Proc. Nati. Acad. Sci. USA Vol. 78, No. 4, pp. 2465-2468, April 1981 Genetics

Gene-amplification model of carcinogenesis (oncogene/tumor promotion/homogeneously staining regions/initiation/sister chromatid exchange) MARTIN L. PALL Program in Genetics and Program in Biochemistry/Biophysics, Washington State University, Pullman, Washington 99164 Communicated by N. H. Horowitz, January 19, 1981 ABSTRACT A two-stage model of carcinogenesis is proposed plification ofa proto-oncogene requires a series ofunequal sister based on recent evidence for the occurrence of proto-oncogenes chromatid exchanges in different cell cycles. Because each un- in the vertebrate genome, evidence for gene amplification during equal crossing-over would be relatively rare and each must oc- carcinogenesis, and studies ofthe action oftumor promoters. The cur in a different cell cycle, considerable time would almost model is based on the view that an increase in the level of gene always elapse between exposure to carcinogen and the appear- product from such proto-oncogenes is sufficient to induce neo- ance of the transformed cell. plastic transformation. It proposes that the initial step in carcin- This model of carcinogenesis involves large tandem dupli- ogenesis (initiation) is a mutation producing a tandem duplication cations, unequal crossing-over, and sister chromatid exchange, of a proto-oncogene. Gene amplification can then occur by suc- cessive unequal sister chromatid crossing-over events in several all of which have been amply demonstrated in various genetic cell cycles until sufficient gene product is produced to transform studies. the cell. It is especially attractive because it provides a reasonable explanation for the long latency following treatment with a car- cinogen. It is in excellent agreement with the two-state mech- No completely satisfactory model has been proposed to explain anism ofcarcinogenesis (4, 5). The initiating process is the pro- the long latency occurring between exposure ofan organism to duction of the initial tandem duplication, and the subsequent a chemical carcinogen or to ionizing radiation and the appear- promoting process involves cell division and unequal sister ance of cancer in that organism. Such carcinogens are thought chromatid crossing-over. to act as somatic cell mutagens (1) and no long lag would be The major types of evidence supporting the model include expected between mutation induction and expression. studies on the existence and properties of proto-oncogenes in The model ofcarcinogenesis proposed here is based, in part, the vertebrate genome, cytogenetic evidence for gene ampli- on recent studies ofcancer-causing retroviruses. Each such vi- fication during carcinogenesis, and evidence that promoters of rus appears to carry a cancer-causing oncogene, the gene prod- carcinogenesis stimulate sister chromatid exchange. uct of which is required for neoplastic transformation. Some- what surprisingly, the nontransformed host cell carries a similar ONCOGENE EVIDENCE or identical gene, a proto-oncogene (2), which of course nor- Several ofthe viral oncogenes have been studied by nucleic acid mally fails to produce neoplastic transformation in that cell (2, hybridization and been found to be similar or identical to DNA 3). The likely explanation, for which there is good evidence in sequences in the host genome (2, 3, 6-10). Most of these vir- the case ofavian sarcoma virus, is that the amount ofgene prod- ulent transforming retroviruses are defective, having replaced uct produced by the proto-oncogene is insufficient to produce a portion of the required genes for virus replication by the on- transformation, but the much larger amount of gene product cogenic sequence (2, 6-12). Both the hybridization studies and produced by the retroviral oncogene is sufficient to transform the isolation of new defective virulent transforming viruses on the host cell (see Oncogene Evidence below). growth of nondefective retroviruses in the laboratory have led The following model is based on the proposal that chemical- to the proposal that each defective virus was formed by replace- or radiation-induced carcinogenesis is due to the production of ment of some viral genetic information by an oncogenic DNA tandem duplications of a proto-oncogene in the cell genome, sequence from the host cell genome (2, 6, 11). producing a large increase (such as 5- to 10-fold) of the proto- From the terminology of Huebner and Todaro (13) and oncogene gene product. The initial event in carcinogenesis (ini- Bishop et al. (2) the viral genes involved in transformation are tiation) is proposed to be a mutation that produces a single tan- called "oncogenes" and the similar or identical sequences in the dem duplication of a proto-oncogene. This should produce a vertebrate genome are designated "proto-oncogenes." As in- 50% increase in gene product in a diploid cell, insufficient to dicated above, there is evidence for such proto-oncogenes cor- transform that cell. However, after DNA replication has oc- responding to several viral oncogenes, and several ofthese have curred, two sister chromatids will be formed, each of which been shown to be transcribed in nontransformed cells (2, 3, carries a tandem duplication (Fig. 1). This cell or one ofits prog- 6-12). The evidence on proto-oncogenes is best in the case of eny is proposed to undergo infrequent unequal sister chromatid avian sarcoma virus oncogene, src, and its homologous proto- crossing-over, generating a chromatid with three tandem genes oncogene, sarc or proto-src (2), and includes the following: and one with one (Fig. 1). After subsequent rounds of cell di- proto-src is present (2, 14-18), transcribed (17, 18), and trans- vision and DNA replication, additional unequal sister chromatid lated (18-22) in the nontransformed cell. The translation prod- exchanges may occasionally occur, generating still higher du- uct is very similar in structure (20-22), immunological activity plications, until the minimum amount of gene product is syn- (20-22), and enzymatic activity (20-22) to the src gene product thesized to transform the cell. By this model, then, gene am- in the avian sarcoma virus-transformed cell. The proto-src gene can be transferred to the avian sarcoma virus genome by re- The publication costs ofthis article were defrayed in part by page charge combination into a mutant deleted for src and, when so trans- payment. This article must therefore be hereby marked "advertise- ferred, appears to be capable ofhigh-level transcription/trans- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. lation and of transforming the host cell (23-25). 2465 Downloaded by guest on September 24, 2021 2466- Genetics: Pall Proc. Natl. Acad. Sci. USA 78 (1981)

All except possibly the extreme 5' end of the new src gene chromosomes appear to be derived from homogeneously stain- in some of these recombinants is derived from the proto-src ing regions in at least some cases (33). Double minute chro- sequence ofthe host cells (26). Thus, it appears that all or almost mosomes have been reported to occur in a wide variety ofneo- all of the genetic information in this oncogene is derived from plastic cells (32, 33, 35, 37-42), but are rare in nonneoplastic the host cell genome. cells (35, 37). These studies suggest that the amplification of a Nontransformed cells have only about 1% as much src-type gene, possibly a proto-oncogene as suggested here, commonly gene product as do transformed cells (2), supporting the sug- accompanies the development ofmalignancies. gestion that the level of this gene product is crucial in deter- mining whether it produces neoplastic transformation. EXPERIMENTAL TESTS AND RELATED APPLICATIONS CYTOGENETIC EVIDENCE Three experimental tests of the model are suggested here. The A model of gene amplification similar to that suggested above first is to use cloned, labeled DNA probes to detect changes in has been proposed in the case ofthe stepwise induction ofmeth- proto-oncogene DNA in transformed cells by using the South- otrexate resistance (27, 28). Resistance to methotrexate appears ern blot (43) method. Changes produced by tandem duplica- to be produced by multiple tandem duplications of the struc- tions should include both an increase ofintensity ofrestriction tural gene for dihydrofolate reductase. Gene amplification is fragments included in such duplications and the possible pro- also reported to be the genetic mechanism of resistance to an- duction of new fragments spawning DNA contained in two ad- other drug (29). jacent duplications. Cloned probes to be used in such studies The tandem gene duplications of the dihydrofolate reductase could be derived from oncogenes from a large variety oftrans- gene are usually found to occur in homogeneously staining re- forming retroviruses. Because there are estimated to be at least gions of a chromosome (28, 30, 31). Presumably, such regions a dozen and quite possibly a much larger number ofoncogenes stain homogeneously because they are genetically homogene- (44), it is likely that many such probes will be required for a ous, representing multiple tandem copies of the same DNA serious test of the model. sequence. They may be viewed (30) as the "cytological conse- A second test would involve using such labeled probes to quence of chromosome amplification." perform in situ hybridization of neoplastic cells possessing ho- Homogeneously staining regions have been widely reported mogeneously staining regions. The model predicts that for the to occur in malignant cells (32-35). They occur in a wide variety proper combination ofprobe and cell line, the homogeneously of tumors and, as reviewed earlier (35), appear to be common staining region will be labeled by the probe. in neoplastic cells and very rare in nonneoplastic cells. In one A third related approach would be to isolate double minute recent study ofhuman tumors, 5 out of 16 tumors had most or chromosomes from tumors and to clone DNA segments from all cells showing a chromosome with a homogeneously staining them. Such cloned segments could then be used as probes to region (34). The occurrence ofhomogeneously staining regions determine if other similar tumors have experienced amplifica- in malignancies may be taken as evidence that gene amplifi- tion for the same sections of the genome. cation commonly accompanies the development of the malig- If the model can be confirmed by these or other tests, it nancy. It is suggested here that the amplification may be of a should lead to important, practical approaches to cancer treat- proto-oncogene. ment. Individual tumors could be screened by Southern blot The other cytogenetic manifestation of gene amplification is tests to determine which proto-oncogene was amplified during the occurrence ofdouble minute chromosomes. These carry the its development. Different tumors with a common molecular amplified dihydrofolate reductase genes in unstable, but not basis may be susceptible to a common strategem of treatment. stable, methotrexate-resistant cell lines (36). Double minute This treatment might involve conventional surgical removal and

PROTO -ONC I PROTO - ONC PROTO-ONCj PROTO-ONCi~1 | PROTO-ONC| PROTO-ONC PROTO -ONC PROTO-ONC Cell Division(s) DNA Replication(s) IPROTO-ONCIPROTO-ONCIPROTO-ONCI |PROTO-ONC PROTO-ONC PROTO-ONCI Cell Division DNA Replication [PROTO-ONC PROTO -ONC PROTO-ONC PROTO -ONC PROTO -ONC PROTO -ONCIPROTO-ONCIP|ROTO-ONCIP|ROTO-ONCI PROTO-ONCI FIG. 1. Unequal sister chromatid exchange leading to gene amplification ofproto-oncogenes (designated proto-onc). Sections ofsister chromatids are shown with the position of crossing-over designated "X". Downloaded by guest on September 24, 2021 Genetics: Pall Proc. Natl. Acad. Sci. USA 78 (1981) 2467 radiation therapy or chemotherapy, or both. Alternatively, im- motion events to occur at higher rates. Along these lines, it munological or other specific attacks on the transformed cells would be interesting to determine if processes leading to dele- might be used, based on specific cell-surface or other changes tion or translocation might also generate tandem duplications produced by amplification of a particular proto-oncogene. The next to the point of the chromosome break. latter approach may be a very promising one. A second category ofmalignancy which does not fit the simple The model also suggests approaches to screening chemicals gene amplification model is that of virus-induced transforma- for initiation or promotion activity. Initiation could best be de- tion. These may be rare in humans (11). tected by developing mutagen screening systems for specifically A third group of tumors which may occur by an unrelated measuring rates ofinduction of large tandem duplications. Pro- mechanism are those thought to be of nongenetic origin. The moters should be screened via their effects on sister chromatid most likely examples of this type are the murine teratocarci- exchange. The methotrexate-resistance system (27, 28) dis- nomas (53, 54). Genetically marked cells derived from terato- cussed above may be useful in both ofthese screening problems. carcinomas, when injected into mouse embryos, can develop into a wide variety of normal mouse cells and tissues and, when PROMOTERS OF CARCINOGENESIS incorporated into the germ line, can produce normal progeny The gene-amplification model of carcinogenesis predicts that (53, 54). The finding that cells derived from teratocarcinoma promoters ofcarcinogenesis should act by stimulating both cell cells often behave quite normally, when placed into a normal division and sister chromatid exchange. There is a large amount developmental situation in a mouse embryo, has led to the in- of data, which will not be reviewed here, that promoters do ference that the teratocarcinoma cells are genetically normal stimulate cell division. In addition, a known promoter (phorbol and that their neoplastic transformation is due to developmental ester) has been shown to stimulate sister chromatid exchange aberrations of gene expression (53). Although this inference is (45, 46), but a chemically similar nonpromoter was inactive (45). attractive, there is an alternative interpretation based on the Certain antipromoters inhibited this stimulation (45, 46). Many gene-amplification model. Unlike most mutations, tandem du- complete carcinogens that act as initiators and promoters stim- plications can revert at high frequencies; the extra gene copies ulate sister chromatid exchange (47, 48). Consequently, a sub- are readily deleted by crossing-over or loss of double minute stantial amount of data exists supporting this prediction. It chromosomes, or both. Thus, it is possible that the teratocar- should also be noted that Bloom syndrome individuals, who are cinoma was generated by gene amplification and that genetically chromatid normal progeny cells were produced from the tumor cells by predisposed to cancer, have high frequencies ofsister loss ofthe extra gene copies. Ifsuch normal cells have a selective exchange (49). advantage in the embryo, they might show up in the mouse tissues to the exclusion of tumor cells. APPLICABILITY OF THE MODEL Because of the many diverse types of malignancies, it is im- I thank B. Gerdes, D. Ward, and R. Reeves for helpful discussions. portant to consider which are most likely and least likely to be 1. Ames, B. N. (1979) Science 204, 587-593. the result of the proposed gene-amplification mechanism. The 2. Bishop, J. M., Courtneidge, S. A., Levinson, A. D., Opper- most likely are those showing homogeneously staining regions mann, H., Quintrell, N., Sheiness, D. K., Weiss, S. R. & Var- or double minute chromosomes because of the evidence for mus, H. E. (1980) Cold Spring Harbor Symp. Quant. Biol. 44, gene amplification in the generation of these chromosomal ab- 919-930. normalities. It should be emphasized, however, that tumors 3. Roussel, M., Saule, S., Largrou, C., Rommens, C., Boug, H., Graf, T. & Stehelin, D. (1979) Nature (London) 281, 452-455. without either of these chromosomal properties may have ex- 4. Berenblum, I. (1941) Cancer Res. 1, 44 48. perienced gene amplification in a chromosome region too small 5. Friedewald, W. F. & Rous, P. (1944)J. Exp. Med. 80, 101-126. to be easily identifiable by standard cytogenetic techniques. 6. Scolnick, E. M., Rands, L., Williams, D. & Parks, W. P. (1973) Consequently, it is possible that a large fraction of malignancies J. Virol. 12, 458-463. are produced by the proposed mechanism. 7. Rasheed, S. (1980) Cold Spring Harbor Symp. Quant. Biol. 44, However, three specific categories of malignancies must be 779-786. 8. Baltimore, D., Shields, A., Otto, G., Goff, S., Besmer, P., considered to be unlikely candidates for generation by this Whitte, 0. & Rosenberg, N. (1980) Cold Spring Harbor Symp. mechanism unless additional features are assumed. One ofthese Quant. Biol. 44, 849-854. are the malignancies characterized by specific deletions or 9. Stehelin, D., Saule, S., Roussel, M., Sergeant, A., Lagrou, C., translocations, such as the deletion commonly found in reti- Rommens, C. & Raes, M. B. (1980) Cold Spring Harbor Symp. noblastomas (50) and the translocation (Philadelphia chromo- Quant. Biol. 44, 1215-1223. some) found in chronic myelogenous leukemia (51, 52). The 10. Sheiness, D. K., Hughes, S. H., Varmus, H. E., Stubblefield, E., Bishop, J. M. (1980) Virology 105, 415424. association between a specific chromosomal change and a spe- 11. Temin, H. M. (1980) Cold Spring Harbor Symp. Quant. Biol. 44, cific type of malignancy suggests, but does not prove, that the 1-8. chromosome change has some causal relationship to the neo- 12. Oskarsson, M., McClements, W. L., Blair, D. G., Maizel, J. V. plastic transformation. Clearly ifthere is such a causal relation- & Vande Woude, G. F. (1980) Science 207, 1222-1224. ship, the gene-amplification model, at least in its basic form, 13. Huebner, R. J. & Todaro, G. J. (1969) Proc. Natl. Acad. Sci. USA cannot be involved in these neoplastic transformations. Modi- 64, 1087-1094. 14. Stehelin, D., Guntaka, R. V., Varmus, H. E. & Bishop, J. M. fication of the model can be suggested, however, which would (1976) J. Molec. Biol. 101, 349-365. require a specific translocation or deletion in order to generate 15. Astrin, S. M. (1978) Proc. Natl. Acad. Sci. USA 75, 5941-5945. gene amplification producing a large increase in gene product. 16. McClements, W., Hanafusa, H., Tilghman, S. & Skalka, A. For example, ifa proto-oncogene is under negative autogenous (1979) Proc. Natl. Acad. Sci. USA 76, 2165-2169. regulation, it might be necessary to remove the structural gene 17. Spector, D. H., Smith, K., Padgett, T., McCombe, P., Roul- away from an adjacent operator site in order for gene amphfi- land-Doussoix, D., Moscovici, C., Varmus, H. E. & Bishop, J. M. (1978) Cell 13, 371-379. cation to generate a large increase in gene product. Alterna- 18. Spector, D. H., Baker, B., Varmus, H. E. & Bishop, J. M. (1978) tively, a chromosomal translocation might move a proto-onco- Cell 13, 381-386. gene to a chromosomal location characterized by higher 19. Collett, M. S., Brugge, J. S. & Erikson, R. L. (1978) Cell 15, frequencies ofsister chromatid exchange, thus allowing the pro- 1363-1369. 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