The Origin of Point Mutations in Human Tumor Cells1 Bernard S

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[CANCER RESEARCH 52. 249-253, January 15. 1992] Perspectivesin CancerResearch The Origin of Point Mutations in Human Tumor Cells1 Bernard S. Strauss2

ICRF Clare Hall Laboratories and Department of Molecular Genetics and Cell Biology, The university of Chicago, Chicago, Illinois 6063 7

The idea that point mutation is central to carcinogenesis is zling since one of the major hypotheses of the 1970s was the an old one (e.g. Ref. l). Until recently, however, specific genetic proposal that carcinogens acted to produce point mutations; changes had not been observed in tumors and a keystone of the that all, or at least the overwhelming majority, of carcinogens belief that genetic change is central to carcinogenesis was what were mutagenic; and that the production of mutations was the appeared to be a close correlation between mutagenicity and mechanism by which carcinogens acted (13). The discovery of carcinogenicity (2). The situation is almost completely reversed transforming oncogenes (14), and particularly the observation today. The class of nonmutagenic carcinogens has been shown by Barbacid et al. (15) that methylnitrosourea-induced breast to be considerable (3). On the other hand, so many genetic tumors in rats were invariably associated with Gâ€”Â»Atransitions changes have been observed in individual cancers that the in the ras oncogene of the tumor, appeared to fulfill one of measured mutation rates in somatic cells seem unable to ac Koch's postulates, that an etiological agent should be invariably count for their appearance. For example, single tumors may associated with the development of disease. Identity of the type contain both ras and p53 point mutations along with chromo of mutation produced in experimental tumors by methylnitro- somal changes (4). A recent contribution to this series postu sourea, i.e., Gâ€”Â»Atransitions,with the mutational specificity lates a hypermutability in tumors to account for the multiple observed in vitro (16, 17) lent credence to the hypothesis that mutations observed (5). However, it may be that no special chemically induced tumors resulted, in the first instance, from mechanisms are required but rather that the estimates of mu point mutation in a growth-controlling gene. It was apparent tation rate thus far reported for somatic mammalian cells and even in these investigations that additional factors were in for neutral mutations in the genome do not reflect the rates in volved, since tumors appeared in animals treated with carcino developing tumors. Such measurements most often use Luria- gen only at a particular phase in the estrus cycle (15), but the Delbruck conditions to estimate preexisting mutations. The role of mutagen appeared to be both necessary and clear. unit in which mutation is measured is the cell generation (6). The ras mutations occurred at only a limited number of Such conditions are not applicable to the situation in tumors. positions and it was possible to look for mutations at these Rather, developing tumors are similar to Escherichia coli kept positions by in situ hybridization techniques using synthetic under selective conditions as described by Shapiro (7), by Cairns oligonucleotides (18). Numerous studies of a variety of human et ai. (8), and by Hall (9) in that mutation is time rather than tumors established that ras mutations could be observed, but replication dependent (9, 10) and that it continues in the only in a portion of tumors. Mutation in ras was neither absence of cell proliferation. The mutations observed in bacteria necessary nor sufficient for all tumor development but it did result in a selective advantage, and the particular mutations occur in some tumors. The base changes involved were limited observed in tumors also appear to give the cells in which they to a few codons and their pattern differed in different types of occur a selective advantage. The mechanism by which these tumor (19). time-dependent mutations occur is unknown, but the specificity That carcinogenesis is a complex process has been under of the mutations observed in tumors implies that at least some stood for some time (see Ref. 20 for a general discussion). In are induced as a result of alterations in the DNA. It is not yet experimental systems, the separation of initiation, promotion, clear how the rate of mutation is to be expressed in quiescent and progression fits a simple hypothesis in which initiation cells held under selective conditions. corresponds to the reaction of DNA with an ultimate carcino gen resulting in a mutation which is converted into a tumor by Mutations in Human Cancer promotion and progression. The age dependence of cancer is in agreement with the hypothesis that multiple events, possibly That genomic change, broadly defined, is a part of the trans mutations, are required for the production of a tumor (21) formation of cells to a malignant phenotype is documented by although opinions differ on the precise number involved. Ac many studies establishing the presence of specific chromosomal cording to the view that malignant tumors are due to the changes in particular tumors (11). Translocations and deletions accumulation of multiple genetic changes (22), tumors would as well as aneuploidy accompany the development and progres result after the accumulation of successive (mutational) events. sion of many tumors (4). Specific translocations are character Each event is supposed to be independent. The last event would istic of some carcinogenic transformation (12). Until recently necessarily occur late and would result in a sudden burst of it was not clear that point mutations (the transitions, transver tumors in old age. Estimates of five to seven events fit the age sions, and simple frame-shifts of experimental genetics) were distribution pattern for many tumors, although for retinoblas- also a part of the carcinogenic transformation. This was puz- toma two events suffice (23). The observation of Fearon and Vogelstein (22) of an accumulation of events accompanying the Received 9/3/91; accepted 10/30/91. 1This paper was prepared during the tenure of a Fogarty Senior International development of malignancy in colon tumors fits the multiple Fellowship from the NIH at the ICRF Clare Hall Laboratories. The work from events hypothesis. the author's laboratory was supported by grants from the NIH and the Depart The oncogenes were defined by an experimental protocol ment of Energy. 2To whom requests for reprints should be addressed, at Department of which required these genes to be dominant (14). A second class Molecular Genetics and Cell Biology, The University of Chicago, 920 E. 58th of relevant genes is recessive, participates in tumorigenesis Street, Chicago, IL 60637. when mutant, and presumably protects against tumorigenesis 249

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. POINT MUTATIONS IN TUMORS when present in the wild type configuration, hence their descrip "generally agreed that mutation rates in mammalian cells occur with a frequency of some 10~5 to 10~6 mutations per cell tion as tumor suppressor genes (24). A wider range of mutations can be detected in such genes, since inactivation can occur by generation" (21). However, it may be that the mutation rates changes at numerous sites, whereas activation of a physiological used for calculation are the wrong rates measured under circum function requires more limited change. The discovery of the stances that are inapplicable to the situation in developing role of the inactivated retinoblastoma gene (25) and of the p53 tumors for reasons presented by Cairns (8) and by Hall (9). The gene (26) has led to a series of studies on the mutations of these mutation rates measured in experimental mammalian systems genes found in human cancers (see references below). The are for selected genes for which "classical" Luria-Delbruck investigations have been made possible by advances in the variance analysis (6) can be used to extract true mutation rates. technology of identifying mutations, particularly by the use of In particular, the rates are measured per cell generation since the polymerase chain reaction which makes determination of it is assumed that this is the appropriate time unit and that the specific mutational change possible without the necessity of mutation requires division. Selection is immediate, in that cells cloning each particular molecule (27). It is this technical devel are plated on media in which they must grow in order to be opment that has made possible the great increase in our knowl scored. [The electrophoretic variants (31) are neutral mutations edge of the mutations present in tumors. The following gener which confer no particular selective advantage.] However, as alizations can be made (a) mutations in p53 can be detected in Cairns (8) and Hall (9) point out, there is another class of tumors of a wide variety of types; (b) in general not all tumors mutations which can be detected when organisms are allowed of a particular type have p53 point mutations (as yet detected); to persist on medium which permits survival but not growth. (c) among those tumors in which p53 mutations are detected, Under these conditions mutations which permit utilization of there is likely to be a loss of heterozygosity, i.e., only the mutant a component in the medium for proliferation can be selected. alÃeleofthe gene is detected (22); and (mutation rate in to be induced by carcinogens and if so, by what carcinogens? these circumstances (33) and it is certainly not yet clear, even These are important questions because the link between carcin in E. coli, what the molecular mechanism of the process may ogenic agents acting as mutagens and carcinogenesis is no be. The process in E. coli depends on the genetic makeup of the longer as clear as it seemed to be to Ames some years ago (13). cells and on the status of genes concerned with DNA repair It might be supposed that an initiating mutation produced a such as at/a, controlling the adaptive response to alkylation, clone of cells, one of which mutated and produced a second and ogt controlling the constitutive C^-methylguanine DNA methyltransferase, since spontaneous mutations from his~ to clone which on mutation of one cell produced a third clone and so on until the eventual production of a clone with a malignant his+ in nondividing cells were increased in ada ogt cells but not tumor phenotype. Loeb (5) and Stein (21) have recently pre in cells carrying only ada or ogt mutant genes (34). Cairns et sented the argument that the spontaneous mutation rate in al. (8) observed that the repair-deficient uvrE mutant produced human somatic cells is not sufficient to account for the number more lac* revertants in nonproliferating cultures than the wild of mutations (or genetic events) that would be required, given type. Some as yet unspecified function of the recA gene seems estimates of five to seven mutations required for (some) cancers. to be involved (35). The argument is based on the estimate of about 1.4 x 10~'Â° Possible mechanisms for the production of such mutations mutation/base pair/cell generation for selected hgprt mutants that need not, in the first instance, be "adaptive" can be sug and 1.2 x 10~10 mutation/base pair/cell generation for unse- gested. The observations that DNA repair, and hence localized lected electrophoretic protein variants (5). Per gene, this cor DNA turnover, depends in many instances on the transcrip- responds to a frequency of 10~5-10~6/cell generation (21). The tional status of a gene (36) may provide an important clue to mutation rates for chromosomal change and for epigenetic the mechanism. For example, inefficient mismatch correction changes are certainly higher than for point mutations (30). The of transcribed genes in stationary cells followed by selection age distribution data indicate that five hits may be required and will result in clones containing one or more "beneficial" muta if these are independent mutations they should occur at a tions. Given a time-dependent mechanism for mutation, mul frequency of (10~5)s = 10~25/cell generation. Stein (21) gives a tiple mutations permitting entry into the cell cycle might ac figure of IO'6 cells in the human per lifetime which corresponds cumulate in single cells. However, as in bacteria (9, 10), such a to (IO16 â€”¿1)generations! He concludes "on a 5-hit model, system would have to eliminate nonselected mutations, since cancer should seldom occurâ€”indeed in not more than 10~9 the rate of such mutations in colorectal cancers remains low down to 10~14 of the populationâ€”that is, never." The argu (Ref. 37, Footnote 35) as is the mutation rate in human colo ments of both Loeb (5) and Stein (21) depend on its being rectal tumor cell lines (38). The observed mutation frequency 250

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. POINT MUTATIONS IN TUMORS for mutations of the 5q21 gene in colorectal cancers was ob ties which may contribute in an important way to their carcin served to be at least 10 times higher than for nonselected ogenic action. For example, carcinogen treatment may result mutations (Ref. 37, Footnote 36). in some epigenetic alteration of the cell population as a whole, The distinction between the conditions for the measurement which makes it possible to respond to subsequent mutagenic of mutation and those required for the changes involved in events. An example of such a (admittedly ill-defined) state is carcinogenesis may provide an explanation for the high rate of the response of populations of rodent cells to doses of ionizing cellular transformation in rodent cells as compared to mutation. radiation which appear to alter all of the cells so that each has Transformation in cell culture is much used as an in vitro model an increased probability of undergoing a mutation-like trans of tumorigenesis (39). The rate of (induced) transformation in forming event (45, 46). The data indicating that whole popu rodent cells is considerably higher than the mutation rate. lations can be induced to a state predisposed to mutate are not Radiation-induced transformation reaches 0.7-0.8% of the vi understood on a molecular level. able cells (39). Landolph and Heidelberger (40) compared mu tation and transformation frequencies induced by acetylami- Induced versus Spontaneous Mutations nofluorene and benzo(a)pyrene in the same cells. They found transformation rates 52 times higher than mutation for acetyl- The discovery that point mutations are often concomitants aminofluorene and 26 times higher for benzo(a)pyrene. Such of carcinogenesis is a confirmation of the early idea that genetic data were never easy to interpret. The cell line used was selected alteration is part of tumorigenesis. Mutations of the retinoblas- for the study of transformation (although it did not transform toma gene are closely enough associated with tumor develop spontaneously) and so a single final event might have been all ment to provide a basis for genetic counseling (47, 48). On the that was required. Although it did not seem likely that mutation other hand, mutations of p53 or ras have not been observed in (or mutations) would occur at such high frequencies, transfor all cases of what appear to be single human tumor types. Insofar mation was brought about by mutagenic agents and for as it is correct that all tumors are idiosyncratic and that "cancer" benzo(fl)pyrene at least, the dose-response curves for mutation is not a single disease, the finding that a particular mutation and transformation had similar slopes. Mutation in these stud does not occur in all cases of what appears to be a single tumor ies was measured to ouabain resistance. Ouabain-sensitive cells type may indicate simply that undifferentiated growth may are killed in the assay. Transformation was measured by allow result from a variety of changes, a concept in line with our ing viable cells to incubate until foci appeared. The similarity increased understanding of the complexities of positive and of these conditions to the bacterial experiments seems clear. It negative growth controls in cells. Nonetheless, the failure to would be interesting to reexamine such transformants using find mutations of a particular oncogene in all cases of what modern techniques for the detection of oncogene mutations. appears to be a particular tumor type makes the necessary association of mutations of these oncogenes with the cancer a Carcinogens as Mutagens hypothesis. A fraction of the particular multiple mutations observed in tumors might yet turn out to be more a result of The results of a long series of experiments to test the hypoth the cancer state than its cause. esis that rodent carcinogens are mutagens (as defined in a Given the existence of numerous point mutations in tumors, Salmonella test system) seems to be "not necessarily." The one can ask whether they occur spontaneously or are induced reverse, that mutagens are (necessarily) rodent carcinogens, is by carcinogens, either endogenous or environmental. [The hy statistically closer to the truth. Ashby and Tennant (3) separate pothesis that endogenous carcinogens might result from normal genotoxic and nongenotoxic carcinogens and conclude that cellular oxidative processes (49) or from hydrolytic decay as a "rodent carcinogenicity can no longer be regarded as a single result of the high concentration of intracellular water (50) entity." Many of the genotoxic and nongenotoxic compounds makes the concept of "spontaneous" mutation harder to define. stimulate cell division, and this stimulation of cell division may If it were possible to eliminate endogenous damage, "sponta itself be a factor which promotes tumor growth (41). One view neous" mutations might be considered as only those due to the is that many otherwise innocuous compounds are toxic at a infidelity of the polymerases and the inefficiency of the mis very high dose and that at such high doses they stimulate match detection systems.] If the mutations are induced, then proliferation. This hypothesis (42, 43) supposes that prolifer their pattern should serve as a "signature" of the mutagens ating cells have a higher mutation rate (because mutation most involved. Although most environmental mutagens produce sev often occurs during cell division) and thus the chance of induc eral kinds of damage in DNA, and therefore different kinds of ing one of the mutations resulting in tumorigenesis is increased. mutation, there are certain hallmarks which distinguish induced Genotoxic compounds are considered to be particularly carcin from spontaneous mutations. Lesions in DNA may be roughly ogenic at higher doses because of the interaction between the classified as instructional or noninstructional depending on the damaged bases produced by these compounds and the increased ability of the altered site to readily form Watson-Crick base replication rate such compounds induce (41, 43). Each round pairs (51). Alkylating agents such as A'-methyl-jV'-nitro-j'V-ni- of replication on a damaged template increases the probability trosoguanidine or methylnitrosourea react with DNA to form of mutation. The argument made by Ames and Gold (42, 43) O^methylguanine as one of their aIkyIat ion products. O6-Meth- and Cohen and Ellwein (41) is complex and in part ad hoc, ylguanine is an instructional lesion and in experimental systems since they find it necessary to postulate additional protective produces mainly G:Gâ€”>A:Ttransitions in DNA (52) as a result factors for cells which are ordinarily proliferative in order to of the mispairing of the altered guanine with T (transition: account for the fact that such dividing tissues do not develop substitution of purine for a purine or a pyrimidine for a pyrim- tumors more often (42). Furthermore, not all tumor promoters idine; transversion: substitution of a purine for a pyrimidine or act by inducing hyperplasia (44). The Ames argument is impor a pyrmidine for a purine.) As indicated above, the finding in tant in emphasizing the finding that carcinogens, including rats that tumors induced by methylnitrosourea all had ras genes those which alter DNA and are mutagenic, have other proper with a Gâ€”Â»Atransitionat codon 12 was an important indication 251

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1992 American Association for Cancer Research. POINT MUTATIONS IN TUMORS that tumors include carcinogen-induced point mutations (15). advantage of such mutations, it is hard to explain their occur Alkylating agents show a peculiar sequence specificity so that rence given the currently accepted rate of somatic cell mutation. when confronted with a run of guanines, e.g., 5' GGG3', It has therefore been postulated that tumor cells must encode mutation tends to occur at the middle or 3' G (53). In contrast, altered enzymes of DNA metabolism that make for an increased the activated forms of polyaromatic hydrocarbons produce mutation rate (5). Such enzymes, if they exist, must maintain mainly noninstructional lesions which along with abasic sites the specificity of somatic cells for induced mutation, since the tend to be replicated by the addition of A opposite the lesion patterns of Gâ€”Â»Ttransversionobserved in lung and certain liver producing a high frequency of Gâ€”Â»TandAâ€”>Ttransversions tumors are reminiscent of those observed for mutation induced (54-56). There are exceptions to this "A rule" (57) but numer by bulky chemical carcinogens in experimental systems (54- ous studies have confirmed that a variety of activated hydrocar 56). It is not yet clear how many point mutations are present bons tend to produce Gâ€”Â»Ttransversions(e.g., Refs. 54-56). in single tumors and we need more information about the Investigations of the specificity of the mutations found in occurrence of ras, p53, and other mutations in the same tumor. human tumors have proceeded far enough to provide a strong If there are indeed multiply (point) mutated tumors, as the inference that at least some of the mutations are induced. The discovery of widespread p53 mutation makes one suspect, then published data indicate that the type of mutagenic change for a an alternative explanation for the high mutation rates is sug particular gene is different for different tumors (28, 58) and gested by analogy with recent studies in bacteria. These studies this difference appears to follow similar patterns in both ras indicate a high mutation rate for selected genes in nondividing and p53 mutations. Tabulation of the changes in K.-ras codon cells, one dependent on time rather than generation. The mo 12 and 13 mutations from 46 tumors indicates 29 transitions lecular mechanism(s) of this adaptive mutation phenomenon is (23 in the 3' G) or 63% of the changes (4, 59). Seventy-nine % unknown although its efficiency does depend on the genetic of p53 colon carcinoma mutations are Gâ€”Â»Atransitions, al makeup of the cell. If such systems operate in mammalian cells, though most of these occur at CpG dinucleotides (28). Many then mutation rates as usually measured are not applicable. of the Câ€”>Ttransitions related to human genetic disease occur This explanation is compatible with the hypothesis of altered within CpG dinucleotides suggesting a role for spontaneous gene products leading to high mutability but only for selected deamination of 5-methylcytosine in their production (60). In genes. What the recent data on mutation in tumors make clear contrast, for both ras and p53 non-small cell lung carcinoma, is that the processes leading to the fixation of mutations in G:C-Â»T:Atransversions are the most common changes. Twelve human cells need further characterization. of 16 (75%) codon 12 or 13 K-ras changes in non-small cell lung carcinoma were transversions (61,62). Twenty-three of 30 Acknowledgments (77%) of p53 mutations in non-small cell lung carcinoma were transversions (28). Two recent studies agree in reporting a high The author would like to thank Drs. Tomas Lindahl, Peter Karran, frequency of Gâ€”Â»Ttransversions at one particular p53 codon Mark Meuth, Barbara Sedgwick, Steven West, and Richard Wood for in hepatocellular carcinomas from particular geographical areas their hospitality and for many helpful discussions. (63, 64). These distributions are not what would be expected from spontaneous change. 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Bernard S. Strauss

Cancer Res 1992;52:249-253.

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