that overall c-onc expression cannot be interpreted given our ignorance of the onc products and their targets. An interesting finding is that a human promyelocytic leukemia cell line,

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HL60, shows an elevated expression of c-myc associated with amplification of c-myc sequences (Collins and Groudine, 1982; Favera et al., 1982). This recalls the c-myc amplification in CSV-induced lymphoma (Section V,C, 1). VI. The Host Response

We have considered so far the interactions between oncogenic retroviruses and host cells that render these cells neoplastic. Whether or not the descendants of such cells succeed in forming a tumor depends on the many host factors mentioned in Section I. Space does not permit a lengthy examination of these factors and, indeed, there is little information on some of them. We should, however, consider briefly the responses of the host reticuloendothelial and hemopoietic systems, since these are germane to avian viral neoplasms and associated diseases. These cells are not only the targets for neoplastic conversion by many viruses but various compartments of these systems may be either stimulated or depressed by retrovirus infection. Interactions between such responses may have a complex outcome, greatly affecting the resultant disease patterns. In Section I1 we mentioned the postulated role of viral antigens in the genesis of murine leukemia and it is clear that avian viral antigens also stimulate lymphocyte mitogenesis (Wainberg and Israel, 1982). Although this is not known to play any part in lymphoid leukosis it is probably important in the frequently recorded regression of RSV-induced sarcomas (Wainberg et al., 1979). The observation that both RSV sarcomas (Wainberg et al., 1977) and ALV lymphomas (Section V,B), frequently contain defective viruses may attest to the effectiveness of this host response. Immune responses against viral and possibly other antigens may also introduce an autoimmune component into many retrovirus-induced syndromes. This aspect has not been well studied in birds, but it may play a role in the anemia and nephritis frequently seen in ALV-infected fowl. A depressant effect on host cell populations is shown particularly by the REV complex and by envelope subgroup B, D, and F ALV, agents that can be cytotoxic in vitro. For instance, immunesuppression by ALV is most marked among B, D, and F subgroups and may be due to cytotoxicity (Rup et al., 1982). In contrast, nondefective REV cause an immunesuppression, that may exacerbate the oncogenic effect of coinfecting REV-T, and that seems to involve activation of spleen suppressor cells (Rup et al., 1979). ALV-induced osteopetrosis provides another example in which, although the disease is primarily caused by osteoblast proliferation, a suppression of monocytes (and hence osteoclasts) may also play some part (Smith and Ivanyi, 1980). The complex effects of these various factors is illustrated by the roles they

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may play in ALV-associated anemia, a disease of unknown pathogenesis. First, the anemia might have an aplastic component, either because a condition such as osteopetrosis obliterates the bone marrow cavity or as a result of an erythroblastosis that imposes additionally a block on erythrocyte maturation. However, anemia can be induced by ALV before any neoplastic changes occur, and again subgroup B, D, and F viruses are the most pathogenic (Graf et al., 1976; Smith and Schmidt, 1982). Thus it seems that hemolytic anemias can occur, either by direct cytotoxicity or by an autoimmune mechanism. The latter, in turn, is clearly subject to influence both by cytotoxic effects and by any concomitant virus-induced hyperplasia or neoplasia of immune reactive cells. These complexities of host response clearly add greatly to the pathological heterogeneity of retrovirus-induced disease. A full understanding of them will commensurately simplify the task of elucidating the role in neoplasia of the basic virus-cell interaction. VII. Conclusions and Prospects

A. THENATUREOF ONCOGENES

We have surveyed the evidence that retrovirus-induced tumors in birds and other species result from the activity of v-onc or c-onc genes. Moreover, it is now apparent that c-onc expression may be important in the genesis of tumors of nonviral origin. This suggests that our knowledge of the pathogenesis of all neoplasia may be furthered by a complete understandingof the cellular roles of oncogenes. The first steps toward this understanding, identifying the structure and primary function of v-onc and c-onc genes, are now in progress and shduld readily be achieved. Our catalogue of onc genes may be expanded by investigations of animal cancers, particularly unusual tumors caused by nontransforming viruses in which a novel onc gene might be captured by a virus or stimulated by proviral insertions. Concurrently, classic genetic and biochemical analyses of the known onc genes will be accelerated by molecular cloning and sequencing of the genes, which will permit (1) use of the deduced amino acid sequences of the onc products to prepare specific probes to study these proteins within the cell and (2) mutation in vitro of the genes to investigate their function.

B. THECONSEQUENCES OF onc ACTIVITY IN THE CELL The next step in understanding onc genes must look at their behavior in normal and neoplastic cells. These studies, now beginning, fall into two categories that comprise the two major questions of contemporary tumor

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virology: (1) where and when are onc genes expressed? (2) What are the cellular processes in which they participate? 1. The Regulation of Oncogene Expression Once probes for onc proteins have been obtained the descriptive part of this question, localizing onc products, will be limited only by the cell biologist’s ability to distinguish cell types and to separate them into subcellular components (see, for example, Miiller et al., 1982). However, more attention will now be paid to elucidating and manipulating the factors that determine onc expression, probably at the transcriptional level, since disruption of such factors could lead to the untoward onc activity implicated in some nonviral neoplasia. Such studies at present are largely with related model systems (Varmus, 1982): for example, regulation of natural or experimentally introduced endogenous proviruses (Breindl et al., 1980;Groudine et al., 1981; Jaenisch et al., 1981; Stuhlmann et al., 1981)or of morphological reversion of virally transformed cells (Chiswell et al., 1982a,b; Dyson et al., 1982). However, it should be possible to study directly control of cell oncogenes, as suggested by the experiments of Cooper et af. (1980) that indicated their regulation by cis-acting factors. The concepts behind such work may be those prevalent in other studies on the control of gene expression but, unlike many other genes popular for such investigations, oncogenes have the attraction of being themselves entities that may be pervasively involved in cell regulatory processes. 2. The Cell Targetsfor Oncogene Activity We have emphasized that, to understand the pathogenesis of neoplasia in molecular terms, it is essential to unravel all the metabolic interactions of oncogenes. Once a function is assigned to an onc product such studies can begin, but the precedent of work on pp60”” (Section IV,B) suggests that numerous potential targets will be revealed and tools will be needed to determine their relevance. Virus mutants with partial transformation defects are proving useful and site-directed mutagenesis will increase the precision of this approach. However, studies with defective viruses have limitations and other strategies must be sought, two of which are now apparent. It may be possible to obtain cell mutants that do not respond to oncogene activity, They may, for example, be detected as revertants of virally transformed cells that are resistant to transformation by superinfecting virus and in which the v-onc product is present and nondefective. Cells that, by some criteria, may be in this category have been described (Lau et al., 1980) but not yet studied in depth. A disincentive to this approach is the view that, since oncogenes are conserved phylogenetically, they are involved in essen-

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tial cell activities in which mutations will be either lethal or unworkably leaky, The paucity of cells with the appropriate mutant phenotype seems to endorse this view and it may be advisable to seek conditional rather than nonconditional mutations of this type. The other lead to important cell processes may come from work on the tumor-transforming genes that are detected by DNA transfection. The almost universal recipient cell for such work, NIH/3T3, apparently transforms in response to single DNA sequences despite the likelihood that the neoplastic phenotype is acquired in stages, involving the activity of several genes. NIH/3T3 may thus be biased in providing a “window” on the final genetic changes in the path to neoplasia. onc gene expression, as in avian lymphomas, is probably an early stage in this progression (Section V,B) and it is thus significant that only a fraction of the transforming DNA revealed by NIH/3T3 transfection has been found homologous to known onc sequences (Parada ef al., 1982; reviewed by Rigby, 1982). Nonetheless, the genes that transform NIH/3T3 can be cloned and their products detected. Since they may represent the end of a sequence of events initiated by oncogene activity, they should facilitate elucidation of the intermediate stages. C. THEOPTIMIST’S VIEW The approaches outlined above should eventually enable retrovirologists to bridge the gap between the pathology and molecular biology of viral neoplasms. Will this knowledge assist in managing human and animal cancers? Information on the expression of tumor genes may help to predict how they are activated and this may be of diagnostic or prognostic use. Nonetheless, since onc genes are probably crucial to basic cell processes, it may be difficult to use our knowledge therapeutically to devise treatments that diminish onc activity in tumors yet do not simultaneously disrupt normal cell functions (Bishop, 1982). However, a sanguine outlook argues that, although essential to the development of the organism as a whole, a given onc activity is unlikely to be important to all cells at every stage of life. We should learn not only which genes are active in which tumors but also which normal adult cells rely on the unimpeded functioning of onc products. Such knowledge may ultimately permit manipulation of the levels of onc products or their proximal or distal targets in tumor cells, thus suppressing tumor growth or promoting differentiation, yet sparing normal cells. This approach, aimed directly at the factors that determine tumor growth, rather than at the consequences of that growth, may be less cytocidal and less subject than other therapies to the selection of nonresponsive variant cells. At the least it may be a useful adjuvant to the present therapeutic rationale of eliminating from the body as many tumor cells as possible.

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ADJUVANT CHEMOTHERAPY FOR COMMON SOLID TUMORS David A. Berstock. and Michael Baurn Kings College Hospital Medical School, London, England

............................... 1. Introduction ............................. ............................... 11. Selection of Patients ..................... 111. Selection of Drugs ............................................................. 1v. V. VI. VII. VIII. IX. X. XI.

Toxicity. ............................................................. .. Breast Cancer.. ................................................................ Malignant Melanoma. ......................................................... Lung Cancer.. ................................................................. Colorectal Cancer. ............................................................. Osteogenic Sarcoma ........................................................... Ovarian Carcinoma.. .......................................................... Discussion ..................................................................... References .....................................................................

315 316 3 16 316 3 17 32 1 323 323 324 325 325 326

I. Introduction

Most clinicians would accept the view that the majority of common solid tumors are systemic diseases at the time of diagnosis. This is borne out by the fact that more than 509/0 of patients presenting with solid tumors which appear to be localized at the time of diagnosis eventually develop metastatic disease however “perfectly” the primary tumor is removed. The possibility that cancer is a systemic disease was first discussed in 1802 at a medical committee meeting where John Abernethy and Matthew Baillie et al. discussed the question “May cancer be regarded at any period or under any circumstances merely as local disease, or does the existence of cancer in one part afford a presumption that there is a tendency to a familiar alteration in other parts of the animal?” The result of their deliberations was published in July 1806 in the Edinburgh Medical Journal and extensively cited by Shimkin (1957). Adjuvant systemic therapy of one form or another is therefore an extremely attractive concept. In theory it represents the only way in which the outlook can be improved in the treatment of patients presenting with neoplastic lesions that appear to be localized. Unfortunately, our present state of knowledge as regards both the behavior of malignant cells and also the drugs available to eradicate these cells from the organism is extremely limited. Because of this a number of problems are encountered when attempting to devise methods of adjuvant systemic therapy: first, the selec*Present address: Clatterbridge Hospital, Bebington, Wirral, Merseyside L63 4JY, England. 315 ADVANCES IN CANCER RESEARCH. VOL. 39

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN-0- 12-006639-4

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tion of patients; second, the selection of drugs; and third, the toxicity, both long and short term, associated with the use of these drugs. 11. Selection of Patients

Because of the nature of the currently available chemotherapeutic agents it is important to select those patients who are at highest risk of developing metastatic disease so as to avold using toxic agents on patients who do not require any further treatment. In general, it would appear that the best indicator of “the high risk” patient is the involvement of regional lymph nodes with metastatic tumor cells. Other methods of selecting high risk patients involve histological criteria. The degree of differentiation of certain tumors correlates well with survival, but this is by no means true of all solid tumors. As our basic knowledge of the neoplastic processes improves it may be possible to select patients for adjuvant therapy on the basis of more sophisticated and sensitive techniques, such as the presence of tumor-associated markers in either blood or urine or the cell surface characteristics identified using batteries of monoclonal antibodies. 111. Selection of Drugs

The selection of drugs for use as adjuvant therapy is usually based on evidence for response in the advanced disease. This would seem a logical approach, but is not necessarily the correct one. It may be that the behavior of circulating clumps of tumor cells or micrometastases is quite different from that of advanced disease and therefore either the response to, or bioavailability of, such drugs may be altered in the different stages of the disease. Furthermore, unlike the chemotherapy employed against bacteria, in vitro assessment of sensitivity of tumor cells to chemotherapeutic agents has proved unrewarding. IV. Toxicity

The toxic effects of chemotherapeutic agents is a major consideration when dealing with patients who have no overt evidence of any disease and who in general feel perfectly well. In addition, no matter how good our selection of high risk patients, there may be a group of patients who will not benefit from any form of systemic therapy and therefore the toxic effects, both long and short term, will have been inflicted without therapeutic gain. The short-term effects of the various drugs in common use range from the mild constitutional upsets to severe debilitating illnesses characterized by nausea, vomiting, diarrhea, alopecia, leukopenia, and thrombocytopenia. A number of studies of adjuvant chemotherapy have reported deaths directly

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attributable to the drugs. In addition to these short-term toxic effects, there is the problem of the long-term toxicity. Adjuvant chemotherapy trials will, however, produce data concerning this problem as their follow-up extends beyond 10 years. However, some of the agents commonly used are known to be carcinogenic (e.g., cyclophosphamide and bladder cancer, Calvert, 1981; L-PAM and acute leukemias, IARC, 1975). Others produce different and specific long-term toxic effects, for example, adriamycin-induced cardiomyopathy and vincristine-induced neurotoxicity. A further question regarding adjuvant chemotherapy must be considered, that is, whether there is any advantage of treating occult metastases “prophylactically” over waiting for clinically detectable metastic disease to become apparent. Were adjuvant chemotherapy proven to be of benefit in terms of survival, then this question would undoubtedly be easy to answer. However, if the results of adjuvant chemotherapy are positive only in terms of increased disease-free survival, then it may be that waiting for the disease to appear would produce just as good overall results, with possibly a better quality of life for the majority of patients in the interim. Many cancer types are undergoing studies involving adjuvant chemotherapy. The most extensively studied of these is undoubtedly breast cancer and the role of adjuvant chemotherapy in the treatment of early breast cancer will be extensively discussed in this article as there are a number of lessons to be learned in studying the way in which adjuvant therapy for breast cancer has evolved which will apply to future studies concering other common tumor sites. V. Breast Cancer

It was not until 1958 that the use of systemic chemotherapy was tested in a scientific manner, i.e., in a controlled, prospective, randomized trial. Since then a minor explosion of clinical trials of adjuvant chemotherapy in early breast cancer has appeared in the literature. Unfortunately in spite of the vast amount of work required to initiate these trials, the majority of them were commenced under an enormous handicap. This proved so overwhelming that many of the trials closed prematurely and others, while continuing on, stood no chance of adding to our knowledge. This handicap was simply the inadequate number of patients who were recruited into the trials, resulting in a failure to detect any difference in population survival even if such differences existed (Peto et al., 1976). It is, therefore, in this context that perhaps the most important lessons with regard to the study of adjuvant chemotherapy can be learned: trials must contain large enough numbers of patients to detect the relatively modest differences in survival that we can realistically hope to achieve with our present chemotherapy

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regimens. Breast cancer is one of the most common tumors, but in spite of this fact it is still a major task to recruit the numbers of patients required to demonstrate important benefits. Therefore, when looking at the less common tumors, it is hardly surprising that trials containing 50 or 100 patients are reported as showing no effect of treatment. The National Surgical Adjuvant Breast Project (NSABP)began studying the role of adjuvant chemotherapy in 1958 when the first patient was entered in a trial ofthiotepa (see Fisher et al., 1975).In this trial women were randomized in a double-blind fashion between two treatment groupsconventional Halsted radical mastectomy plus thiotepa and radical mastectomy alone. The drug was given intravenously on the day of the operation and on the following 2 days to a total of either 0.8 or 0.6 mg/kg body weight. Eight hundred and twenty-six women with early carcinoma of the breast were randomized in this study, but no difference was found in the overall survival rate between the control and the treatment group. An interesting finding, however, was that in premenopausal patients with four or more positive nodes in their axilla there was a delay in the appearance of recurrent disease. It was apparent only up until the thirty-sixth postoperative month, following which the recurrence-free rate was no longer significantly different. In addition, in this group of premenopausal patients with four or more positive nodes there was a significantly greater survival rate than in the control group at 5 years, but the differences, while still being present at 10 years, no longer attained statistical significance. On completion of this thiotepa trial in I96 1 the same investigators began a new study. This was designed to compare the value of 5-fluorouracil(5FU) with thiotepa as an adjuvant to surgery (Fisher, 1977). Again, only a short course of thiotepa, 0.8 mg/kg body weight, was employed as in the first trial and this was compared with 5FU, 25 mg/kg intravenously, on each 4 consecutive days beginning 7 days postoperatively. The results again demonstrated the benefits for a subgroup treated with thiotepa. Premenopausal patients with greater than four positive axillary nodes had less recurrence than untreated patients. The addition of 5FU,however, did not improve the results in the recurrence rates or survival rates which were equivalent to placebo. In addition, 42%of the patients receiving 5FU became leukopenic and a number of deaths were reported in the 5FU group within 60 days of operation. These disappointing results with 5FU did not deter the NSABP from continuing their work with adjuvant chemotherapy and in I972 a trial was designed to compare the effectivenessof a single agent, L-phenylalanine mustard (L-PAM),with placebo. These patients assigned to L-PAMreceived 0.15 mg/kg/day orally for 5 consecutive days every 6 weeks, starting their treatment between 2 and 4 weeks postoperatively. The drug was continued for 2 years or until there was evidence of treatment failure, with dosage

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modification according to the presence oftoxicity. On completion of patient accrual into the trial, a further study was undertaken in which patients were randomized so that they receive either L-PAMor L-PAM plus 5FU. In 1977 a third drug was added, so that the patients were randomized between L-PAM plus 5FU or L-PAM plus 5FU and methotrexate (Fisher et al., 1980). Analysis of the disease-free survival in the three protocols demonstrated a significant improvement in the single agent, L-PAM, compared with the control group. The differencein survival was only of the order O f 5% at 5 years and a further slight improvement in survival when one or both of the extra agents were used. This difference in the disease-free survival was more marked when patients less than or equal to 49 years of age were analyzed separately. In this group the difference between placebo and single agent L-PAM was of the order of 20%at 5 years, but the addition of further agents failed to enhance the survival significantly. It is interesting to note that with this younger group of patients the difference in survival when there were four or more nodes found to be positive was greatly reduced, but still attained statistical significance, both with the single agent and multiple agent chemotherapy. The NSABP group were not the only ones involved in trials of adjuvant chemotherapy in the 1960s. The Scandinavian adjuvant study group began a cooperative clinical trial in 1965 which involved 11 hospitals in Finland, Norway, and Sweden (Nissen-Meyer, 1982). This trial was designed to evaluate the effect of a short course of cyclophosphamideafter mastectomy in early breast cancer. Patients were randomized to either cyclophosphamide, 5 mg/kg body weight, intravenously daily for 6 days starting on the day of operation or no treatment. This protocol was followed in 10out of the 1 1 hospitals. In the eleventh hospital the patients received postoperative radiotherapy after surgical treatment and randomization and administration of chemotherapy took place between 2 and 4 weeks after surgery. One criticism of the trial was that each hospital was permitted to employ its own standard method of surgical, radiological,or endocrinologicaltreatment, so variations in management between hospitals (but not within) was found among the patients studied. The trial organizers argued, not unreasonably, that these variations were of minor importance in view of the fact that each hospital was providing an equal number of patients to the chemotherapy and control groups and, therefore, the overall analysis would be evaluating only the effect of cyclophosphamide. When all the patients were analyzed (1 136) there was a highly significant difference with regard to disease-free survival with a significant improvement in actual survival of the order of 10% at 15 years. If the results of this study are analyzed with regard to menopausal status, it appears that the effect of this short-term chemotherapy course is as good in the postmenopausal patients as in the younger

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patients, suggesting that as far as a short course of cyclophosphamide is concerned, then a putative chemotherapy-induced ovarian suppression could not account for the improved survival. The next important study to consider was started by the National Cancer Institute in Milan in 1973 (Bonadonna et al., 1976).This was designed to evaluate the effects of cyclophosphamide, methotrexate, and 5FU combined (CMF) as long-term cyclical therapy. Patients were randomized following radical mastectomy, initially to receive either 12 cycles of CMF or no treatment and later to receive either 12 cycles ofCMF or 6 cycles of CMF. The early results of this trial were extremely promising and by 14 months after surgery 2090 of the control patients demonstrated treatment failure but only 5.3% of the treatment group showed any recurrence. At the time these results were heralded as a major advance in the treatment of early breast cancer but, as will be seen, the premature reporting of trial data can be very misleading and, in addition, this improvement in recurrence-free survival was not attained without severe side effects. Alopecia occurred in more than 50% of patients, cystitis in 309/0, and nausea and vomiting in the majority. Also there were some quite marked myelosuppression, although this approached dangerous levels only in a small percentage of patients. Furthermore, it was not surprising to find that many of the patients could not tolerate this regimen and a large number discontinued therapy or continued at a reduced dosage. More recent results from this study (Bonadonna and Valagussa, 198 I) show that the adjuvant chemotherapy was significantly superior to control in the premenopausal group only (64Yo disease-free survival for treatment group versus 43%disease-free survival for the control group). There was no significant difference in survival in the postmenopausal women. In the latest publication from this study patients have been stratified into those receiving 85% or more of the prescribed drug, those receiving 65- 84% of the prescribed drug, and those receiving less than 65% of the prescribed drug (Bonadonna and Valagussa, I981). By employing this retrospective stratification, the authors have identified a group of both premenopausal and postmenopausal women who have done particularly well, that is, those women who received 85% or more of the prescribed therapy. Such retrospective stratification is potentialy hazardous when analyzing the results of randomized prospective clinical trials and will often generate misleading information, viz. Coronary Drug Research Group ( 1980). For instance, one could argue that failure to tolerate chemotherapy may be an early manifestation of visceral or bone marrow metastases. If this were the case, then those patients tolerating the majority of the drug would continue to exhibit improved survival characteristics and this difference would become more apparent as the years went by. In order to demonstrate conclusively that the benefits of adjuvant chemotherapy are dose respon-

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32 1

sive, trials need to be designed to compare low-dose and high-dose regimens randomly. One further reference to adjuvant chemotherapy in early breast cancer concerns the work of Cooper et af. (1979). This study does not contain a large number of patients, nor is it a randomized trial. It is considered here because the work is frequently quoted by the proponents of aggressive combination chemotherapy used after mastectomy and it serves to illustrate how little we may learn from a study which uses historical controls. Cooper’s group studied a total of 100 patients with four or more positive axillary nodes. They were treated for 9 months postoperatively with vincristine. prednisolone, cyclophosphamide, and methotrexate and 5FU. Seventythree of these women received adjuvant chemotherapy starting immediately after surgery and the remaining 27 patients began their chemotherapy after radiotherapy. The disease-free survival curve was compared with the large numbers of historical controls and the group of patients who started their chemotherapy immediately after surgery fared far better than the historical controls. The authors confine their argument in favor of this chemotherapy regime to those 73 women whose chemotherapy began immediately after surgery, who appear to be doing remarkably well. What is of equal importance is that the remaining 27 behaved considerably worse than the historical controls. It must be emphasized at this point that some form of selection must have been operating in choosing patients who were not to have postoperative radiotherapy, making them the more favorable group to begin with. There have been a large number of other adjuvant chemotherapy trials in early breast cancer, but the majority of these contained small numbers of patients and have failed to demonstrate a clear benefit from one form of treatment or another. Of course, this does not imply that such a benefit does not exist and in many ways one could regard those patients involved in these smaller studies as having been wasted in terms of clinical material with which to advance our knowledge in the subject. In addition there have been large numbers of studies using historical controls which demonstrate to their authors’ entire satisfaction the ineluctable truth concerning the benefits of aggressive adjuvant therapy with multiple drugs with or without “immunotherapy” (Buzdar et al., 1978; Cooper et al., 1979; Jones and Salmon, 1979). VI. Malignant Melanoma

Because of the extremely poor prognosis associated with the more advanced stages of malignant melanoma, it would appear to be an ideal candidate for the study of adjuvant chemotherapy. However, it is not a very common disease worldwide and it is therefore difficult to include a large

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number of patients in any one study. In spite of this, there have been a number of studies of adjuvant chemotherapy in this condition. The drug most commonly used, because of its “effectiveness” in disseminated disease, has been dimethyltriazemoimidazole carboxamide (DTIC). The Central Oncology Group evaluated the use of DTIC in a prospective trial of 194 postoperative patients with melanoma (Hill et al., 1981). The treatment group received four doses of DTIC in 12 months at a dose of 4.5 mg/kg over 10 days. The control group had no further treatment. At a median follow-up of 2.5 years 4590 of the control patients were disease free compared with 28% of the DTIC patients. In addition, 4490 of the control group patients had died compared with 5 1% of the DTIC group. Nearly all the patients receiving DTIC exhibited toxicity, including nausea and vomiting, although there were no fatalities directly ascribed to the drug theapy. It is difficult to draw any conclusions from this study owing to the small number of patients involved and, while at first glance one may jump to the conclusion that DTIC was of no benefit or even harmful to the patients, such a conclusion may well be false. Another drug which has shown to be of benefit in disseminated melanoma is methyl-CCNU. In 1975 a prospective randomized trial to determine whether adjuvant immunotherapy or chemotherapy would prolong the disease-free interval and survival of patients with melanoma was begun by the National Cancer Institute (R. I. Fischer et al., 1981). The patients were randomized to receive either no further treatment, Bacillus CalmetteG d r i n (BCG), BCG plus neuraminidase-treated allogeneic tissue cultured melanoma cells, or methyl-CCNU. One hundred and eighty-one patients have entered the study with a median follow-up time of approximately 29 months. Only 166 of these patients have been considered to be fully evaluable. The remaining 15 patients were excluded because they developed documented recurrence within 2 months of randomization and it was assumed therefore that they may have had recurrent disease at the initiation of the study. Exclusion of patients on these grounds is of dubious validity, but the authors do state that the conclusions of the study were unchanged whether or not the 15 unevaluable patients were included in the final analysis. No statistical difference in survival or time to recurrence was found between the four treatment groups. This, again, is to be expected when such small number of patients are randomized in four separate ways. The toxic effects of methyl-CCNU were experienced by nearly all the patients taking the drug and, while no patients died of toxicity, many had enough marrow suppression to cause the next dose of methyl-CCNU to be delayed by 1 or 2 weeks until the blood count returned to normal. In addition, 3 of the 25 patients who received greater than 1200 mg/m3 of methyl-CCNU developed renal failure reauirine. dialysis.

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VII. Lung Cancer

This tumor, being one of the commonest tumors in males and carrying with it an extremely poor prognosis, is an ideal candidate for study in the adjuvant setting. However, a survey done by Tate et al. (1 979) showed that in the United Kingdom only 2% of patients presenting with lung cancer were included in clinical trials of any nature and the majority of work done on adjuvant chemotherapy in this disease has come from the Veterans Administration Group (Higgins, 1972; Shilds and Keehn, 1977).They have studied single agent chemotherapy, such as cyclophosphamide, nitrogen mustard, and methotrexate, as these drugs have been shown to have a modest benefit in the advanced disease. The results of the various trials have on the whole been negative. However, combination chemotherapy regimens have been developed to produce much higher response rates in the advanced disease and it is important that these or similar regimens are subjected to appropriate trials. Some encouraging results from Takita et al. ( 1979)have shown that combination chemotherapy given preoperatively to patients with inoperable disease has converted the lesion to a resectable mass. VIII. Colorectal Cancer

This disease is the commonest malignant tumor in man (Silverberg, 1978) and is the second most common cause of death from cancers in the United States. Unfortunately, one of the major problems in determining whether adjuvant chemotherapy is of value in this condition is the lack of agents to which the tumor is sensitive. Over the years 5FU has been used in the management of advanced disease, but with rather poor overall response rates of approximately 20%. In addition, there is little evidence that this drug increases overall survival time when used in this situation (Moertel, 1975). Early adjuvant trials using thiotepa (Dixon et al., 1971) and 5FU (Dwight et al., 1969) given in small doses perioperatively showed no significant differences in survival rates between treatment and randomized control groups. The Veterans Administration Surgical Oncology Group (Higgins et al., 1981) then began a further study, referred to as the prolonged intermittent therapy trial, which included only patients in whom pathological study of the specimen suggested a high possibility of postoperative recurrence. These involved patients falling mainly into Duke’s grade C tumors. The patients were then divided into two groups- those that were thought to have had a curative resection and those that were thought to have had a palliative resection. Six hundred and seventy-seven patients were included in the study and randomized either to be given 5FU in sequential courses at 6- to 8-week intervals or to a control group. Patients in the “curative” group received the drug course over an 18-month period and those in the “pallia-

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tive” group continued until death or until evidence of disease progression. Survival at 5 years in the curative group was 49% for the patients receiving the drug compared with 44.7%for those treated by surgery alone. Survival at 18 months in the group having palliative resection was 38%for the patients receiving the drug compared with 27% for the controls. Neither of these differences reached statistical significance but here again we see an encouraging but far from conclusive result in favor of adjuvant chemotherapy in a study with the likelihood of an important type I1 error. A further trial by the same group was then commenced ( H i e n s et al., 198 l), randomizing their high risk colorectal cancer patients (mainly colon cancer) to a combination of methyl-CCNU and 5NU. Six hundred and fifty-four patients have been entered into this trial, with approximately 5 1% reaching the 5-year follow-up period. No therapeutic benefit has as yet been reported from the use of this combination and should this negative trend continue then this would of course make results of the previous 5FU trials (using single agents) even more difficult to interpret. IX. Osteogenic Sarcoma

Although this is a relatively rare tumor it is discussed in some detail here because it is interesting to see how the use of adjuvant chemotherapy in this disease has become accepted practice in the absence of convincing scientific data to justify its use. A number of trials have been performed using adjuvant chemotherapy and comparing the treated group with historical controls (Cortes et al., 1974; Jaffe et al., 1974). It is this disease more than any other which emphasizes the fallacy of the utilization of historical controls because in a study performed at the Mayo Clinic (Rab et al., 1976; Taylor et al., 1978) it was found that a recent change in the natural history of osteogenic sarcoma between 1963 and 1974 had occurred and could explain the apparent benefit of the new treatment. The 2-year disease-free survival rate of their untreated controls in this period improved from 25 to 50%.The reasons for this were not clear, but they seemed unrelated to patient selection or modern preoperative methods of evaluation. A further problem when studying osteogenic sarcoma and its response to adjuvant chemotherapy is that various factors have been identified in patients that have a marked bearing on prognosis, including histological classification and site of the primary tumor. It is therefore a tumor with many different prognostic variables and one whose natural history has been shown to be changeable and yet it is a disease where adjuvant chemotherapy has in general been adopted. The only possible explanation for this phenomenon is the uncritical adoption of an attractive but unproved new hypothesis for a disease with tragic consequences occurring in fit young people. Activity cannot be equated with progress.

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X. Ovarian Carcinoma

Here again, the role of adjuvant chemotherapy has not been clearly defined. Several large trials have been undertaken, but long-term follow-up is inadequate at this point to establish whether or not chemotherapy has a place in the management of this disease. The Ovarian Cancer Study Group in conjunction with the Gynaecological Oncology Group has two studies in progress at the moment (Longo and Young, 1981). First, a comparison between melphalan and a watch policy in Stage I patients with well or moderately differentiated grades of tumor and, second, comparing melphalan to intracavity radioactive phsophorus in Stage I patients with ascites or tumor outside the ovarian capsule or with poorly differentiated tumor. No survival data are yet available from these studies, although if translation from response rates in the advanced disease can be applied, then there is certainly good reason to continue adjuvant chemotherapy trials, particularly in Stage I1 disease. XI. Discussion

Throughout this article on the role of adjuvant chemotherapy in the different solid tumors, one overriding theme is self-evident. In general there appears to be a modest but as yet unproved benefit to be derived from adjuvant chemotherapy in the majority of these tumors. Perhaps the main reason for the minimal benefit is simply that the agents currently in use are not specific enough in their toxicity to cancer cells and the reason that the benefits still remain in doubt stems from the fact that the trials on the whole have contained too few patients to show them with statistical confidence. Therefore, the overriding question must be, what is the place of adjuvant chemotherapy in present day clinical practice? We reluctantly conclude that adjuvant chemotherapy is still experimental and therefore has a place only in randomized clinical trials until its worth is demonstrated beyond reasonable doubt. Future trials must set out specifically to answer the question of the costs and benefits of adjuvant chemotherapy and, therefore, must, in the context of our present pharmaceutical armamentarium, contain large numbers of patients. In order to attain these numbers it is necessary for centers to join together under the umbrella of single protocols to collect these numbers swiftly. While investigating the role of adjuvant chemotherapy, it is essential to bear in mind the cost, and that not only includes the cost in terms of the patient, i.e., toxicity, patient’s time away from work, etc., but also the cost to the service itself in terms of manpower and resources. One also has to bear in mind the safety factor involved, especially in the use of complicated drug regimens outside the specialist centers. Having considered all these costs,

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they can finally be equated with the magnitude of benefit adjuvant chemotherapy affords the patient. It will then be incumbent on the clinician to make up his own mind about the use of adjuavent chemotherapy, but until these data are at hand he cannot be expected to make a rational judgment. Meanwhile, there are a number of ways in which the costs can be reduced. The most important consideration has to be the improved selection of patients. Our present methods involve selection of patients on the basis of lymph node involvement, vascular invasion, or histological grading. Tumor markers, such as CEA in colorectal cancer and human chorionic gonadotropin in chorion carcinoma, have been used to identify patients with residual disease after surgery. Unfortunately, in the majority of solid tumors reliable tumor markers are not available. Again, looking into the future, perhaps monoclonal antibodies may help in the identification of clinically significant tumor markers and they may also play a role on the therapeutic side when linked with cytotoxic agents. A further reduction in the “costs” may well result from the continuous search for alternative agents to be used in adjuvant regimens. A prime example of this appears in the adjuvant treatment of breast cancer, whereby the antiestrogen, tamoxifen (Nolvadex), is being compared with cytotoxic chemotherapy. A drug such as Nolvadex, if proven to be of equal benefit in terms of survival, would represent a major advance in view of its negligible toxicity. In summary, therefore, the immediate future of adjuvant chemotherapy must rest with the introduction of well-designed randomized trials to clarify and quantify the therapeutic benefits. Second, our efforts must be directed toward better selection of patients and, finally, improvement in the selection of treatment schedules with the ultimate aim of reducing toxicity while maintaining any therapeutic benefit.

REFERENCES Bonadonna, G., and Valagussa, P. (198 I ) . Dose-response effect ofadjuvant chemotherapy in breast cancer. N. Engl. J. Med. 304, 10- 15. Bonadonna, G., Valagussa, P., Rossi, A., et ul. (1976). Combination chemotherapy as an adjuvant treatment in operable breast cancer. N . Engl. J. Med. 294,405-410. Buzdar, A. U., Gutterman, J. U., Blumenschein. G. R., et al. (1978). Intensive post-operative chemoimmunotherapy for patients with stage I1 and stage 111 breast cancer. Cancer 41, 1064- 1075. Calvert, A. H. (1981). The long term sequelae of cytotoxic therapy. Cancer Top. 3,77- 79. Cooper, R. G., Holland, J. F., and Glidewell, 0. (1979). Adjuvant chemotherapy of breast cancer. Cancer 44,793-798. Coronary Drug Project Research Group (1980). Influence of adherence to treatment and response of cholesterol on mortality in the Coronary Drug Project. N . Engl. J. Med. 303, 1038- 1041. Cortes, E. P., Holland, J. F., Wang, J. J., eta/. (1974). Amputation and Adriamycin in primary osteosarcoma. N. Engl. J. Med. 291,998. Dixon. W. J., Longmire, W. P., and Holden, W. D. (197 I). Use of triethylthiophosphoramide

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as an adjuvant to the surgical resection of gastric and colorectal carcinoma. Ann. Surg. 173,26. Dwight, R. W., Higgins, G. A., and Keehn, R. J. (1969). Factors influencing survival after resection in cancer of the colon and rectum. A m . J. Surg. 117,5 12. Fisher, B. (1977). Attempts at combined modality therapy. In “Breast Cancer Management Early and Late” (B. A. Stoll, ed.), pp. 53-66. Heinemann, London. Fisher, B., Slack, N., Katrych, D., and Wolmark, N. (1975). Ten year follow-up results of patients with carcinoma of the breast in a co-operative clinical trial evaluating surgical adjuvant chemotherapy. Surg. Gynaecol. Obstet. 140, 528 -534. Fisher, B., Redmond, C., Fisher, E. R., and participating NSABP investigators. (1980). The contribution of recent NSABP clinical trials of primary breast cancer therapy to an understanding of tumor biology-an overview of findings. Cancer 46, 1009- 1025. Fisher, R. I., Terry, W. D., Hodes, R. J., et a/. (1981). Adjuvant Immunotherapy or Chemotherapy for Malignant Melanoma. Surg. Clin. N. A m . 61, 1267- 1288. Higgins, G. A. ( 1 972). Use of chemotherapy as an adjunct to surgery for bronchogenic carcinoma. Cancer 30, 1383. Higgins, G. A., Donaldson, R. C., and Humphrey, E. W. (1981). Adjuvant therapy for large bowel cancer- Update of Veterans Administration Surgical Oncology Group Trials. Surg. Clin. N. A m . 61, 13 I I - 1320. Hill, G. T., Moss, S . E., Golomb, F. M., et al. (1981). DTIC and combination therapy for melamona. 111. DTIC (NSC 45388) Surgical Adjuvant COG Protocol 7040. Cancer 47, 2556. IARC ( 1975). “Monograph on the Evaluation of the Carcinogenic Risk of Chemicals to Man,” Vol. 9. International Agency for Research on Cancer, Lyons. Jaffe, N., Frei, E., 111, Traggis, D., and Bishop, Y. (1974). Adjuvant methotrexate and citrovorum factor treatment of osteogenic sarcoma. N. Engl. J. Med. 291,994. Jones, S. E., and Salmon, J. E., eds. (1979). “Adjuvant Therapy of Cancer 11.” Grune & Stratton, New York. Longo, D. L., and Young, R. C. (198 I). The natural history and treatment of ovarian cancer. Annu. Rev. Med. 32,475-490. Moertel, C. G. ( 1 975). Clinical management of advanced gastrointestinal cancer. Cancer 36, 675. Nissen-Meyer, R., Kjellgren, K., and Mansson, B. (1982). Adjuvant Chemotherapy in Breast Cancer. Recent Results Cancer Res. 80, I42 - 148. Peto, R., Pike, M. C., Armitage, P., ef al. (1976). Design and analysis of randomized clinical trials requiring prolonged observation of each patient. Part I. Introduction and Design. Br. J. Cancer34,585-612. Rab, G. T., Irvins, J. C., Childs, D. S., ef al. (1976). Elective whole lung irradiation in the treatment of osteogenic sarcoma. Cancer 38,939. Shields, T. W., and Keehn, R. J. (1977). Postresection stage grouping in carcinoma of the lung. Surg. Gynaecol. Obstet. 145, 725 - 728. Shimkin, M. B. (1957). Thirteen questions: Some historical outlines for cancer research. J. Natl. Cancer Inst. 19,295-305. Silverberg, E. ( 1978). Cancer Statistics. Cancer J. Clin. 28, 17. Takita, H., Hollingshead, A. C., Rizzo, D. J., et al. (1979). Treatment of inoperable lung carcinoma: A combined modality approach. Ann. Thorac. Surg. 28, 363 - 368. Tate, H. C., Rawlinson, J. B., and Freedman, L. S. (1979). Randomized comparative studies in the treatment of cancer in the United Kingdom: Room for improvement? Lancet ii, 623 -625. Taylor, W. F., Irvins, J. C., Dahlin, D. C., et al. (1978). Trends and variability in survival from osteosarcoma. Mayo Clin. Proc. 53, 695.

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INDEX A Adjuvant chemotherapy for solid tumors, 315-326 breast cancer, 317-321 colorectal cancer, 323-324 drug selection, 3 I5 - 326 lung cancer, 323 malignant melanoma, 32 1 - 322 osteogenic sarcoma, 324 ovarian carcinoma, 325 patient selection, 3 I6 toxicity. 3 16-3 17 Airway epithelium, neoplastic development in, 1-70 Airways, heterotopic transplantation of, 8 - 9 Antimitotic factors, role in concomitant tumor immunity, 109- I12 Avian leukosis virus, insertional mutagenesis in, 295 -296 Avian retroviruses, pathogenesis of, 269-3 14 Avian sarcoma viruses, v-oncgenes, 283-284

B B e l l leukemias in mice, chromosome changes, 156- 158 B-cell lymphomas from avian leukosis viruses, 299 -30 I Breast cancer, adjuvant chemotherapy of, 3 17-32 1

Chromosome 4 in mice, tumorigenicity and, 168- I70 Chromosome 15, malignancy and, 157 Chromosomes in mouse, cancer and, 153-183 Cigarette smoking, lung cancer and, I - 3 c-mos and c-ras, properties of, 302 Colorectal cancer, adjuvant chemotherapy Of, 323-324 Complementation analysis of genes . involved in murine tumorigenesis, 170-171 c-onc genes activation by avian leukosis viruses, 30 1 - 302 role in nontransforming retroviruses, 292 - 305 Concomitant tumor immunity. 71 - 120 antigenic specificity, 88 - 89 immunologic reactivity of host, 89-90 mechanisms of tumor resistance in, 103- I13 metastase inhibition and, 93- 103 mechanisms, 1 12- I 1 3 nutrient factors, 107- 109 sinecomitant immunity vs., 90-93 tumor-cell immunogenicity, 86- 88 tumor-size effects, 82 - 86

D

C

DNA fragments, suppression of tumorigenicity in murine hybrids, 172 DTIC, immunogenic tumor cells from, 136- 138 DTlC lines, protective effect, chemotherapy and, 140-141

Cancer in mouse, chromosomes and, 153- I83 Cancer cells, altered hexose transport in, I77 Carcinogens, intratracheal instillation of, 5-6

Cell culture, tracheal carcinoma studies using, 16-18 Cell populations in airway neoplasms, 44-59 Chemotherapy, DTlC lines and, 140- 141

E Epithelial focus assay of neoplastic development, 1 I - 13, 5 I -59 329

330

IN1)EX

Epithelial hyperplasia, ultrastructural pathology of, I9

G Genes, amplification of, in mouse tumorigenesis, 159- 162

H Hexose transport, altered, in malignant cells, 177 hrl mutants of polyomavirus, 225-227 Humans, cancer in. mutagen-induced variants in therapy of, 146- 147

I Immunity, concomitant tumor type, 7 I - 120 Immunogenic tumor cells from mutagens, 136- I38 chemotherapy and, 140- I4 I new antigens on, 138- 140, 141- 144 Immunologic mechanisms in resistance to second tumor graft, 103- I13 Intratracheal instillation of carcinogens, 5 -6

L Late mutants of polyomavirus, 235 -236 Leukemia viruses, defective, v-onc genes, 284-285 Lung cancer adjuvant chemotherapy of, 323 cytological criteria for development of, 25 etiology of, 2 Lytic cycle of polyoma virus, 252-258

M Malignant melanoma, adjuvant chemotherapy of, 32 1- 322 Metastases inhibition of, concomitant immunity and, 93- I03 mechanisms, 112- 113 Mice chromosomes and cancer in, 153- I83 genetic basis for transformation in, 176-177 viruses in, role in tumorigenesis, 158- 159 m/I mutants of polyomavirus, 227-230

Mouse cell lines chromosome loss, 167- 168 complementation analysis, 170- 17 I general characteristics, I63 - 166 suppression of tumerigenicity, I67 mechanisms, I75 - I76 Mutagens, antigenic tumor cell variants obtained with, 12 I - 15 I

N ncr mutants of polyomavirus, 230-235 Neoplasms, phases in development of, 4 Neoplastic development in airway epithelium. 1-70 acute phase response role in, 23-24 analysis of, 9- 13 “carcinogen-altered” epithelial cells in, 45-47 cell culture studies, 16- I8 cell population studies, 44-59 chronic phase responses, 24-44 in animal models, 29-44 in humans, 24- 29 morphologic characterization, 30- 33 quantitation, 40-43 sequential changes, 33-40 cytologic and ultrastructural abnormalities, 18 epithelial focus assay of, 1 1 - I3 experimental approaches and methodologies, 4- I8 heterotopic transplantation of airways, 8-9 histologic changes, 19-20 in vilro studies, 2 I -23 in vivo exposure systems and tumor induction models, 5-9 intratracheal instillation of carcinogens. 5-6 lesion reversal, 43 -44 modifiers of, 59-62 morphology of acute phase responses in. 18-24 neoplastic variant in, 48-50 organ-culture studies, 13- 16 pellet implantation technique, 6 tracheal explant-outgrowth system of assay, 10- 11 tracheal lavage method, 6 - 8

33 1

INDEX

Nitrosoquanidine derivative, antigen variant tumor cells from, 125- 136 Nutrients, role in concomitant tumor immunity, 107- 109

0 Oncogene($ cell specificity of, 290-292 cell targets for, 307-308 in naturally occurring tumors, 303-305 nature of, 306 regulation of expression of, 307 targets for activity of, 285-290 viral and cell types compared, 30 I -305 Organ culture, tracheal carcinoma studies using, 13-16 “Operation immunity” to tumors, 77 Osteogenic sarcoma, adjuvant chemotherapy of, 324 Ovarian carcinoma, adjuvant chemotherapy of, 325

P Pellet implantation of carcinogens, 6 Plasmacytomas in mice, chromosome changes, 157- I58 Polyomavirus, 183-268 background, 186- 188 genome comparison with SV40 genome, 189-195 structural organization, 188 - 189 hrt mutants, 225-227 late mutants, 235-236 lytic cycle, 252-258 rnlt mutants, 227-230 mutants, 221 -236 ncr mutants, 230-235 oncogenes, 280-281 structure of genome, 188- 189 T antigens of, 195-221 transformation by, 236 -252 free viral DNA, 243-245 models, 237-241 T-antigen role, 245-252 viral gene integration, 241 -243 tsa mutants, 223-225

R Retroviruses (avian) diseases caused by, 274-277 genome structure, 272 host response to, 305 - 306 nononcogenic, reasons for, 299 nontransforming, pathogenesis, 292 - 306 oncogenesis by direct virus infection, 270-273 indirect, 273-274 possible mechanisms, 270-274 pathogenesis of, 269 - 3 14 role of v-onc genes, 278 - 292

S Second tumor challenge, concomitant tumor immunity and, 71 - 120 Sinecornitant tumor immunity, 77-80 Squamous cell carcinoma, ultrastructural pathology of, 19 SV40 virus genome, comparison with polyomavirus genome, 189- 195 tumongenicity in murine hybrid cells and, 172-175

T T antigens (of polyomavirus) characterization, 196- 200 definition, 195-221 in vitro functions, 209-217 large T-antigen, 210-213 middle T-antigen, 2 I3 - 2 16 small T-antigen, 216-217 in vivo functions, 217-221 large T-antigen, 218-219 middle T-antigen, 2 19- 22 1 location within cell, 207-209 properties, 204 role in transformation, 245,252 T-cell leukemias in mice, chromosome changes, 154- 156 Tobacco smoke, lung cancer and, 1-3 Tracheal explant-outgrowth assay of neoplastic development, 10- 1 1, 45-50 Tracheal lavage method for tumor induction, 6 - 8

332

INDEX

Transformation by polyomavirus, 236 -252 Triazenylimidazole derivative, immunogenic tumor cells from, I36 - I38 Trisomy I5 in murine tumorigenic hybrids, 171-172 fsa mutants of polyomavirus, 223-225 “tum-” variants immunogenic, from mutagens, 125- 136 in vifro analysis, I28 - I 34 in vivo analysis, I27 - 128 protection against, 134- 136 Tumor cells antigenic variants from mutagens, 121-151 fusion with host cells, 177 immunogenic, from mutagens, I36 - 138

Tumor immunity, concomitant, 7 I - 120 Tumors chromosome changes in, 159- 160 excision of, effect on second tumor challenge, 77 - 80 solid, adjuvant chemotherapy for, 3 15-326

V Viruses in mice, role in tumorigenesis. 158-159 v-onc‘ genes diversity and structure, 278 -283 functions, 283-292 nature of products of. 283-285 role in retrovirus pathogenesis, 278-292

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C . A . Coirlson Epidermal Carcinogenesis E. V. Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski Hormonal Aspects of Experimental Tumorigenesis T. U . Gardner Properties of the Agent of Rous No. I Sarcoma R . J . C. Harris Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism Cliarles Heidelberger The Carcinogenic Aminoazo Dyes James A. Miller und Elizabeth C . Miller The Chemistry of Cytotoxic Alkylating Agents M. C. J . Ross Nutrition in Relation to Cancer Albert Tonnenbarrm and Herbert Silverstone

Plasma Proteins in Cancer Riclicird J . Winzler AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexunder Chemical Constitution and Carcinogenic Activity G. M. Badger

333

Carcinogenesis and Tumor Pathogenesis 1. Berenblum Ionizing Radiations and Cancer Austin M. Bnres Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger und G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin T. Klopp and J m n n e C. Bateman Genetic Studies in Experimental Cancer L. w. Law The Role of Viruses in the Production of Cancer C. Oberling and M . Guerin Experimental Cancer Chemotherapy C. Chester Stock AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullman and B. Pidlmcin Some Aspects o f Carcinogenesis P. Rondoni Pulmonary Tumors in Experimental Animals MicliueI B. Shitnkin

334

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G. Gallon The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Abraham Godin Some Recent Work on Tumor Immunity t? A . Corer Inductive Tissue Interaction in Development Clifford Grohstein Lipids in Cancer Frances L. Haven and W. R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . L a c a s s a g n e , N . P. bit^ HoI, R . Daudel, and E Zujdelu The Hormonal Genesis of Mammary Cancer 0.Muhlhock AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R . W. Begg Primary Carcinoma of the Liver Charles Berman Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal t? N . Campbell The Newer Concept of Cancer Toxin Waro Nakuharu and Fumiko Fukrroku Chemically Induced Tumors of Fowls t? R . Peacock

Anemia in Cancer Vincent E. Price and Robert E. Green,field Specific Tumor Antigens L. A . Zilher Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K . Weisburger and John H . Weisburger AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar Bodansky The Plant Tumor Problem Armin C . Brairn and Henry N . Wood Cancer Chemotherapy by Perfusion Oscar Creech, Jr. and Edward T. Krementz Viral Etiology of Mouse Leukemia Ludwick Gross Radiation Chimeras t? C. Koller, A . J . S . Davies, and Sheilu M . A . Doak Etiology and Pathogenesis of Mouse Leukemia J . E A. P: Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G . M. Timmis Behavior of Liver Enzymes in Hepatocarcinogenesis George Weher AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J . W. Beard Mechanisms of Resistance to Anticancer Agents R . W.Brockman

CONTENTS OF PREVIOUS VOLUMES Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M . Court Brown and Ishbel M . Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L . Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT INDEX

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A . E Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M . J . Kopac and Gladys M. Mazeyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H . E Kraybill and M . B. Shimkin Experimental Tobacco Carcinogenesis Ernest L . Wynder and Dietrich Hoifman AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard Stamhaugh and Sidney Weinh014.S6>

The Relation o f the Immune Reaction to Cancer Louis V. C a w Amino Acid Transport in Tumor Cells R. M . Johnstone and P. G. Scholefield

335

Studies on the Development, Biochemistry, and Biology of Experimental Hepatomas Harold t? Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells I. E Seitz AUTHOR INDEX-SUBJECT INDEX

Volume 10 Carcinogens, Enzyme Induction, and Gene Action H . V. Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A . Clark Griffin The Enzymatic Pattern of Neoplastic Tissue W. Eugene Knox Carcinogenic Nitroso Compounds t? N . Magee and J . M . Barnes The Sulfhydryl Group and Carcinogenesis J . S. Harrington The Treatment o f Plasma Cell Myeloma Daniel E. Bergsagel, K . M . Griffith, A . Haut, and W. J . Stuckley, Jr. AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethran Sidney S . Mirvish Runting Syndromes, Autoimmunity, and Neoplasia D. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Suul Kit The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelson

336

CONTENTS OF PREVIOUS VOLUMES

Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos und Mary E Argus CUMULATIVE INDEX

Role of Cell Association in Virus Infection and Virus Rescue J . Svobodu and I . Hloirinek Cancer of the Urinary Tract D . B . Cluyson ond E . H . Cooper Aspects of the EB Virus M . A . Epstein AUTHOR INDEX-SUBJECT INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G . Pusternuk Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . I . Deichmun Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hanqfirsu Cellular Immunity against Tumor Antigens Kurl Erik Hellstriiin and Iiigegrrd Hellsfrom Perspectives in the Epidemiology of Leukemia Irving L. Kessler rind Abruham M . Lilien,feld AUTHOR INDEX-SUBJECT INDEX

Volume 13 The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata I? Alexander and J . C. Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswuld Jurrett The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajunan V. Sherbet The Characteristics of Animal Cells Transformed in Vitro Ion Mucpherson

Volume 14 Active Immunotherapy Georges Math6 The lnvestigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events George Meyer Passive lmmunotherapy of Leukemia and Other Cancer Rolund Motta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalf Complement and Tumor Immunology Kusuya Nishioku Alpha-Fetoprotein in Ontogenesis and It5 Association with Malignant Tumors G . I . Abelev Low Dose Radiation Cancers in Man Alice Stewurt AUTHOR INDEX-SUBJECT INDEX

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J . S . Rirtel, S . S . Teverhia. und J . L . Melnick Nasopharyngeal Carcinoma (NPC) J . H . C . Ho Transcriptional Regulation in Eukaryotic Cells A . J . M u c C i l l i v r a y . J . Purrl, rind G . ThrelJhll

CONTENTS OF PREVIOUS VOLUMES

337

Atypical Transfer RNAs and Their Origin in Neoplastic Cells Ernest Borek and Sylvia J . Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . Fialkow Electron Spin Resonance Studies of Carcinogenesis Harold M . Swartz Some Biochemical Aspects of the Relationship between the Tumor and the Host V. S . Shapor Nuclear Proteins and the Cell Cycle Gary Stein and Renato Baserga

Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis o n the Transkei, South Africa Gerald I? Warwick and John S . Harington Genetic Control of Murine Viral Leukemogenesis Frank Lilly and Theodore Pincus Marek's Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K . Nazerian Mutation and Human Cancer Alfred G. Knudson, Jr. Mammary Neoplasia in Mice S . Nandi and Charles M . McGrarh

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 16

Volume 18

Polysaccharides in Cancer Immunological Aspects of Chemical CarcinVijai N . Nigam and Antonio Cantero ogenesis Antitumor Effects of Interferon R. W. Baldwin Ion Gresser Isozymes and Cancer Transformation by Polyoma Virus and SimiFanny Schapira an Virus 40 Physiological and Biochemical Reviews of Joe Sambrook Sex Differences and Carcinogenesis Molecular Repair, Wound Healing, and Carwith Particular Reference to the Liver cinogenesis: Tumor Production a PossiYee Chi4 Toh ble Overhealing? Immunodeficiency and Cancer Sir Alexander Haddow John H . Kersey, Beatrice D. Spec'tor, and The Expression of Normal HistocompatibilRobert A . Good ity Antigens in Tumor Cells Recent Observations Related to the ChemoAlena Lengerovu therapy and Immunology of Gestational 1,3-Bis(2-ChloroethyI)-l-Nitrosourea Choriocarcinoma (BCNU) and Other Nitrosoureas in K. D. Bagshave Cancer Treatment: A Review Glycolipids of Tumor Cell Membrane Stephen K . Carter, Frank M . Schahel, Jr., Sen-iriroh Hakomori Lawrence E. Broder, and Thomas I? Chemical Oncogenesis in Culture Johnston Charles Heidelherger AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 17 Volume 19 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijai N . Nigam and Antonio Cantero

Comparative Aspects of Mammary Tumors J . M . Hamilton

338

CONTENTS OF PREVIOUS VOLUMES

The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M . Temin Cancer, Differentiation. and Embryonic Antigens: Some Central Problems J . H . Coggin, Jr. irnd N . G . Anderson Simian Herpesviruses and Neoplasia Fredrich W. Deinhnrdt. Lawrence A . Falk, and Lauren G. Wove Cell-Mediated Immunity to Tumor Cells Ronald B. Herherman Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastun and George S . Johnson Tumor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michael B. Shimkin and Gary D. Stoner Cell Death in Normal and Malignant Tissues E. H. Cooper, A . J . Bedford, nnd T. E. Kenny The Histocompatibility-Linked Immune Response Genes Baruj Benacerraf and David H . Katz Horizontally and Vertically Transmitted Oncornaviruses of Cats M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keef A . Rujferty, Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G . B . Clernents The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E. Trosko and Ernest H . Y. Chu SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M . C . Rapin and Max M . Burger Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. J . V. Nossol The Role of Macrophages in Defense against Neoplastic Disease Michael H. Levy rind E. Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis P. Sims and R 1.. Grover Virion and Tumor Cell Antigens of C-Type RNA Tumor Viruses Heinz Bauer Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?” Sir Alexander Haddow SUBJECT INDEX

Volume 22 Renal Carcinogenesis J. M. Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M . Sieber and Richard H . Adamson

Interrelationships among RNA Tumor Viruses and Host Cells Raymond V. Gilden Proteolytic Enzymes, Cell Surface Changes. and Viral Transformation Richard Rohlin, lih-Nan Chou, and Paul H . Black Immunodepression and Malignancy Osias Stutman SUBJECT INDEX

CONTENTS O F PREVIOUS VOLUMES

Volume 23 The Genetic Aspects of Human Cancer W. E. Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S. Weinstein, Frederick B. Merk, and Joseph Ahoy Genetics of Adenoviruses Harold S . Ginsberg and C . S. H . Young Molecular Biology of the Carcinogen, 4-Nitroquinoline 1-Oxide Minako Nagao and Takashi Sugimura Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection A . Frank, W. A . Andiman, and G. Miller Tumor Progression and Homeostasis Richmond T. Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill and Jana Hillova SUBJECT INDEX

Volume 24 The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? J . R Levy and J . C . Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E. Gri&n P,-Microglobulin and the Major Histocompatibility Complex Per A . Peterson. Lars Rusk, and Lars Ostberg Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Claudio Basilico

339

Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H. Clark, Jr., Michael J . Mastrangelo, Ann M. Ainsworth, David Berd, Robert E. Bellrt, and Evelina A. Bernardino SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA I;: L. Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harvey L. Ozer and Krishna K . Jha Tumor-Bound Immunoglobulins: In Situ Expressions of Humoral Immunity Isaac rl Witz The Ah Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S. Thorgeirsson and Daniel W. Nebert Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Anthony E. Pegg Immunosuppression and the Role of Suppressive Factors in Cancer lsao Kamo and Herman Friedman Passive lmmunotherapy of Cancer in Animals and Man Steven A. Rosenberg and William D. Terry SUBJECT INDEX

Volume 26 The Epidemiology of Large-Bowel Cancer Pelayo Correa and William Haenszel Interaction between Viral and Genetic Factors in Murine Mammary Cancer J . Hilgers and R Bentvelzen Inhibitors of Chemical Carcinogenesis Lee W. Wattenberg

340

CONTENTS OF PREVIOUS VOLUMES

Latent Characteristics of Selected Herpes- Structure and Morphogenesis of Type-C Reviruses troviruses Jucli G . Stevens Ronald C . Montc~laround Drctzi P. BoAntitumor Activity of Corynr~brrc~tc~rirtm logni>.si pnrvritn BCG in Tumor lmmunotherapy Lirka Milas and Murtin T. Scott Robert W. Buldiiin und Malcolm V. Pitnm The Biology of Cancer Invasion and MetasSUBJECT INDEX tasis Isuiuh J . Fidler, Douglus M . Gerstrn. rind Iun R . Hart Volume 27 Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis Translational Products of Type-C RNA TuA . Bnrny, E Brx, H . Chuntrentw, Y. C/o//mor Viruses t e ~ D. : D~keg~l, J . G k V s d ~ ~Rl. , KPIJohn R . Stephenson, S~isl~ilknmrrr G. tmann. M. Leclercq, J . Leirtwtz. M . Dpvure, and Fred H. RiJynolds,Jr. Mammerickx, and D. Portrtelle Quantitative Theories of Oncogenesis Molecular Mechanisms of Steroid Hormone Alice S. Whittc~more Action Gestational Trophoblastic Disease: Origin of Stepken J . Higgins und Ulrich Geliring Choriocarcinoma. lnvasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . 1. Brewer, E. E. Torok. 6.D . Kcillan, C . R . Stanhopr. und B. Halpern The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Hurold B . Hewitt Mass Spectrometry in Cancer Research John Rohoz Marrow Transplantation in the Treatment of Acute Leukemia E. Donnall Thomas, C. Dean Buckner, Alexander Fcfer, Parrl E. Neimun, utid Ruiner Storh Susceptibility of Human Population Groups to Colon Cancer Murtin Lipkin Natural Cell-Mediated Immunity Ronald B . Herhermuti and Howurd T. Holden SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Considerations E M. Burnet Tumors Arising in Organ Transplant Recipients Isruel Penn

SUBJECT INDEX

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J . E A . R Miller Suppressor Cells: Permitters and Promoters of Malignancy'? Duvid Naor Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer JosP Uric4 The Role of Glutathione and Glutathione STransferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L . E Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Rcroslahti and Murkkci Seppiilti Mammary Tumor Viruses Dun H . Moore. Carole A . Long, Akhil B. Vaidya, Joel B. Sheflield, Arnold S . Dion, und Etienne Y. Lusfi-rrgireA Role of Selenium in the Chemoprevention of Cancer A . Clark Griffin SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

34 I

Volume 30

Volume 32

Acute Phase Reactant Proteins in Cancer E . H . Cooper und Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Ghero and Alpha Peled On the Multiform Relationships between the Tumor and the Host K S.Shapoi Role of Hydrazine in Carcinogenesis Joseph Bulb Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kazymir M . Pozharisski, AIexei J . Likhavchev, Valeri E Klimashevski. nnd Jacob D. Shaposhnikov The Molecular Biology of Lymphotropic Herpesviruses Bill Siigden. Christopher R . Kintnrr, and Willie Murk Viral Xenogenization of Intact Tumor Cells Hiroshi Kohayushi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C. Austin and Charles W. Boone

Tumor Promoters and the Mechanism of Tumor Promotion Leila Diumond, Thomas G. O'Brien, und William M . Baird Shedding from the Cell Surface of Normal and Cancer Cells Paul H . Black Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNAand RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd W. Law, Michael J . Rogers, and Ettore Appella Nutrition and Its Relationship to Cancer Bandaru S . Reddy. Leonard A . Cohen, G. David McCoy, Peter Hill, Johri ff. Weisburger, and Ernst L . Wynder INDEX

Volume 33

INDEX

The Cultivation of Animal Cells in the Chemostat: Application to the Study of Tumor Cell Multiplication Michael G. Tovey Ectopic Hormone Production Viewed as an Volume 31 Abnormality in Regulation of Gene Expression The Epidemiology of Leukemia Hiroo lmura Michael Alderson The Role of Viruses in Human Tumor5 The Role of the Major Histocompatibility Haruld zur Huirsen Gene Complex in Murine Cytotoxic T The Oncogenic Function of Mammalian SarCell Responses coma Viruses Hermiinn Wugner. Klaris Pfizenmciii~r, P o d Andersson and Murtin Riillinghojy The Sequential Analysis of Cancer Develop- Recent Progress in Research on Esophageal Cancer in China ment Li Mingxin (Li Min-Hsin). Li Ping, trnd Li Emmanrrel Furher and Ross Cumeron Baororig ( L i Pao-.Iring) Genetic Control of Natural Cytotoxicity and Mass Transport in Tumors: Characterization Hybrid Resistance and Applications to Chemotherapy Edwwrd A . Cltrrk und Richurci C . Harmon Rakesh K . Jain, Jonas M . Weissbrod. wid Development of Human Breast Cancer James Wei Sefton R . Wellings INDEX

INDEX

342

CONTENTS OF PREVIOUS VOLUMES

Volume 34 The Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G. Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The Tumor Dormant State E. Federick Wheelock, Kent J . Weinhold. and Judith Levich Marker Chromosome 14q- in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retroviral Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability Jurnes W. Guutsch, John H . Elder, Fred C. Jensen, und Richard A . Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A . Fleischman Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model David T. Purtilo INDEX

Volume 35 Polyoma T Antigens Walter Eckhart Transformation Induced by Herpes Simplex Virus: A Potentially Novel Type of Virus-Cell Interaction Berge Hampar Arachidonic Acid Transformation and Tumor Production Lawrence Levine The Shope Papilloma-Carcinoma Complex of Rabbits: A Model System of Neoplastic Progression and Spontaneous Regression John W. Kreider w i d Gerald L. Btrrtlett

Regulation of SV40 Gene Expression Adolf Gruessmun, Monika Gruessmunn, and Christian Mueller Polyamines in Mammalian Tumors, Part 1 Giuseppe Scalubrino and Maria E. Feriolo Criteria for Analyzing Interactions between Biologically Active Agents Morris C . Berenbuum INDEX

Volume 36 Polyamines in Mammalian Tumors, Part I1 Giuseppe Sculahrino and Maria E. Ferioli Chromosome Abnormalities in Malignant Hematologic Diseases Janet D. Rowley and Joseph R . Testa Oncogenes of Spontaneous and Chemically Induced Tumors Robert A. Weinberg Relationship of DNA Tertiary and Quaternary Structure to Carcinogenic Processes Philip D . Lipetz. Alan G. Gulsky, rind Ralph EStephens Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes Tore Godal and Steinur Funderud Evolution in the Treatment Strategy of Hodgkin's Disease Giunni Bonudonna and Armando Santoro Epstein-Barr Virus Antigens-A Challenge to Modern Biochemistry David A . Thorley-Lawson. Clark M. Edson. and Kathi Geilinger INDEX

Volume 37 Retroviruses and Cancer Genes J . Michriel Bishop Cancer, Genes, and Development: The Drosophila Case Elisuheth Grrtqfy Transformation-Associated Tumor Antigens Arnold J . Leviric)

CONTENTS O F PREVIOUS VOLUMES

343

Neoantigens, Host-Tumor Interactions, and Regulation of Tumor Growth Benjamin Bonavidu The Initiation of DNA Excision-Repair George W. Teehor and Krystynci Frenkel Steroid Hormone Receptors in Human Breast Cancer G e o r g e W. Sledge, Jr. und William L . McGuire Relation between Steroid Metabolism of the Host and Genesis of Cancers of the Breast, Uterine Cervix, and Endometrium Mitsrio Kodumu and Toshiko Kodumu Fundamentals of Chemotherapy of Myeloid Leukemia by Induction of Leukemia Cell Differentiation INDEX Motoo Hozrimi The in Vifro Generation of Effector Lymphocytes and Their Employment in Tumor lmmunotherapy Eli Keilur untl DulYd W. Weiss Volume 38 Cell Surface Glycolipids and Glycoproteins in Malignant Transformation G . fi)gee.swurcin The SJL/J Spontaneous Reticulum Cell Sarcoma: New Insights in the Fields of INDEX

Pericellular Matrix in Malignant Transformation Kuri Alitulo and Antti Vuheri Radiation Oncogenesis in Cell Culture Curmia Borck M h c Restriction and If Genes J u n KItin untl Zoltan A . N a g y Phenotypic and Cytogenetic Characteristics of Human B-Lymphoid Cell Lines and heir Relevance for the Etiology of urkitt’s Lymphoma KennPtli Nilsson trntl G e o r g ~Kli& Translocations Involving lg Locus-Carrying Chromosomes: A Model for Genetic Transposition in Carcinogenesis George Kloin r i n d Gilhi,rt Lenoir

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