Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5669-5673, June 1995 Genetics

Adaptive in as a model for the multiple mutational origins of tumors (cancer) BARRY G. HALL Biology Department, River Campus, University of Rochester, Rochester, NY 14627 Communicated by Evelyn M. Witkin, Rutgers University, Piscataway, NJ, March 9, 1995

ABSTRACT The cells in most tumors are found to carry evidence led to the dogma that selection and were multiple mutations; however, based upon mutation rates completely separate processes in which mutations occurred determined by fluctuation tests, the frequency of such multi- completely randomly with respect to their eventual effect on ple mutations should be so low that tumors are never detected the organism, and selection acted only to alter the frequencies within human populations. Fluctuation tests, which determine of those random mutations in subsequent generations. the cell-division-dependent mutation rate per cell generation The universality of that dogma has recently been strongly in growing cells, may not be appropriate for estimating challenged by a series of studies which have shown that during mutation rates in nondividing or very slowly dividing cells. periods of prolonged, intense selection, microbial cells can Recent studies of time-dependent, "adaptive" mutations in specifically generate mutations that are useful to them at the nondividing populations of suggest that sim- time. The findings of these recent studies do not conflict with ilar measurements may be more appropriate to understanding those of Luria and Delbruck and others because all those the mutation origins of tumors. Here I use the ebgR and ebgA earlier studies employed lethal selections that could detect genes of Escherichia coli to measure adaptive mutation rates only preexisting mutations. All of the recent studies have where multiple mutations are required for rapid growth. employed nonlethal selection, which can detect both preexist- Mutations in either ebgA or ebgR allow very slow growth on ing mutations and mutations that may be caused by the lactulose (4-0-fJ-D-galactosyl-D-fructose), with doubling times selection itself. of 3.2 and 17.3 days, respectively. However, when both muta- Adaptive mutations (sometimes called directed, selection- tions are present, cells can grow rapidly with doubling times induced, or Cairnsian) are spontaneous mutations that occur of 2.7 hr. I show that during prolonged (28-day) selection for during periods of prolonged stress as specific responses to growth on lactulose, the number oflactulose-utilizing mutants environmental challenges and that occur more often when they that accumulate is 40,000 times greater than can be accounted are selectively advantageous than when they are selectively for on the basis of mutation rates measured by fluctuation neutral. The key feature that distinguishes "adaptive" from tests, but is entirely consistent with the time-dependent adap- "random" mutations is their specificity; i.e., they appear to tive mutation rates measured under the same conditions of occur only in the genes that are under intense selection. prolonged selection. Adaptive mutations have been shown to occur in at least eight loci in E. coli (11-15), at one locus in Clostridium thermocellum The origins of many cancers and tumors involve progressive (16), and at three loci in (17, 18). chromosomal aberrations and multiple mutations in different They have been shown to arise by base substitutions (11, 14, 17, genes (1-5). About 25% of human colon cancers contain more 19), by both positive and negative frameshifts (18-20), and by than nine mutations (6), and Loeb (7) argues that most tumors precise excision of mobile genetic elements (IS150 and Tn3) contain at least four mutations, while Stein (8) argues for five (12, 15). A report purporting to show that excision of IS150 is mutations. Whether four, five, or more mutations are required, not adaptive (21) has now been refuted (22). Adaptive muta- Loeb and Stein agree that if the mutations occur indepen- tions are thus a general phenomenon in terms of the loci at dently at about 1.4 x 10-10 per base pair per cell division (7), which they can occur, in terms of the nature of the mutations, the frequency of human tumors should be so low that tumors and in terms of their occurrence in both prokaryotes and are never detected (7-9). eukaryotes. The evidence that multiple mutations play a causal role in In a typical experiment a strain of E. coli that requires a the development of human tumors is so strong that the failure nutrient, such as , is grown on solid medium that of measured mutation rates to account for the observed contains a limiting supply of that nutrient. When the nutrient number of mutations indicates a fundamental failure in un- is exhausted, there is a sparse population present on each plate. derstanding the origins of mutations in humans. Either the Mutants, typically revertants that no longer require the nutri- measurements of spontaneous mutation rates have been off by ent, continue to grow and form small colonies, or papillae, on several orders of magnitude, which seems very unlikely, or the the sparse bacterial lawn. The number of mutant colonies that wrong thing has been measured; i.e., the measured rates are appear within a day after the nutrient is exhausted is consistent irrelevant to the phenomenon of tumorigenesis. with the number that would be expected based on the rate at Luria and Delbruck (10) showed that, in Escherichia coli, which that allele spontaneously mutates as estimated by the mutations could occur in the absence of the environmental Luria-Delbruck fluctuation test (10). During continued incu- challenge that selected for the mutant phenotype. This meant bation new mutant colonies appear continuously over periods that those mutations had not been caused by selection, but that ranging from a few days up to several weeks (for reviews see selection had acted only to reveal the existence of preexisting refs. 23 and 24); i.e., the measured random mutation rate does mutations. As additional studies in both bacteria and higher not correctly predict the ultimate number of mutants that organisms confirmed that view, a stronger interpretation of the appear in the population. Adaptive mutations appear to be specific to the gene that is The publication costs of this article were defrayed in part by page charge under selection; i.e., during selection for Trp+ revertants in a payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: IPTG, isopropyl ,B-D-thiogalactopyranoside. 5669 Downloaded by guest on September 29, 2021 5670 Genetics: Hall Proc. Natl. Acad. Sci. USA 92 (1995) trpB cysB double mutant, no Cys+ revertants were detected, density, which varied from experiment to experiment, was 5 x nor were Trp+ revertants detected when the same strain was 108 to 2.5 x 109 cells per plate, after which lactulose-utilizing subjected to selection for Cys+ revertants (14). mutants appeared as papillae (small colonies on the thin lawn) Strauss (9) has discussed the potential relationship between over the next few weeks. adaptive mutations in bacteria and the origin of mutations in Measurement of Adaptive Mutation Rates. The number of tumor cells. He has pointed out that spontaneous rates are lactulose-utilizing papillae was counted each day. Because measured by fluctuation tests in growing cell populations and reconstruction tests have shown that papillae are visible within are probably irrelevant to the development of most tumors. 48 hr, each papilla was scored as a mutation that occurred 2 Instead, he argues, developing tumors are similar toE. coli cells days previously. The number of viable cells per plate was kept under nondividing conditions during prolonged selection. estimated by suspending plugs from lactulose selection plates Under those conditions the relevant measure is the time- and plating appropriate dilutions onto rich medium or, when dependent adaptive mutation rate, and mutation continues in X342LD scavenger cells were present, onto CMSD-met me- the absence of cell proliferation. dium upon which the metB- X342LD cells cannot grow. The The purpose of this paper is to examine further the parallels adaptive mutation rate was calculated as the average number between adaptive mutations in microorganisms and the origins ofmutations per plate divided by the number ofviable cells per of mutations that ultimately lead to the development of tumors plate and is expressed as the number of mutations per cell per in higher organisms. In particular, the purpose is to ask day. whether adaptive mutations in E. coli may serve as a useful model to explain the multiple mutational origins of tumors. RESULTS System for Detecting Multiple Mutations. The ebg operon of MATERIALS AND METHODS E. coli, located on the opposite side of the chromosome from E. coli K-12 Strains. The strains employed in this study were the lac operon, encodes a second ,B-galactosidase that is an DS4680A (genotype, AlacZ4680 spc HfrC; Ebg phenotype, alternative to the better known lacZ ,B-galactosidase system. wild type), lB1 (genotype, ebgR AlacZ4680 spc HfrC; Ebg Mutations in the ebg operon can allow lacZ deletion strains of phenotype, constitutive synthesis of wild-type enzyme), 5A1 E. coli to utilize lactose and other ,3-galactoside sugars as sole (genotype, ebg451 AlacZ4680 spc HfrC; Ebg phenotype, in- carbon and energy sources (26, 29-31). The ebgAC genes ducible synthesis of Ebg+I" enzyme), and X342LD (genotype, encode the a and 83 subunits, respectively, of Ebg enzyme, AlacZ4680 metBI HfrC; Ebg phenotype, wild type). while the ebgR gene encodes a repressor that controls expres- Media. MS (mineral salts) medium (25) contained glucose sion of the ebgAC genes (26, 32). The wild-type Ebg enzyme (2 mg/ml), lactulose (4-0-f3-D-galactosylfructofuranoside, 1 (ebgA0 gene product) hydrolyzes lactulose (galactosyl-3-1,4- mg/ml), or glycerol (0.1 ,ul/ml) as carbon sources. When fructose) very inefficiently, and the Ebg repressor (ebgR+ gene lactulose was the carbon source isopropyl f-D-thiogalactopy- product) is virtually insensitive to lactulose as an inducer (26, ranoside (IPTG, 0.2 mM) was included to induce expression of 33). As a result, AlacZ strains are unable to utilize lactulose as the lacY-encoded ,B-galactoside transport system. IPTG does a carbon and energy source. not induce expression of the ebg operon (26). A mutation from G to either C or T at base pair 4223 of the CMSD-met medium (25) is a defined, complete medium ebg operon (ebgA+'I allele) results.in the replacement of Trp977 that includes each of the 20 common amino acids, except by Cys in the a subunit of Ebg enzyme (32) and increases the methionine, at 20 ,ug/ml. activity toward lactulose by 30-fold at physiological substrate L agar (27) was used as a rich medium. concentrations (34). Mutations to ebgR- result in constitutive Growth Rates. The number ofviable cells per plate (N,) was synthesis of Ebg enzyme as about 5% of the soluble protein of estimated by suspending plugs from lactulose selection plates the cell (26). Together these two mutations permit growth on. and plating appropriate dilutions onto rich medium. The lactulose at the rate of 0.26 hr-1 (a doubling time of 2.7 hr). growth rate, k, was estimated from a least-squares fit of ln(N,) Neither of these mutations alone permitted detectable vs. t, based upon the relationship N, = Noekt, where t is growth on lactulose (growth rate < 0.03 hr-1, doubling time expressed in days and k is expressed in days-1. >35 hr) (refs. 26 and 32; unpublished results). Selection for Fluctuation Tests. Random mutation rates were determined growth on lactulose by the AlacZ strain DS4680A provides a by fluctuation tests. A set of independent 10-ml cultures were means to study adaptive multiple mutations. grown in glycerol mineral salts medium containing IPTG. Growth Rates of the Single-Mutant Intermediates. Al- Samples (10 ,l) were removed from 10 cultures, diluted, and though neither single mutant (ebgR- ebgA0 or ebgR+ ebgA+II) spread onto rich medium to determine the number of viable permitted detectable growth on lactulose as measured by cells per culture, after which the entirety of each culture was growth in liquid medium over a period of about 24 hr, it was concentrated by centrifugation and spread onto a plate con- important to determine the growth rates of those intermedi- taining lactulose minimal medium plus IPTG. ates during prolonged incubation on plates-the conditions The average number of mutations that occurred during the under which the multiple-adaptive-mutation experiment growth of each culture (A) was determined from the distribu- would be carried out. Fig. 1 shows the growth of each single tion of the number of colonies per culture according to the mutant on lactulose plates. The growth rate of strain lB1 method of Stewart et al (28) for analysis of fluctuation tests, (ebgR- ebgA0) was 0.04 ± 0.022 day-1 (growth rate ± 95% as implemented by Stewart's DATAFIT program. The average confidence limits), which is a doubling time of 17.3 days; the mutation rate during growth of the cultures was calculated as growth rate of strain SA1 (ebgR+ ebgA4+1) was 0.222 ± 0.053 ,u/mean number of cells per culture and is expressed as day-1, which is a doubling time of 3.2 days. Note that the points mutations per cell division. in this particular experiment could be interpreted as strain 5A1 Selection of Lactulose-Utilizing Mutants During Prolonged lagging for 5 days, then beginning to grow at the rate of 0.286 Incubation on Plates. Approximately 105 cells were spread day-1, and strain lB1 lagging for 8 days, then beginning to onto lactulose selection medium, which was MS medium grow at 0.116 day-'. This interpretation would affect only the containing a limiting supply of glycerol (0.01%), an excess of expected number of double mutants in Fig. 4, where it slightly lactulose (0.1%), IPTG, and (when X342LD scavenger cells improves the agreement between observed and expected were used) methionine (100 ,ug/ml). The plates were incu- values. bated at 30°C in a humidified chamber to prevent dehydration Mutation Rates. Random mutation rates. The random mu- of the medium. Glycerol was exhausted when the population tation rates in growing cultures were determined by fluctua- Downloaded by guest on September 29, 2021 Genetics: Hall Proc. Natl. Acad. Sci. USA 92 (1995) 5671 strains. Fig. 2A shows the mutation rates from ebgR+ to ebgR- strainS 5A1 (ebgR+ ebgA+H) 1 06 in three experiments in which the number of initial SA1 cells 0 - *.I -'3~ -strain lB1 (ebgR7 ebgA°)| was varied, and also shows the "combined" rates that were obtained by averaging the common results of the three exper- 0L. iments. The mutation rate to ebgR- increased from 2 x 10-9 0. per cell per day to 10-5 per cell per day over a period of 10 days. a(o _ In contrast, the mutation rate to ebgA+"I remained fairly 0 1 05 0 constant over a period of 4 weeks (the change in mutation rate was 7.3 ± 19.7 x 10-11 per cell per day per day, and the z 0,<;~~~~ probability that the slope was not different from zero was 0.23), 0 _q__ with the average mutation rate over that period being 2.5 ± 104 0.45 x 10-9 per cell per day (Fig. 2B). 0 5 10 15 20 The mutations that occurred during prolonged selection on Day lactulose medium cannot be accounted for on the basis of the random mutation rates that were measured in growing cells. FIG. 1. Growth of single-mutant intermediates on lactulose. The Fig. 3 shows the observed accumulation of lactulose-utilizing growth rates of strains SA1 (0) and lB1 (o) on lactulose minimal mutants compared with that which would be expected from medium were measured as described. random, replication-dependent mutations. tion tests (10) as described in Materials and IWethods. The The mutations to ebgR- during prolonged selection for mutation rate (,u) from ebgR+ to ebgR- was mleasured in 61 lactulose utilization were nonrandom also in the sense that independent cultures of strain SA1 and was 7.6 x:10-8 per cell they were specific to the presence of lactulose in the medium. division. The mutation rate from ebgA0 to ebgA1+11 was mea- Strain SA1 was spread onto medium containing only a limiting sured in 100 independent cultures of strain lBi zand was 1.8 x concentration of glycerol (0.01%), and the plates were incu- 10-10 per cell division. bated for 17 days. At intervals during that period cells were Adaptive mutation rates. Mutation rates durin ng prolonged washed from those plates, concentrated, and spread onto selection on lactulose medium were determinecd in the same lactulose plates to determine the frequency of ebgR- (lactu- lose-utilizing) mutants. If the mutation rate to ebgR- were the A io- * Experiment 1 same during carbon starvation in the absence of lactulose as it * Experiment 2 * was in the presence of lactulose, then the of * Experiment 3 frequency ebgR- * * * mutants should increase dramatically over that period. In + Combined contrast to that expectation, the frequency remained constant - -0.10 (the change in frequency was -1.4 ± 9.1 x 10-5 per cell per 00o 106 day per day, and the probability that the slope was not different S from zero was 0.75). ~o1C 10 0 These observations indicate that the observed mutations to O1 o-7 ebgR- and to ebgA"II during prolonged selection on lactulose 0 * are not random, replication-dependent mutations but are 'S E 1io-8 indeed adaptive mutations. This is evidence that adaptive -6 mutations occur in growing cells. 1o4109 1000 0 2 4 6 8 10 12 14 ,l 0 100 B 104 0 7~~~~ S 0 .o~~~~~~~~L * n A~~~AA * *2** 10 *~v .~~~~~~~~~0co 'a0 * * o * U. S+-++-+++ : * * z 6 . 0 0 .. Ea 00oooooooo O 0.1 *r 0 oooeoogA a I l0 .~~~~ Is io1 * * ~~~~~~~~d AA 0 ~~~~z : a

. 1 ...... 0 ~~0 0 0.01 6 0 5 10 15 20 25 C ~~~~I Day 0 5 10 15 20 25 30 FIG. 3. Observed and expected accumulation of lactulose-utilizing Day mutants during prolonged selection. The expected accumulations were calculated from the number of cells in the experiment, the random FIG. 2. Mutation rates during prolonged selection on lactulose. (A) mutation rates (,u) as measured by fluctuation tests, and the measured Mutation rates from ebgR+ to ebgR- in strain SAL. The number of SA1 growth of the cells during the experiments shown in Fig. 2. The cells was varied by mixing strain SA1 in various proportions with observed accumulation of ebgR- mutants is based upon the data X342LD scavenger cells. When the glycerol was exhausted the number shown in Fig. 2A, and the observed accumulation of ebgA+II mutants of 5A1 cells per plate was 9.6 x 106 in experiment 1 (48 plates used), based upon the data in Fig. 2B. Solid symbols are observed values and 1.3 x 105 in experiment 2 (25 plates used), and 5.5 x 103 in experiment open symbols are expected values. Squares, circles, and triangles are 3 (25 plates used). (B) Mutation rates from ebgA0 to ebg4A+" in strain values for mutations from ebgR+ to ebgR- in strain 5A1 in experiments lB1. When glycerol was exhausted the number of cells per plate was 1, 2, and 3, respectively. Diamonds are mutations from ebgA0 to 4 x 108 (48 plates used). ebgA4+I in strain lBi. Downloaded by guest on September 29, 2021 5672 Genetics: Hall Proc. Natl. Acad. Sci. USA 92 (1995)

Multiple Mutations. The results at this point can be sum- by fluctuation tests, and from the growth rates of each of the marized as follows. Wild-type cells cannot grow on lactulose. single-mutant intermediates (Fig. 4). A total of 28 lactulose- Mutations that allow constitutive synthesis of wild-type Ebg utilizing double mutants were obtained in this experiment, enzyme (ebgR-) permit cells to grow very slowly (doubling which is 40,000 times more than the 0.0007 that would have time, 17.3 days) on lactulose, and mutations that permit been expected, based solely upon random mutations, by day 28. inducible synthesis of class II mutant enzyme (33) (ebgA+41) In contrast, when the adaptive mutation rates each day were also permit cells to grow slowly (doubling time, 3.2 days) on used, the expected number of mutants was very close to the lactulose. While those mutants are growing slowly, selection observed number each day (Fig. 4). It is important to note that can induce secondary mutations at either a constant rate the adaptive mutation rates from ebgR+ to ebgR- and from (secondary mutations to ebgA +II) or a rapidly accelerating rate ebgA+ to ebgA+I1 can, for experimental reasons, be measured (secondary mutations to ebgR-), and those secondary muta- only in strains that already have the other mutations. While it tions allow rapid growth on lactulose (doubling time, 0.11 day). is possible that these mutation rates might be different in a With respect to growth on lactulose, ebgR- ebgA +'I cells can nonmutant background, the close agreement between the be considered as corresponding to full-blown cancer cells that observed kinetics with which double mutants appear and the can divide rapidly and without constraint, while each of the predicted kinetics based on the adaptive mutation rates sug- single mutants can be considered as corresponding to the gests that the mutation rates do not depend upon the presence primary mutants that initiate tumors. We can now ask whether of the other mutation. These results suggest that although wild-type cells can give rise directly to rapidly growing double random, replication-dependent mutations are insufficient to mutants, and whether they can do so solely on the basis of produce multiple mutations during prolonged selection, adap- random mutation rates as measured by fluctuation tests. tive mutations may do so quite effectively. The wild-type strain DS4680A was spread onto lactulose selection plates as described in Materials and Methods, and the DISCUSSION nlates were monitored for the annearance of lactulose-utilizing papillae. The first lactulose-utilizingThe firstlactulse-utlizinmutatapparWhethermutant appeared on day we are considering spontaneous (36), x-ray-induced 13, and nrnutants continued to accumulate through day 28, when (37),, or oncogene-induced (38) transformation of cultured the expe:rimetntwas terminated (Fig.cumu4).e Eaoughof te mant cells, the parallels with adaptive mutations are striking. In both was isola tenwan purmifed(.All mutantsw cases a mutation or series of mutations occurs within a as was the parent strain DS4680A.asashearetsraiDS680.The base at of cells, and after one or more muta- resistant, . Thtionspopulationthe cells dividenondividingwithout constraint. In both cases there is position 4223 of the ebg operon was determined by simplified evidence that the appearance of transformed foci or ofpapillae allele-spiecific amplification (35), and in each case the wild- does not follow the patterns expected from purely random type G hlad been replaced by either a C or a T. All but two of events. In both cases the spectrum ofmutations is unusual (39): the mutants were ebgR-, based upon their forming blue there is a strong tendency toward deletion mutations in tumors colonies on medium with 5-bromo-4-chloro-3-indolyl /-D- (3, 40), whereas adaptive mutations have a spectrum different galactopnyranoside. The remaining two presumably carried the from that of random mutations in E. coli (19). In both cases much rar*er ebgR+L mutations that make the repressor sensitive secondary mutations have been observed in "outside" loci to lactul4ose as an inducer (33). Based upon these tests, all of among the "successful" mutants: about 10% of tumors showed the mutaints were authentic ebgR- ebgA double mutants. alterations in variable-number tandom repeat (VNTR) se- The 1405 cells that were initially spread onto the lactulose quences (3), while about 2% of E. coli Trp+ revertants carried selective plates grew at the expense of the glycerol until a additional auxotrophic mutations (14). These parallels suggest populatiion of 2.5 x 109 cells per plate was achieved by day 3. that similar processes may be involved in oncogenesis and During tthe next 7 days of carbon starvation, the population adaptive mutations in microorganisms. declined at the rate of 0.72 day-1 so that by day 10 the The first step in tumorigenesis is thought to be a mutation populati4on was 1.4 x 107 cells per plate, after which the that frees a cell from normal growth constraints and allows that populati4on size remained constant. The number of lactulose- cell to grow more rapidly than normal cells of the same type utilizing mutants expected each day, based only upon random in that tissue. Subsequent mutations are thought then to mutationis in growing cells, was estimated with the aid of a increase the growth rate further and eventually lead to rapid spreadsheet from the number of viable cells in the population, cell proliferation. There are now three examples of adaptive from the mutation rates to ebgR and to ebgA4II as estimated mutations that show a striking parallel with the multiple- sequential-mutations model for the origins of tumors. First, 30 0 * Observed there is the trpA trpB double reversion example described c l Expected i * above (41, 42), in which reversion of trpB permits slow and co 25 I Expectedif randoml limited growth as the result of cross-feeding from the majority

-WE 0 trpAB population, followed by a second trpA reversion within 20 the expanded population of trpB+ cells. -W Second, there is the example in which an initial adaptive 15 a0. mutation involves excision of an insertion sequence from 0 D within the bglF gene, which allows very slow growth on salicin 0 10I to occur, followed by a second mutation that activates full 0 expression of the bgl operon (22). 5 0 Finally, there is the above example of ebgA ebgR double W* mutants that allow on lactulose. 0 growth 6 The key feature that distinguishes adaptive from random z -5 l l is that adaptive mutagenesis allows beneficial, but D 5 10 15 20 25 30 not neutral or deleterious, mutations to enter populations Day (except as occasional hitchhikers in cells that have acquired useful mutations). The ability to produce adaptive mutations FIG. 4. Observed and expected accumulation of ebgR- ebgA+II would thus be very advantageous in terms of adaptive evolu- double miutants in strain DS4680A during prolonged selection on tion, and it seems likely that as soon as such an ability arose lactulose. See text for discussion of basis of expected values. there would be powerful selection for both its retention and its Downloaded by guest on September 29, 2021 Genetics: Hall Proc. Natl. Acad. Sci. USA 92 (1995) 5673

improvement. The finding that both bacteria and yeast can 6. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C. produce adaptive mutations is consistent with the notion that (1991) Science 253, 49-53. the ability evolved early and is widely distributed. The ma- 7. Loeb, L. A. (1991) Cancer Res. 51, 3075-3079. chinery that produces adaptive mutations would be very useful 8. Stein, W. D. (1991) Adv. Cancer Res. 56, 161-213. 9. Strauss, B. S. (1992) Cancer Res. 52, 249-253. in single-cell organisms but might be deleterious in multicell 10. Luria, S. E. & Delbruck, M. (1943) Genetics 28, 491-511. organisms where the requirement for organizing cells into 11. Cairns, J., Overbaugh, J. & Miller, S. (1988) Nature (London) 335, tissues means that programmed growth constraints must be 142-145. applied to most cells. In multicellular organisms the activation 12. Hall, B. G. (1988) Genetics 120, 887-897. of that adaptive-mutagenesis machinery, which would be ad- 13. Hall, B. G. (1989) 31, 265-271. vantageous to the individual cell in that it would allow that cell 14. Hall, B. G. (1990) Genetics 126, 5-16. to divide rapidly, would be very disadvantageous to the 15. Boe, L. (1990) Mol. Microbiol. 4, 597-601. organism, where the rapidly growing population would be 16. Nochur, S. V., Roberts, M. F. & Demain, A. L. (1990) FEMS described as a tumor. Microbiol. Lett. 71, 199-204. 17. Hall, B. G. (1992) Proc. Natl. Acad. Sci. USA 89, 4300-4303. Given the similarities between adaptive mutations in E. coli 18. Steele, D. F. & Jinks-Robertson, S. (1992) Genetics 132, 9-21. and tumorigenesis, it is not unreasonable to expect that further 19. Hall, B. G. (1991) Genetica 84, 73-76. studies of adaptive mutations may shed additional light upon 20. Cairns, J. & Foster, P. L. (1991) Genetics 128, 695-701. the processes by which cancers arise. For instance, Loeb's (7) 21. Mittler, J. E. & Lenski, R. E. (1992) Nature (London) 356, suggestion that the initial step in tumorigenesis may be the 446-448. occurrence of mutator mutations (8) did not appear to be 22. Hall, B. G. (1994) Mol. Biol. Evol. 11, 159-168. supported by the observations that tumor cells do not seem to 23. Foster, P. L. (1992) J. Bacteriol. 174, 1711-1716. have increased mutation rates (39, 40). Recently, however, 24. Hall, B. G. (1994) FEMS Microbiol. Lett. 117, 237-242. Loeb (43) has made a convincing case for mutator phenotypes 25. Hall, B. G. (1995) J. Mol. Evol. 40, 86-93. in 26. Hall, B. G. & Clarke, N. D. (1977) Genetics 85, 193-201. some cancers on the basis of instability of microsatellite 27. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold DNA in some tumor cell lines (44) and on the finding that Spring Harbor Lab. Press, Plainview, NY). lesions in human genes that are homologous to the mismatch 28. Stewart, F. M., Gordon, D. M. & Levin, B. R. (1990) Genetics repair gene mutS (45) and mutL (46) ofE. coli account for the 124, 175-185. majority of hereditary nonpolyposis colorectal cancers. On the 29. Hall, B. G. & Hartl, D. L. (1974) Genetics 76, 391-400. other hand, it is clear that not all tumor lines show a mutator 30. Hall, B. G. & Hartl, D. L. (1975) Genetics 81, 427-435. phenotype. The failure to find elevated mutation rates in 31. Hall, B. G. (1976) J. Mol. Biol. 107, 71-84. earlier studies (39, 40) and in 11 of the 14 colorectal carcinoma 32. Hall, B. G., Betts, P. W. & Wootton, J. C. (1989) Genetics 123, cell lines studied by Bhattacharyya et aL (44) may itself be 635-648. 33. Hall, B. G. (1978) Genetics 90, 673-691. misleading because mutator mutations that increased the 34. Hall, B. G. (1981) Biochemistry 20, 4042-4049. mutation rates in only nondividing cells would not have been 35. Xu, L. & Hall, B. G. (1994) BioTechniques 16, 44-45. detected in those studies. A recent study (25) has shown that 36. Rubin, A. 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