Recent evidence for oncogenesis by insertion mutagenesis and gene activation HAROLD E. VARMUS Department of Microbiology and Immunology, University of California, San francisco, California 94143 1 Introduction 309 If Identifying potential cellular genetic contributions to oncogenic mechanisms 310 1 Cellular homologues of viral oncogenes 310 2 Cellular genes that transform cultured cells 312 fi Oncogenesis by viruses lacking onc genes 313 Keywords: Retroviruses, Proviruses, Viral oncogenesis, Oncogenes, Inser- tion mutagenesis, Transformation, Avian leukosis, Enhancement of transcription , Summary Putative cellular oncogenes have recently been identified by their homology vith known viral oncogenes and by their capacity to transform the behaviour Of cultured cells. Retroviruses lacking oncogenes may induce tumotrs by tering or activating such cellular oncogenes. Analysis of such tumours reveals novel mechanisms for modulating expression of eukaryotic genes, identifies new candidate oncogenes, and suggests possible mechanisms for oncogenesis by non-viral agents. Introduction For many years, tumour virologists paid lip service to the possibility that the chromosomal sites at which viral DNA integrates might have a determining influence upon the neoplastic process. Until recently, this notion has had few adherents, for several good reasons. (i) Insertion of viral genomes into host DNA seemed most likely to affect host genes by disrupting them; it is difficult io envisage a cancer arising from the inactivation of one gene in a diploid genome. (ii) Although more promising consequences of viral insertions were plausible (e.g., gene activation, creation of a hybrid gene, or inactivation of a pene at a locus for which the host was heterozygous), such speculations were nsupported by experimental observations. (iii) The integrated (proviral) forms of DNA and RNA tumour viruses appeared to be inserted randomly in Cancer Surveys Vol. 1 No. 2 1982 310 Harold E. Varmus the host genome, reducing the likelihood of introducing viral DNA at the presumably rare positions conducive to oncogenesis. (iv) Mounting evidence’ for transforming genes (oncogenes) carried by many DNA and RNA tumour viruses seemed to obviate the need for more complicated and unsubstantiateq mechanisms of viral oncogenesis. Events of the past couple of years have refocused attention upon inser. tional mechanisms for viral oncogenesis and have provided some specific experimental reagents with which to pursue the idea that many types of cancer of unknown cause—not only certain virus-induced cancers— might have their roots in the disordered expression of host genes. My aim in this brief review is to provide the nonspecialist with an understanding of the experimental findings that have contributed to the current viewpoint. Two overlapping areas of study are central to the ensuing discussion: (i) efforts to define cellular genes whose altered expression might contribute to tumour formation and (ii) efforts to decipher the mechanisms by which tumours are caused by RNA tumour viruses (retroviruses) lacking their own oncogenes. Il Identifying potential cellular genetic contributions to oncogenic mechanisms At least two classes of host genes can now be considered candidates for some role in oncogenesis—a group of genes identified by their homology with retroviral oncogenes and another group identified by their capacity to induce " transformation of a cultured mouse fibroblast cell line. 1 Cellular homologues of viral oncogenes The first of these groups came into view during a search for the origins of the genetically defined transforming gene (src) of Rous sarcoma virus (RSV). Molecular hybridization reagents specific for src annealed to normal cellular DNA from the natural host for RSV (chickens) and to DNA from all other vertebrates, suggesting that the homologous cellular sequences had been rela- tively well conserved throughout evolution (Stehelin et al., 1976; Spector et al., 1978a). Tests for RNA and protein products of the src-related sequences demonstrated that they constituted a competent coding domain normally expressed at a low level (Spector et al., 1978b; Collet et al., 1978; Opper- mann et al., 1979). Additional tests for genetic linkages and polymorphisms, and for the structure of the domain, indicated that the src-related domain was a structurally conventional cellular gene (now called c-src), with multiple introns, little or no variation among members of a species, and no apparent linkage or resemblance to the endogenous proviruses that inhabit the germ lines of many animals (Hughes et al., 1979a, b; Parker et al., 1981; Shalloway et al., 1981). - Subsequent studies of a large number of other RNA tumour viruses cap- able of transforming cultured cells and inducing tumours rapidly in animals have unearthed over fifteen distinct viral oncogenes, each of which is closely Oncogenesis by insertion mutagenesis and gene activation 311 elated to (and presumably derived from) a cellular gene (Bishop, 1981; Bishop and Varmus, 1982). For convenient communication, these genes have been generically labelled ‘v-onc’s’, the viral oncogenes, and ‘c-onc’s’, their cellular counterparts (Coffin et al., 1981). Each member of these classes has been assigned a trivial name (e.g., src, myc, mos; see Table 1) according to recently published rules. We may be approaching the limit of the number of -onc’s that will ultimately be identified, since two related sets of oncogenes (fes and fps, bas and ras) have been isolated in viruses arising in different host species (Shibuya et al., 1980; Andersen et al., 1981); moreover, some pncogenes have appeared repeatedly in independent virus isolates within the ame species (Table 1). Nevertheless, there may be additional cellular genes of similar potential that, for one reason or-another, are not susceptible to ransduction by retroviruses. Other means will be required to identify those yenes. _ An account of the functions and products of the several c-onc’s is beyond he purview of this short essay (see Bishop and Varmus, 1982). For our Lurposes, it is sufficient only to outline the evidence supporting the simple nference that at least some cellular homologues of v-onc’s do themselves have oncogenic potential. Such potential could be achieved in two ways: by utations that alter the nature of the gene products or by modulations of bene expression that raise the level above a threshold required for neoplastic effects. For at least two c-onc’s (c-mos and c-ras), the latter possibility has been found sufficient by using a strong promoter of transcription to enhance expression of the cellular genes (Oskarsson et al., 1980; Blair et al., 1981; fable 1. Cellular sequences identified as probable oncogenic sequences in the genomes of Number of virus isolates Example Animal origin >3 Rous sarcoma virus (Prague strain) Chicken >3 Fujinimi sarcoma virus : Chicken 2 Y73 sarcoma virus Chicken 1 UR-2 virus Chicken 4 Avian myelocytomatosis virus-29 Chicken 1 Avian erythroblastosis virus Chicken 2 Avian myeloblastosis virus Chicken 1 Reticuloendotheliosis virus, strain T Turkey 2 Moloney murine sarcoma virus Mouse 1 Abelson murine leukaemia virus ’ Mouse 1 BALB murine sarcoma virus Mouse >3 Harvey murine sarcoma virus Rat 2 Snyder-Theilin feline sarcoma virus Cat 1 McDonough feline sarcoma virus Cat 1 Simian sarcoma virus _ Woolly monkey t least fifteen distinguishable sets of sequences (v-onc’s) have been found in the genomes f transforming retroviruses and shown to be homologous to host sequences (c-onc’s) with roperties of cellular genes. Arbitrary names have been assigned to the onc sequences (Coffin et al., 1981). The number of probably independent virus isolates containing each onc sequence, vith an example of each, is listed with the host from which the onc sequences have apparently Sriginated. 312 Harold E. Varmus DeFeo et al., 1981). These experiments required the molecular cloning ang subsequent joining in vitro of two components: a cellular oncogene and a region of retroviral DNA, the long terminal repeat unit (LTR), which encodes a strong promoter. When reintroduced into cultured cells, the ‘acti. vated’ c-onc’s are capable of transforming them to a neoplastic Phenotype. Earlier experiments indicated that c-src could contribute at least Partially to the production of an oncogenic protein, since competent transforming Viruses could be isolated from tumours induced in chickens with deletion mutants of RSV lacking part of v-src (Hanafusa et al., 1977). Biochemical studies of the recovered viruses suggested they were generated by recombination between c-src and the defective v-src (Wang et al., 1979; Vigne et al., 1980; for a dissenting view, see Lee et al., 1981). It should be emphasized, however, that for most c-onc’s there is as yet no direct evidence for their oncogenic potential; in these cases, the structura] differences between v-onc’s and c-onc’s may prove to be critical determinants of function. Even in the provocative situations considered below, in which c-onc’s are activated in tumours induced by retroviruses that lack v-onc’s, it is premature to conclude that the enhanced expression of a c-onc is directly ‘responsible for tumour growth. 2. Cellular genes that transform cultured cells The second class of putative cellular tumour genes has been defined by using mouse NIH/3T3 fibroblasts to assay the transforming potential of naked DNA from a variety of sources, including chemically induced tumours, tumours induced by viruses lacking onc genes, spontaneous tumours, and normal tissues (Shih et al., 1979, 1981; Cooper et al., 1980; Cooper and Neiman, 1980; Lane er al., 1981). Although the efficiency of transformation is quite low, DNA from the transformed cells can be used, in turn, to trans- form fresh cells; the transforming elements can be classified with respect to their susceptibility to restriction endonucleases or their linkage to highly repeated cellular sequences (Shih et al., 1981; Murray et al., 1981; Perucho et al., 1981); and, in a few cases, the transforming components have been molecularly cloned in prokaryotic host-vector systems (Goldfarp et al., 1982, G. Cooper, R. Weinberg, personal communications). Although little is pres- ently known about the structures, products, and normal functions of these putative oncogenes, they are likely to be genetically altered in the tumour cells from which they are derived, since DNA similarly prepared from normal cells is inactive in the transformation assay. As in the case of c-onc’s and v-onc’s, it is for the most part uncertain whether the important differences reside in structural or in regulatory domains. The observation that the shear- ing of DNA from normal cells may confer transforming potential upon it Suggests that regulatory changes may be more important, since the shearing might remove the restraining influence of cis-dominant controlling elements and allow potential transforming genes to be inserted within highly active transcriptional units (Cooper et al., 1980). However, the experiments with normal cell DNA may be misleading: it is not known whether the transform- Oncogenesis by insertion mutagenesis and gene activation 313 ng activity of DNA from tumour cells is derived from the same set of genetic ie as the activity in sheared DNA from normal cells. It is possible that transforming genes defined by the NIH/3T3 cell assay are fon-overlapping with c-onc’s, however, there is reason (described below) for proposing that c-onc’s and transforming genes may play different roles in at feast certain types of neoplasia. The number of potential transforming genes is Still elusive, but it is striking that DNA from certain classes of tumour cells fe-g., mammary tumours [Lane et al., 1981], neural tumours and carcinomas aShih et al., 1981], chemically transformed cell lines [Shilo and Weinberg 7981], B or T cell leukaemias [Lane et al., 1982] and human lung and ladder carcinomas [Perucho et al., 1981; R. ‘Weinberg, personal communi- gation]) exhibit class- “specific patterns using restriction endonucleases. fumour type and that the number of potential transforming genes may be less finan the number of distinguishable types of cancers. | Oncogenesis by viruses lacking onc genes a the late 1970s, amidst the proliferation of viral oncogenes and their pro- cts, increasing attention was also given to the previously elusive question of ow some retroviruses can cause tumours despite the apparent absence of ansforming genes from the viral genomes. There were several reasons for is rekindled interest. First, the long latency between infection and the Bcerance of tumours suggested that viruses lacking v-onc’s might be more gvealing than v-onc* viruses about the pathogenesis of human tumours Weichetal., 1982). Second, the application of restriction mapping to proviruses tumours induced by v-onc™ viruses indicated that these tumours were clonal for at least dominated by clones), so that the sites of proviral insertion could & conveniently studied (Steffen and Weinberg, 1978; Cohen et al., 1979). fhird, the newly perceived structure of proviral DNA—with identical Eoulatory domains (long terminal repeats or LTRs) present at both ends gProvoked hypotheses in which proviruses exerted ° 1980. Rens influences e& B cell lymphomas in the bursa of Fabricius by avian leukosis viruses ih RLV provirus (Neiman et al., 1980; Payne et al., 1981; Neel et al., 1981; ming et al., 1981); (ii) in several instances the provirus is structurally defec- ve and no viral genes are expressed, implying that viral gene products are {ot necessary to maintain the tumour state (Payne et a/., 1981); (iii) virtually fil tumours bear proviruses within the same cellular genetic domain (Neel et 314 Harold E. Varmus al., 1981; Payne et al., 1981), a region first identified by Hayward and his colleagues (1981) as the c-onc called c-myc; (iv) all the tumours bearin insertions in the c-myc region exhibit levels of expression of c-myc RNA 10. to 100-fold above levels in normal B cells (Hayward et al., 1981; Payne et aj. 1982); and (v) DNA from the tumours contains activated transforming genes, as determined by assay in NIH/3T3 cells, with the transforming DNA free of ALV or c-myc sequences (Cooper and Neiman, 1980, 1981). How should these findings be viewed in relation to the pathogenesis of the disease? It is likely that ALV spreads efficiently through the bursal cell popu- lation in a newly infected susceptible animal, inserting proviral DNA more or less at random in the genomes of large numbers of cells. In the rare cell (about 1 in 10°) that acquires a provirus in the c-myc domain, the neoplastic process can presumably be initiated by overproduction of c-myc RNA and protein, (The c-myc protein has not been identified, and there is no proof that it is inherently oncogenic; furthermore, the v-myc domain is usually expressed in virally transformed cells as a hybrid protein also containing peptides encoded by viral structural genes [Bister et al., 1977].) It is probable that additional mutations or rearrangements of cellular genes occur subsequent to activation of c-myc, including one change that activates a gene capable of transforming | NIH/3T3 cells. As the tumour nodule enlarges and the genetically evolving - cellular population competes for dominance of the tumour mass, advantage © belongs to tumour cells in which all proviruses, including the inciting provirus in the c-myc domain, are inactivated (e.g., by partial deletions), since any immune response against viral antigens will no longer operate against such cells. However, retention of at least a single LTR near c-myc may be necessary to maintain the tumour state, even though viral gene products are not required (Payne et al., 1981). An immediate question raised by the study of chicken lymphomas is rele- vant to understanding gene regulation in eukaryotes, as well as tumorigenesis: how does an insertion of ALV proviral DNA amplify the expression of c-myc? The initial premise, and the simplest, was that in each case an ALV LTR would be positioned ‘upstream’ from the genetic target, in an orienta- - tion that would permit it to act as a promoter for transcription (Neel et al., 1981; Payne er al., 1981). Although the majority of tumours do contain this arrangement of c-myc and an ALV LTR, with RNA transcripts that appear to have been initiated within the LTR and extended into c-myc (Hayward et al., 1981), there is also a substantial number of tumours in which other arrangements are observed (Payne et al., 1982). In several tumours, the ALV provirus is upstream from c-myc, but in the transcriptional orientation opposite that of c-myc; in at least one tumour the provirus is downstream _ from c-myc. With these two unexpected arrangements, the ALV LTR cannot act simply as a promoter to enhance the expression of c-myc. The most obvious possibility is that the ALV LTRs indirectly potentiate the transcriptional activity of the chromosomal domain in which they have been inserted, affecting the strength of promoters normally used or recruiting promoters normally inactive. Although the biochemical bases for such phenomena are not understood, Oncogenesis by insertion mutagenesis and gene activation 315 potentially related findings in more malleable contexts suggest that the phenomena are experimentally accessible. Thus, regions near the origin of replication in SV40 DNA and the LTR of RSV (but not the LTR of the non-tumorigenic chicken virus, RAV-0) markedly increase the frequency of {ransformation of thymidine kinase-deficient (tk~) mouse cells to a tkt phenotype when present in a micro-injected plasmid containing the complete herpes simplex tk gene in either orientation (Capecchi, 1980; P. Luciw and M. (Capecchi, unpublished). Enhancing properties of SV40 DNA upon the expression of linked promoters have been mapped within a 72 bp repeat normally positioned upstream from the promoter for the early genes; this peduence has a strong influence upon transcription of the early genes (Gruss et al.; 1981; Benoist and Chambon, 1981); it can stimulate expression of linked heterologous genes using their natural promoters or adjacent promoter-like sequences (Banerji et al., 1981; M. Fromn and P. Berg, per- sonal communication; M. Botchan, personal communication); and it can be replaced during SV40 replication by a functionally homologous domain of the murine sarcoma virus LTR (Levinson et al., 1982). The findings with ALV-induced lymphomas raise a host of additional ques- tions, the most approachable being whether similar events occur in tumours nduced by other viruses without onc genes. Several pieces of evidence encourage belief that they do. (i) In avian B cell lymphomas induced by a retrovirus unrelated to ALV (the reticuloendotheliosis virus called chicken syncytial virus) and by a virus differing only modestly from ALV (the myeloblastosis-associated virus, MAV), proviral insertions near c-myc have been identified, sometimes accompanied by deletions within and amplification of the interrupted domain (Noori-Daloii et al., 1981; D. Westaway and C. Moscovici, unpublished). However, the level of c-myc expression has yet to be determined in these cases and tumour DNA has not been tested for trans- forming activity in NIH/3T3 cells. (ii) Preliminary evidence implicates pro- viral insertion near another cellular oncogene (c-erb), the progenitor of the ransforming gene of avian erythroblastosis virus, in the rare induction of erythroblastosis by ALV (T. Fung and H. J. Kung, personal communication). iii) A significant proportion (about 50%) of mammary carcinomas induced by the mouse mammary tumour virus (MMTV) in C3H mice contain pro- viruses in the same 20 kb region of the host genome (R. Nusse, unpublished), However, this region has been defined only by the presence of proviral DNA: no known cellular oncogenes reside within it, and an activated transcriptional unit has not been found. The analogy with ALV-induced lymphomas is streng- thened by the observation that DNA from MMTV-induced mammary lumours transforms NIH/3T3 cells; the pattern of inactivation of the trans- orming principle by restriction endonucleases suggests that the same oncogene may be involved in each of five cases and in a chemically induced mouse Mammary tumour and a spontaneous human mammary carcinoma Lane et al., 1981). As in the case of ALV-induced lymphomas, the trans- forming sequences seem to be unlinked to proviral DNA. It would be premature to conclude from these provisional data that all retroviruses without oncogenes induce tumours in similar ways. On the one 316 Harold E. Varmus hand, we are far from understanding how oncogenic transformation occurs in the most successfully explored example, ALV-induced lymphoma; on the other hand, some of the most important viruses in this category— including murine, bovine, and feline leukaemia viruses, as well as the newly described human isolates associated with T cell lymphomas and leukaemias (Poiesz et al., 1980; Hinuma et al., 1981)}—have yet to be adequately assessed from these new perspectives. Some insight into the significance and function of putative oncogenes affected in various tumours may be provided by asking whether viruses, such as ALV, that induce several kinds of neoplasm do so by activating the same or different oncogenes in each target cell. Conversely, it will be of interest to determine whether the same oncogene is affected by different viruses in a single target cell, as may be the case for c-myc in B cell lymphomas (see above). It is possible that the answers to these questions will depend upon the normal functions of the various cellular oncogenes (e.g., whether they are general regulators of growth or determinants of differentiation); in any case, the answers may serve as guides for exploring the involvement of such genes in spontaneous tumours and in tumours induced by non-viral agents. A profound understanding of neoplastic mechanisms involving putative cellular oncogenes will doubtless demand detailed descriptions of the pro- ducts of these genes. There is reason to be optimistic about achieving these immediate goals, judging from recent successes with viral oncogenes (reviewed in Tooze, 1980 and Bishop and Varmus, 1982). 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