Proc. Natl. Acad. Sci. USA Vol. 84, pp. 2117-2124, April 1987 Biochemistry Cancer genes: Rare recombinants instead of activated oncogenes (A Review) (retroviral oncogenes/protooncogenes/illegitimate recombination/clonal chromosomal abnormalities) PETER H. DUESBERG* Department of Molecular Biology, University of California, Berkeley, Wendell M. Stanley Hall, Berkeley, CA 94720 Contributed by Peter H. Duesberg, December 22, 1986 ABSTRACT The 20 known transforming (onc) genes of retroviruses are defined by sequences that are transduced from cellular genes termed protooncogenes or cellular oncogenes. Based on these sequences, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes that can be activated from within the cell to cause virus-negative tumors. The hypothesis is popular because it promises direct access to cellular cancer genes. However, the existence of latent cancer genes presents a paradox, since such genes are clearly undesirable. The hypothesis predicts (i) that viral onc genes and proto-onc genes are isogenic; (it) that expression of proto-onc genes induces tumors; (iii) that activated proto-onc genes transform diploid cells upon transfection, like viral onc genes; and (iv) that diploid tumors exist. As yet, none of these predictions is confirmed. Instead: (i) Structural comparisons between viral onc genes, essential retroviral genes, and protoonc genes show that all viral onc genes are indeed new genes, rather than transduced cellular cancer genes. They are recombinants put together from truncated viral and truncated proto-onc genes. (ii) Proto-onc genes are frequently expressed in normal cells. (iii) To date, not one activated proto-onc gene has been isolated that transforms diploid cells. (iv) Above all, no diploid tumors with activated proto-onc genes have been found. Moreover, the probability of spontaneous transformation in vivo is at least 109 times lower than predicted from the mechanisms thought to activate proto-onc genes. Therefore, the hypothesis that proto-onc genes are latent cellular oncogenes appears to be an overinterpretation of sequence homology to structural and functional homology with viral onc genes. Here it is proposed that only rare truncations and illegitimate recombinations that alter the germ-line configuration of cellular genes generate viral and possibly cellular cancer genes. The clonal chromosome abnormalities that are consistently found in tumor cells are microscopic evidence for rearrangements that may generate cancer genes. The clonality indicates that the tumors are initiated with, and possibly by, these abnormalities, as predicted by Boveri in 1914. In order to understand cancer, it is necessary to identify cancer genes. The search for such genes and for mechanisms that generate such genes must take into consideration that, at the cellular level, cancer is a very rare event. The kind of cellular transformation that leads to cancer in vivo occurs only in about 1 out of 2 x 1017 mitoses in humans or animals. The basis for this estimate is that most animal and human cancers are derived from single transformed cells and hence are monoclonal (1-4), that humans and corresponding animals represent about 1016 mitoses (assuming 1014 cells that go through an average 102 mitoses), and that about one in five persons dies from cancer (5). The only proven cancer genes are the transforming (onc) genes of retroviruses. These are autonomous transforming genes that are sufficient for carcinogenesis (6, 7). They transform susceptible cells in culture or in animals with the same kinetics as they infect them (6, 7). Therefore, these viruses are never associated with healthy animals and are by far the most direct and efficient natural carcinogens. However, tumors with retroviruses that contain onc genes are very rare in nature; less than 50 cases are recorded from which such viruses were isolated (4, 6-8). Moreover, these viruses never have been reported to cause epidemics of cancer. The probable reasons are that viral onc genes arise naturally only with great difficulty, via two or more illegitimate recombinations, and that once arisen they are very unstable because they are not essential for virus replication (6, 7). Nonessential genes are readily lost due to spontaneous deletion or mutation. Indeed, onc genes were discovered by analysis of spontaneous deletions of the src gene, the onc gene of Rous sarcoma virus (RSV) (9, 10). Subsequently, about 20 other viral onc genes were identified in retroviruses (6, 7, 8, 11). All of these viral onc genes were originally defined by "transformation-specific" sequences that are different from the known sequences of essential virus genes (12). Since onc genes are unstable, they must also be recent additions to retroviruses. Indeed, the cellular genes from which the transformation-specific sequences of oncogenic retroviruses were transduced have been identified in normal cells. This was done initially by liquid hybridization of transformation-specific viral sequences with cellular DNA (13-17) and later by comparing cloned viral onc and corresponding cellular genes (18). The cellular genes from which viral onc sequences are derived have since been termed proto-onc genes (6). The cellular origin of the transformation-specific sequences of retroviral onc genes is frequently presented as a particular surprise (8, 11). However, cells are the only known source of genetic material from which viruses could transduce genetic information, and viral transduction has been canonical knowledge ever since phage X was first shown to transduce 1-galactosidase in the 1950s. Indeed, viruses are themselves derivatives of cellular genes that have evolved away from their progenitor genes as they acquired their capacity of self-replication. The Hypothesis That Proto-onc Genes Are Latent Cancer Genes. On the basis of the sequence homology between viral onc genes and proto-onc genes, viral onc genes have been postulated to be transduced cellular cancer genes, and proto-onc genes have been postulated to be latent cancer genes or oncogenes (19-27, 103). According to this view, termed the oncogene concept (27), proto-onc genes are not only converted to transforming genes from without by transducing viruses but also converted from within the cell by increased dosage or increased function (19-27, 103). Activation of latent oncogenes from within the cell is postulated to Abbreviation: RSV, Rous sarcoma virus. *Present address: Fogarty Scholar-in-Residence, John E. Fogarty International Center, National Institutes of Health, Bldg. 16, Bethesda, MD 20892 2117 2118 Biochemistry: Duesberg follow one of five prominent pathways: (i) point mutation (28, 29), (ii) chromosomal translocation that brings the latent oncogene under the control of a heterologous enhancer or promoter (23, 30), (iii) gene amplification (26, 27), (iv) activation from a retroviral promoter integrated adjacent to the latent oncogene (8, 22-27), or (v) inactivation of a constitutive suppressor (31). Thus, this view predicts that latent cancer genes exist in normal cells. However, the existence of latent cancer genes is a paradox, because such genes would obviously be undesirable for eukaryotic cells. The oncogene concept was a revision of Huebner's oncogene hypothesis, which postulated activation of latent oncogenic viruses instead of latent cellular oncogenes as causes of cancer (32). Nevertheless, Huebner's hypothesis remained unconfirmed because most human and animal tumors are virus-negative (8, 11). Moreover, the retroviruses and DNA viruses that have been isolated from tumors are not directly oncogenic (4), except for the less than 50 isolates of animal retroviruses that contain onc genes (7, 8, 11). The revised oncogene hypothesis was at first sight highly attractive because it derived credibility from the proven oncogenic function of retroviral onc genes, the viral derivatives of proto-onc genes, and because it promised direct access to the long-sought cellular cancer genes in virus-free tumors by use of previously defined viral onc genes as hybridization probes. Predictably, the hypothesis has focused the search for cellular cancer genes among the 106 genes of eukaryotic cells on the 20 known proto-onc genes (7, 8, 23-27). The hypothesis makes four testable predictions: (i) that viral onc genes and proto-onc genes are isogenic; (ii) that expression of proto-onc genes would cause cancer; (iii) that proto-onc genes from tumors would transform diploid cells as do proviral DNAs of viral onc genes, and, above all, (iv) that diploid tumors exist that differ from normal cells orily in activated proto-onc genes. Despite record efforts in the last 6 years, none of these predictions has been confirmed. Instead: (i) Genetic and biochemical analyses that have defined essential retroviral genes, viral onc genes, and proto-onc genes during the last 16 years showed that viral onc genes and proto-onc genes are not isogenic (6, 7) (see below). (ii) Further, it turned out that most proto-onc genes are frequently expressed in normal cells (7). (iii) Contrary to expectation, none of the 20 known proto-onc genes isolated from tumors functions as a transforming gene when introduced into diploid cells. The apparent exceptions of proto-ras and proto-myc are discussed below. By comparison, proviral DNAs of retroviral onc genes transform normal cells exactly like the corresponding viruses (8, 11). (iv) As yet, no diploid tumors with activated proto-onc genes have been found, except for those caused by viruses with onc genes (33, 34). Instead of activated oncogenes (7), clonal chromosome abnormalities are a consistent feature of virus-negative tumors (1-3, 35) and also of all those tumors that are infected by retroviruses without onc genes (4). The Claim That Proto-ras Genes Become Cancer Genes Due To Point Mutations. Harvey proto-ras is the cellular precursor of the ras genes of Harvey, BALB, and Rasheed murine sarcoma viruses, and Kirsten proto-ras is the cellular precursor of the ras gene of Kirsten murine sarcoma virus (8, 11). Both proto-ras and the viruses encode a colinear protein, termed p21, of 189 amino acids (Fig. 1). In 1982 it was discovered that Harvey proto-ras extracted from a human bladder carcinoma cell line, but not from normal cells, would transform the morphology of a few aneuploid murine cell lines, in particular the 3T3 mouse cell line (28, 29). Subsequently, proto-ras DNAs from some other cell lines and from some primary tumors were also found to transform 3T3 cells (7, 43-45). Since such proto-ras genes behave like dominant and autonomous cancer genes in this morphological assay, Proc. Natl. Acad. Sci. USA 84 (1987) they were claimed to be cellular cancer genes (8, 28, 29). The 3T3 cell-transforming function of the Harvey proto-ras gene from the bladder carcinoma was reduced to a single point mutation that changed the 12th ras codon of p21 from the normal glycine to valine (28, 29). In the meantime, more than 50 different point mutations in five different ras codons have been identified that all activate 3T3 cell-transforming function (39, 46). Since the viral ras genes and proto-ras genes encode the same p21 proteins, whereas most other viral onc genes encode proteins that are different from those encoded by proto-onc genes (Fig. 1; refs. 6 and 7), this system has been considered a direct support for the hypothesis that viral onc genes and proto-onc genes are indeed isogenic and hence can become functionally equivalent by point mutations or enhanced expression (25-29, 46, 103). However, the following arguments cast doubt on the claims that point mutations are necessary or sufficient to convert proto-ras to a dominant cancer gene. (i) The observations that most, but not all (see below), proto-ras genes with point mutations have been found in tumors or in certain cell lines appear to support the proposal that point mutations convert proto-ras genes to dominant cancer genes. However, the case is significantly weakened because, in most spontaneous tumors, ras mutations are very rare (7, 43-45). In fact, the glycine-*valine mutation that was originally found in the human bladder carcinoma cell line (28, 29) has never been found in a primary tumor. Even in certain chemically induced or spontaneous tumors in which ras mutations are relatively frequent, no consistent correlation between ras mutations and tumors has been observed (7, 47-49). Further, it is not known whether, in animals, the origin of a ras mutation coincides with the origin of the tumor. For example, the ras mutation of the human bladder carcinoma (28, 29) was only found in a cell line 10 years after this line was derived from the original tumor (50). On the basis of a numerical argument it is also unlikely that point mutations are sufficient to convert proto-ras genes to dominant cancer genes. The frequency of point mutations of eukaryotes is 1 in 108-1010 nucleotides per mitosis (51, 52). Thus, about 1 in 107 mitoses is expected to generate mutant Harvey ras genes with dominant transforming function, since the diploid human cell contains about 6 x 109 nucleotides and since 50 different mutations can activate each of two sets of ras genes of diploid cells. By contrast, spontaneous transformation that leads to clonal tumors occurs in less than 1 out of about 2 x 1017 mitoses, and only a small minority of the resulting cells contain mutant ras genes. It may be argued that indeed 1 out of 107 mitoses generates a tumor cell with activated proto-ras but that the immune system eliminates most of these cells. However, this is unlikely, since a point mutation is not an easy target for immunity. Further, animals or humans that are tolerant to ras point mutations would be expected to develop tumors at a very early age, if point-mutated proto-ras genes were dominant cancer genes as the 3T3 assay suggests. Instead, spontaneous human tumors with activated proto-ras are very rare and all were observed in adults (7, 43-45). Moreover, the argument that cellular oncogenes exist that can be activated by point mutation and then controlled by immunity is hard to reconcile with the existence of athymic or nude mice, which do not develop more spontaneous tumors than other laboratory mice (53). Furthermore, this view is inconsistent with the evidence that immunosuppressive therapy or thymectomy does not increase the cancer rate of humans (54). Finally, one would predict that in the absence of immunity, as in cell culture, 1 out of 107 normal cells should spontaneously transform due to point mutation of Harvey proto-ras alone, and probably the same number due to mutation of Kirsten Biochemistry: Duesberg proto-ras (8). Yet spontaneous transformation of diploid cells in culture is clearly a much less frequent event. In an effort to directly test the hypothesis that ras genes are activated to dominant cancer genes by point mutation, we (39) analyzed whether the transforming function of ras genes does indeed depend on point mutations. Using site-directed mutagenesis, we found that point mutations are not necessary for transforming function of viral ras genes and proto-ras genes that have been truncated to be structurally equivalent to viral ras genes (39). (ii) Contrary to expectation, the same proto-ras DNAs from human tumors that transform aneuploid 3T3 cells do not transform diploid human (55) or diploid rodent (56-58) cells, the starting material of natural tumors. Thus, transformation of 3T3 cells does not appear to be a reliable assay for transforming genes of diploid cells. Instead of initiating malignant transformation, mutated proto-ras genes just alter the morphology and enhance the tumorigenicity of aneuploid 3T3 cells. Apparently, they activate one of the many morphogenic programs of eukaryotic cells. Observations that untreated 3T3 cells are turnorigenic in nude mice (59-61) are consistent with this view. Thus, proto-ras genes with point mutations are not sufficient to initiate malignant transformation. They only appear as dominant cancer genes in certain aneuploid cells like the 3T3 cells because of unknown biochemical effects that alter the morphology of these cells. Furthermore, morphological transformation of 3T3 cells is not ras gene-specific. It occurs spontaneously (62) and also upon transfection with other DNA species derived from tumors or tumor cell lines that, like proto-ras, do not transform diploid cells (26, 47, 48). Such DNAs are now widely considered as cellular cancer genes (26, 47, 48), although they are not related to viral onc genes and do not transform diploid cells. (iii) Assuming that mutated proto-ras genes are cancer genes like viral onc genes, one would expect diploid tumors that differ from normal cells only in ras point mutation. Contrary to expectation, chromosome abnormalities are consistently found in those tumors in which proto-ras mutations are occasionally found (1, 3). The human bladder carcinoma cell line in which the first proto-ras mutation was identified is a convincing example. This cell line contains more than 80 chromosomes instead of 46 and includes rearranged marker chromosomes (50). In view of such fundamental chromosome alterations, a point mutation seems to be a rather minor event. Indeed, among diploid hamster cells transfected with mutated ras genes, only those that developed chromosomal abnormalities upon transfec- tion were tumorigenic (63). Thus, proto-ras genes with point mutations are neither sufficient nor proven to be necessary for carcinogenesis and are not autonomous cancer genes like viral ras genes. In addition, there is no kinetic evidence that the origin of the mutation coincides with the origin of the tumors in which it is found. It is consistent with this view that proto-ras mutations that register in the 3T3 cell transformation assay have been observed to occur in vivo in benign hyperplasias, as for example in benign murine hepatomas (64) or in benign, purely diploid mouse skin papillomas that differentiate into normal skin cells (65-69). ras mutations have also been observed to arise after carcinogenesis in aneuploid cancer cells (70-72), rather than to coincide with the origin of cancer. By contrast, viral ras genes are sufficient for transformation and thus initiate transformation of diploid cells in vitro and in vivo with single-hit kinetics and concurrent with infection (7, 73, 74). This then raises the question why viral ras genes are inevitably carcinogenic under conditions where proto-ras genes with point mutations are not. A sequence comparison between proto-ras genes and the known viral ras genes has Proc. Natl. Acad. Sci. USA 84 (1987) 2119 recently revealed a proto-ras-specific exon that was not transduced by any of the known retroviruses with ras genes (39). It is not clear whether the untransduced exon has a regulatory or a coding function (39). It follows, however, that proto-ras and viral ras genes are not isogenic (Fig. 1). Since four different viral ras genes have been shown to lack the same proto-ras exon, and since point mutations are not necessary for transforming function, we have proposed that proto-ras genes derive transforming function for diploid cells by truncation of an upstream exon and recombination with a retroviral promoter (ref. 39, see below). The Claim That the Proto-myc Gene Becomes a Cancer Gene Under the Influence of a Heterologous Cellular Enhancer. Proto-myc is the cellular precursor of the directly oncogenic myc genes of four avian carcinoma viruses (MC29, MH2, CMII, and 0K10) (7). The transforming host range of viral myc genes appears to be limited to avian cells, as murine cells are not transformed by cloned proviral DNAs (56, 57, 75). Nevertheless, it is thought that proto-myc, brought under the control of heterologous cellular enhancers or promoters by chromosome translocation, is the cause of human Burkitt lymphoma or mouse plasmacytoma (30, 67, 76). The following arguments cast doubt on whether such activated proto-myc genes are indeed necessary or sufficient for carcinogenesis. (i) The human proto-myc gene is located on chromosome 8. This chromosome is typically rearranged in B-cell lines derived from Burkitt lymphomas (7, 30, 67). However, although chromosome 8 is subjected to translocations, protomyc is frequently not translocated, and when translocated it is frequently not rearranged (7, 30, 67). Moreover, no rearrangements of chromosome 8 were observed in about 50% of primary Burkitt lymphomas; instead, other chromosome abnormalities were recorded (77). Thus, proto-myc translocation is not necessary for lymphomagenesis. (ii) Expression of proto-myc is not consistently enhanced in lymphomas (7). (iii) No proto-myc gene isolated from any tumor has been shown to transform any cells (7). In an effort to assay transforming function in vivo, a proto-myc gene that was artificially linked to heterologous enhancers was introduced into the germ line of mice (76). Several of these transgenic mice developed lymphomas after 1-5 months, implying that activated proto-myc had transformed diploid cells. However, the lymphomas of the transgenic mice were all monoclonal (76). Thus, if the activated proto-myc gene were indeed responsible for the lymphomas, it would be an extremely inefficient carcinogen, because only 1 of about 108 "control" B cells of the same mouse (78) with the same transgenic myc gene was transformed. There is no deletion or mutation analysis to show that the activated proto-myc indeed played a direct role in the tumors of the transgenic mice (76). In contrast, viral myc genes transform all susceptible cells directly and inevitably (7). (iv) If translocated proto-myc were the cause of Burkitt lymphomas, one would expect all tumors to be diploid and to carry only two abnormal chromosomes-namely, number 8 and the chromosome that was subject to reciprocal translocation with number 8. Instead, primary Burkitt lymphomas exist with two normal chromosomes 8 that carry other chromosome abnormalities (77). Thus, translocated protomyc genes are neither sufficient nor proven to be necessary for carcinogenesis. The Probability of Spontaneous Transformation in Vivo Is At Least 109 Times Lower Than Predicted from Proto-onc Gene Activation. It was estimated above that the probability of spontaneous transformation that leads to monoclonal tumors in humans is 2 X 10-17 per mitosis. One would expect activation of a preexisting, latent proto-onc gene to be a much more frequent event. For a given proto-onc gene, the prob- 2120 Biochemistry: Duesberg ability of activation per mitosis would be the sum of the probabilities associated with each of the five putative pathways (26, 27, 31) of proto-onc activation. (i) Since the probability of a point mutation per nucleotide per mitosis is -10-9 (51, 52), the probability that any one of the 20 known proto-onc genes is activated would be 2 x 20 x 10-9, assuming only one activating mutation per proto-onc gene. However, it would be 10-7 for Harvey ras alone, since 50 different mutations are thought to activate this gene to a dominant cancer gene (see above). (ii) The probability of a given proto-onc gene to be activated by amplification is 10-8, considering that about 1 in 103-10W mitoses leads to gene amplification in vitro (and possibly in vivo) and that about 103 out of the 106 kilobases (kb) of eukaryotic DNA are amplified (79, 80). The probability that any one of the 20 known proto-onc genes would be activated by amplification would then be 2 x 10-7. (iii) The probability of oncogene activation by chromosome translocation depends largely on what the distance is between a proto-onc gene and a heterologous enhancer, and on which enhancers are considered sufficient for activation. Since distances >50 kb of DNA have been considered sufficient for activation of proto-myc (8, 67) and proto-abl (8, 81), the proto-onc gene of murine Abelson leukemia virus (8), and since an enhancer is likely to be found in every 50 kb of cell DNA, nearly every translocation within 50 kb of a proto-onc gene should be activating. Thus the probability that a given proto-onc gene is activated per translocation would be 5 x 10-5 (50 kb out of 106 kb). The probability that one of the 20 known proto-onc genes is activated would then be 10-3 per translocation. Translocation frequencies per mitosis are not readily available. In hamster cells, translocations are estimated to occur with a probability of 10-6 per mitosis (82, 83). In cells directly derived from mice and humans, even higher frequencies (0.01-0.3) have been observed upon study in vitro (84-86). The probability of a translocation per meiotic cell division in humans has been determined to be 10-3-10-4, based on chromosome abnormalities in live births (87). Assuming one translocation in 104 mitoses, the probability that 1 out of the 20 known proto-onc genes is activated per mitosis by translocation would then be about 10-7. (iv) The probability that a proto-onc gene would be activated from without by the promoter or enhancer of a retrovirus integrated nearby is even higher than those associated with the intrinsic mechanisms. Since retrovirus integration within 1-10 kb of a putative latent cancer gene is considered sufficient for activation (8, 22-26), and since retrovirus integration is not site-specific (8, 11) and eukaryotes contain about 106 kb of DNA, a given proto-onc gene would be activated in at least 1 out of 106 infected cells (4, 7). The probability that any one of the 20 known proto-onc genes would be activated would be 2 x 10-5 per infected cell. The sum of these probabilities should reflect the spontaneous transformation frequency of cells per mitosis in vivo and in vitro. It would be between lo-5 and 10-. It should be at least 10-7 due to Harvey proto-ras mutations alone. Nevertheless, the actual number may be 10 times lower, or about 10-8, depending on whether all or only some of these four putative mechanisms could activate a proto-onc gene and depending on whether a given cell is susceptible to transformation by a given onc gene or to a given retrovirus. Instead, spontaneous transformation per mitosis that leads to monoclonal tumors is only about 2 x 10-17 in vivo. Thus, the expected probability of spontaneous transformation due to activation of preexisting oncogenes differs at least by a factor of 109 from that observed in diploid cells in vivo. Again, it may be argued that spontaneous malignant transformation does indeed occur at the above rates but that immunity eliminates nearly all transformants. However, in Proc. Natl. Acad. Sci. USA 84 (1987) this case athymic or nude mice should not exist and the cancer incidence should increase significantly upon immunosuppressive therapy or thymectomy, yet this is not the case (53, 54). Moreover, diploid cells in culture have not been observed to transform at the above rates. (v) Certain cancers (e.g., retinoblastomas) are thought to be caused by activation of oncogenes that are normally suppressed by two allelic suppressor genes (31). Cancers caused by such genes would be the product of inactivations of two allelic suppressors and thus very rare (31). In individuals with genetic defects in one putative suppressor allele, tumors such as retinoblastomas possibly occur due to inactivation of the second suppressor allele with frequencies similar to those estimated above for point mutation, translocation, and retrovirus insertion (31). However, in more than 80% of retinoblastomas that occur in individuals without prior genetic defect, the putative suppressor genes appear to be normal as judged by chromosome analysis (31), arguing either that other suppressors inhibit the putative retinoblastoma oncogene or that it does not exist. Instead, other chromosomal abnormalities that are always seen in such tumors (31) may be relevant to carcinogenesis (see below). Further, this activation hypothesis predicts that normal cellular DNA would cure retinoblastoma cells upon experimental transfection, yet this has not been reported. Likewise, it would be expected that experimental, human-nonhuman heterokaryons that lack chromosomes with suppressor genes would be transformed, but this has also not been reported. Thus there is as yet no proof for suppressed cancer genes in normal cells. The Hypothesis That Activated Proto-onc Genes Require Unknown Complementary Genes for Carcinogenesis. Because of the consistent difficulties in demonstrating oncogenic function of proto-onc genes, a revision of the oncogene concept has recently been favored. It proposes that "activated" proto-onc genes, like proto-ras or proto-myc, are not autonomous onc genes like their viral derivatives but are at least necessary for the kind of carcinogenesis that requires multiple cooperating oncogenes (30, 57, 67, 68, 88, 89). Thus, activated proto-onc genes are proposed to be functionally different from, yet structurally equivalent to, viral onc genes. According to this theory, activated proto-onc genes would not be expected to register in transformation assays that detect single-hit carcinogens like viral onc genes (6, 7). However, this hypothesis fails to provide even a speculative explanation for why activated proto-onc genes are no longer to be considered functionally equivalent to viral onc genes (7). Clearly, until the postulated complementary cancer genes are identified, this hypothesis remains unproven (7). The hypothesis also fails to explain why among certain tumors, such as the human carcinomas, individual carcinomas are only distinguishable from each other by the presence or absence of activated, putative oncogenes (7, 43-45). This implies either (a) that unknown oncogenes that do not register in the 3T3 cell assay would cause the same tumors as the putative oncogenes that do or (b) that the putative oncogenes are not necessary for these tumors. Viral onc Genes as Specific Recombinants Between Truncated Viral and Cellular Genes. Genetic and structural analyses of retroviral genes, viral onc genes, and proto-onc genes and direct comparisons between them have shown that viral onc genes and proto-onc genes are different both structurally and functionally. Therefore, it has been proposed that viral onc genes are indeed new genes that do not preexist in normal cells, rather than being transduced cellular genes (refs. 6, 7, 12, and 18; Fig. 1). The original basis for this proposal was the definition of the transforming gene of avian carcinoma virus MC29 (90) as a genetic hybrid rather than a transduced cellular oncogene (37). It consists of 5' regulatory and coding elements (Agag) from an avian retrovirus linked to 3' coding Proc. Natl. Acad. Sci. USA 84 (1987) Biochemistry: Duesberg proto-onc genes 5'?? S DZ = Retrovirus 8 to 9 kb FIG. 1. The generic, recombinant structures of retroviral onc and their relationship to viral onc genes (stippled) and cellular proto-onc genes (unshaded). The genes are compared as transcriptional units or mRNAs. All known viral onc genes are tripartite hybrids of a central sequence derived from a cellular proto-onc gene, which is flanked by 5' and 3' elements derived from retroviral "fproto-onc" genes. Actual size differences, ranging from >1 to 7 kilobases (kb) (8), are not recorded. The map order of the three essential retrovirus genes, gag, pol, and env, and the splice donor (SD) are indicated. Four groups of viral onc genes are distinguished based on the origins of their coding sequence (C:ID). (Group 1) The coding unit has a tripartite structure of a central proto-oncderived sequence that is initiated and terminated by viral coding sequences. Avian myeloblastosis virus (AMV) is an example (8, 36). (Group 2) The coding unit is initiated by a viral sequence and terminated by a proto-onc sequence. The Agag-myc gene of avian carcinoma virus MC29 is an example (7, 8, 18, 37). The hybrid onc genes of Fujinami avian sarcoma virus (FSV) (38) and Abelson murine leukemia virus (AbLV) are other examples (8). (Group 3) The coding unit of the viral onc gene is colinear with a reading frame of a cellular proto-onc gene. The ras gene of the Harvey and BALB murine sarcoma viruses (HaSV and BaSV) (39) and the myc gene of the avian carcinoma virus MH2 are examples (40, 41). (Group 4) The coding unit is initiated by a proto-onc-derived domain and terminated by a viral reading frame. The src gene of Rous sarcoma virus (RSV) is an example (6, 8). The transcriptional starts and 5' nontranscribed regulatory sequences (?) of all proto-onc genes are as yet not, or not exactly, known (7, 8). There is also uncertainty about 5' translational starts and open reading frames in some proto-onc genes (?) that are not transduced into viral onc genes, as in proto-myc (42), proto-src (6), or proto-ras (39). It is clear, however, that proto-onc-specific regulatory elements are always replaced by viral promoters and enhancers and that proto-onc coding sequences are frequently recombined with viral coding sequences. Thus, all viral onc genes are tripartite recombinant genes of truncated viral and proto-onc genes. genes elements from cellular proto-myc (ref. 37; Fig. 1). Initially this became evident from comparison of the structure and map order of MC29 with that of the three essential retrovirus genes, 5'gag-pol-env 3' (refs. 91 and 92; Fig. 1). Sequence comparison of the viral Agag-myc gene with the chicken proto-myc gene provided direct proof that only a truncated proto-myc gene was present in MC29. Indeed, a complete 5' proto-myc exon was missing from the viral Agag-myc gene (18). This was apparently not an accident, since the same 5' proto-myc exon was also missing in the three other myc-containing avian carcinoma viruses MH2 (40, 41), CMII, and OK10 (7, 93). Thus, a viral and a cellular gene functioned as progenitors or proto-onc genes of each of the viral recombinant myc genes (Fig. 1). More recently, the four known viral ras genes were each also shown to lack a 5' proto-ras exon (ref. 39; see above; Fig. 1). 2121 Comparisons between the onc genes of other retroviruses and the corresponding proto-onc genes proved that all viral onc genes, defined as transcriptional units, are new genes that are recombinants of proto-onc genes and retroviral genes (refs. 6-8; Fig. 1). Most but not all viral genes also encode new recombinant proteins. Based on the origin of their coding elements, the viral onc genes can be divided into four groups (Fig. 1). Group I includes those with amino- and carboxylterminal domains from retroviruses and central domains from proto-onc genes. The onc gene of avian myeloblastosis virus is the prototype (8, 36). Group 2 includes those with aminoterminal domains from viral genes and carboxyl-terminal domains from proto-onc genes. The Agag-myc gene of MC29 is the original example (see above). The onc genes of Fujinami sarcoma virus (38) and Abelson leukemia virus (8) also have the generic Agag-X structure. Group 3 includes those that are colinear with a reading frame of a proto-onc gene. The ras genes of Harvey and BALB murine sarcoma virus (39) and the myc gene of avian carcinoma virus MH2 (40, 41) are examples. Group 4 includes those with an amino-terminal domain from a proto-onc gene and a carboxyl-terminal domain from the virus. The src gene of RSV is the prototype (6, 8). Since three of the four groups of recombinant viral onc genes also encode recombinant proteins, their specific transforming function can be directly related to their specific structure compared to that of proto-onc gene products. The transforming function of the recombinant onc genes of group 3, which encode transforming proteins that are colinear with proteins encoded by proto-onc genes, cannot be explained in this fashion. However, all viral onc genes of this group each lack at least one proto-onc-specific 5' exon, like the avian carcinoma viruses with myc genes (7, 18, 40, 41, 93) or the murine sarcoma viruses with ras genes (39). Conceivably, elimination of transcribed or untranscribed suppressors or elimination of an upstream proto-ras cistron (39) or protomyc cistron (42) and recombination with viral promoters are the mechanisms that generate transforming function (Fig. 1). It follows that viral onc genes and the corresponding proto-onc genes are not isogenic. Viral onc genes are hybrid genes that consist of truncated proto-onc genes recombined with regulatory and, frequently, with coding elements from truncated retroviral genes. These consistent structural differences must be the reason why viral onc genes inevitably transform and why proto-onc genes are not transforming although they are present in all and are active in most normal cells (6, 7). Clearly, if cellular oncogenes preexist in normal cells, it would be much more likely to find retroviruses with intact cellular oncogenes than retroviruses with new onc genes put together from unrelated and truncated viral and cellular genes by illegitimate recombination. However, it may be argued that proto-onc gene truncations reflect packaging restrictions of transducing retroviruses, rather than conditions to activate proto-onc genes. Such restrictions would have to be mostly sequence-specific, as most retroviruses with onc genes can accommodate more RNA [at least 10 kb as in RSV (94)] than they actually contain [3-8 kb (8)]. But there is no evidence that retroviruses discriminate more against certain transduced or artificially introduced sequences (8) than against others, because retroviruses can accommodate very heterogenous sequences, such as the 20 different transformationspecific sequences (6, 7, 8, 12). Yet all nonessential sequences of retroviruses are unstable and hence lost unless selected for a given function (6, 7). Moreover, the fact that the same exons were selectively truncated from proto-onc genes in independent viral transductions that have generated active onc genes indicates that specific truncations are necessary for transforming functions. Examples are selective truncations of proto-myc, the precur- 2122 Biochemistry: Duesberg sor of four avian carcinoma viruses (7, 18, 40); proto-ras, the precursor of three murine sarcoma viruses (39); proto-myb, the precursor of avian myeloblastosis and erythroblastosis viruses (8, 95); proto-erb, the precursor of three avian sarcoma and erythroblastosis viruses (8); proto-fes, the precursor of three feline sarcoma viruses (8); proto-fps, the precursor of three avian sarcoma viruses (8, 96); proto-abl, the precursor of Abelson murine leukemia virus and a feline sarcoma virus (8); proto-mos, the precursor of several Moloney sarcoma viruses (8, 97); and proto-src, the precursor of RSV and two other avian sarcoma viruses (98). In some cases of such selective transductions, the same exons were even truncated at exactly the same breakpoints, as for example in two different avian sarcoma viruses derived from proto-fps (96). The existence of at least five retroviruses containing proto-onc sequences that had already been truncated by recombination with other cellular genes prior to transduction lends further independent support to this view. Examples are the onc genes of avian carcinoma virus MH2 (7, 40, 41), of avian erythroblastosis and sarcoma virus AEV (8), of avian erythro- and myeloblastosis virus E26 (95), of feline sarcoma virus GR-FeSV (8, 99), and of RSV (6, 8). Certainly the odds against transduction of rare rearranged proto-onc genes instead of normal proto-onc genes are overwhelming. Yet five out of the less than 50 known isolates of retroviruses with onc genes (8) contain previously rearranged proto-onc sequences, most likely because truncation is necessary for transforming function. Indeed, it may be argued that these viruses have transduced these rearranged proto-onc genes from a preexisting tumor that was generated by these rearrangements. Thus, the rearranged proto-onc genes of these five oncogenic retroviruses may be "transduced cellular oncogenes" after all. Therefore, truncation of proto-onc genes by recombination with retroviral or cellular genes appears to be necessary to convert proto-onc genes to transforming genes. A definitive assessment of why viral onc genes transform, and cellular proto-onc genes don't, requires more than comparisons of primary structures and transforming tests with DNAs. It will be necessary to know what proto-onc genes do and whether they encode proteins that function alone or as complexes with other proteins. I propose, then, that proto-onc genes that are transcriptionally activated or have undergone point mutations, but retain a germ-line structure, are not cellular cancer genes. I suggest that the hypothesis that proto-onc genes are latent cellular cancer genes that can be converted to active transforming genes, by increased dosage or function, is an overinterpretation of sequence homology to structural and functional homology with viral onc genes. This proposal readily resolves the paradoxes posed by the hypothesis that proto-onc genes are latent cellular cancer genes that can be activated by enhanced expression or point mutation. The proposal accounts for the frequent expression of proto-onc genes in normal cells (7). The proposal is also entirely consistent with the lack of transforming function of "activated" proto-onc genes from tumors. The observation that mutated proto-ras changes the morphology and enhances tumorigenicity of aneuploid and tumorigenic 3T3 cells is important, but not an exception to the experience that native proto-onc genes from tumors analyzed to date do not transform diploid cells. The proposal also provides a rationale for the chromosome abnormalities of tumor cells, as these appear to be microscopic evidence for cancer genes (see below), instead of the "activated" proto-onc genes identified to date. The Hybrid onc Genes of Retroviruses as Models of Cellular Cancer Genes. The proposal that proto-onc genes derive transforming function by truncation and recombination with Proc. Natl. Acad. Sci. USA 84 (1987) retroviral or cellular genes predicts that recombinations among cellular genes could also generate transforming genes. The view that cellular cancer genes are rare recombinants of normal cellular genes is in accord with the fact that rearranged and abnormal chromosomes are the only consistent, transformation-specific markers of tumor cells (1-4, 35). Further, the clonality of chromosome alterations [e.g., the marker chromosomes of tumors (1-4, 35)] indicates that tumors are initiated with, and possibly caused by, such abnormalities, as originally proposed by Boveri in 1914 (100). The generation of retroviral onc genes from viral genes and proto-onc genes could indeed be a model for this process. Less than 50 isolates of retroviruses with onc genes have been documented (7, 8, 11), although both potential parents of retroviral onc genes are available in many animal or human cells because retroviruses are widespread in all vertebrates (4, 8, 11). This extremely low birth rate of retroviruses with onc genes must then reflect the low probability of generating de novo an oncogenic retrovirus from a proto-onc gene and a retrovirus by truncating and recombining viral and cellular genes via illegitimate recombinations (6, 7, 12). Clearly, at least two illegitimate recombinations are required (Fig. 1): one to link a 3' truncated retrovirus with a 5' truncated proto-onc gene, and the other to break and then splice the resulting hybrid onc gene to the 3' part of the retroviral vector. The first of these steps would generate a "cellular" cancer gene that ought to be sufficient for carcinogenesis. The birth of such a gene would be more probable than that of an oncogenic retrovirus that requires two illegitimate recombinations, but it would be harder to detect than a complete replicating retrovirus with an onc gene. Nevertheless, even this would be a rare event. Given that such a recombination would have to take place within the 8-9 kb of a retrovirus (Fig. 1) integrated into the 106-kb genome of a eukaryotic cell and also within an estimated 1-2 kb of a proto-onc gene (Fig. 1), and assuming that translocation or rearrangement occurs with a probability of lo-4, the probability of such a recombination per mitosis would be (8 x 10-6) x (2 X 10-6) X (lo-4) 10-'5. That a second illegitimate recombination is required to generate a retrovirus with an onc gene would explain why the occurrence of these viruses is much less frequent than spontaneous transformation due to recombinant cancer genes. This probability may, nevertheless, be higher than the square of 10-15, since the two events may be linked and since multiple integrated and unintegrated proviruses exist in most infected cells. The probability that illegitimate recombination would generate cancer genes from normal cellular genes would also be very low, since most illegitimate recombination would inactivate genes. Inactivation of certain growth-control genes may in fact be necessary for tumorigenesis. The above estimates for the probability of spontaneous transformation (2 x 10-17) per mitosis and of translocation (10-4), which would be a minimal estimate for illegitimate recombination, suggest that 10P translocations or rearrangements are needed to generate a transforming gene that causes a monoclonal tumor. This could be a single autonomous transforming gene that is like a viral onc gene, or it could be a series of mutually dependent transforming genes (101, 102) that would each arise with a higher probability than an autonomous onc gene. The facts that multiple chromosome alterations are typically seen in tumors (1-3, 35, 77) and that as yet no DNAs have been isolated from tumors that transform diploid cells with single-hit kinetics suggest that most cellular cancer genes are indeed not autonomous carcinogens like viral onc genes. It is consistent with this view that most cellular genes are also not converted to autonomous cancer genes by retroviral transduction via illegitimate recombination and truncation. Only about 20 cellular genes, the proto-onc genes, have been Biochemistry: Duesberg converted to autonomous viral onc genes, although viral transduction via illegitimate recombination is a random event that does not benefit from sequence homology between retroviruses and cells (6, 7, 12). Thus, viral onc genes have not as yet fingered preexisting cellular cancer genes. No cellular gene is a structural or functional homolog of a viral onc gene, but the viral onc genes appear to be models for how cancer genes may arise from normal cellular genes by rare truncation and recombination. I thank S. A. Aaronson, S. Blam, M. Kraus, M. Pech, K. Robbins, S. Tronick and others from the Laboratory of Cellular and Molecular Biology (National Cancer Institute, Bethesda, MD) for critical and amusing discussions and generous support during a sabbatical leave and B. Witkop (National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD) for asking many of the basic questions that I try to answer in this manuscript. I also thank my colleagues H. Rubin for encouragement and K. Cichutek, R.-P. Zhou, D. Goodrich, S. Pfaff, and W. Phares (University of California, Berkeley, CA) for inspiring comments and their work. 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