Reprinted from Proc. Nati. Acad. Sci. USA Vol. 75, No. 7, pp. 3297-3301, July 1978 Cell Biology Relationship between somatic mutation and neoplastic transformation (chemical carcinogenesis/Syrian hamster/anchorage independence/morphological transformation/neoplastic progression) J. CARL BARRETT* AND PAUL 0. P. Ts'o Division of Biophysics, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Communicated by James Bonner, April 10,1978

ABSTRACT Somatic mutation and neoplastic transforma- which are associated with transformed cells have been studied tion of diploid Syrian hamster embyro cells were examined extensively (5). Thus, somatic mutation and neoplastic trans- concomitantly. Mutations induced by benzo[a pyrene and N- methyl-N'-nitro-N-nitrosoguanidine were quantitated at the formation can be investigated by the same experimental ap- hypoxanthine phosphoribosyltransferase and Na+/K+ ATPase proach-i.e., by studying the heritable alterations of cells in loci and compared to phenotypic transformations measured by culture. The elucidation of the significance of these cellular changes in cellular morphology and colony- formation in agar. changes to tumorigenicity is crucial to an understanding of Both cellular transformations ad characteristics distinct from neoplastic transformation (5). To date, however, somatic the somatic mutations observed at the two loci. Mohological mutation and neoplastic transformation have not been transformation was observed after a time comparale to that studied of somatic mutation but at a frequency that was 25- to 540-fold quantitatively in the same cellular system, thus preventing higher. Transformants capable ofcolony formation in agar were direct comparisons of the two processes. detected at a frequency of 10-5-106, but not until 32-75 pop- Recently, we reported the development of a mammalian ulation doublings after carcinogen treatment. Although this cellular system, utilizing early passage, diploid Syrian hamster frequency of transformation is comparable to that of somatic mutation, the detection time required is much longer than the embryo cells, that is amenable to concomitant studies of neo- optimal expression time of conventionally studied somatic plastic transformation (6, 7) and somatic mutation (8). We mutations. Neoplastic transformation of hamster embryo cells described the parameters involved in the quantification of has been described as a multistep, progressive process. Various mutants of Na+/K+ ATPase and hypoxanthine phosphori- phenotypic transformations of cells after carcinogen treatment bosyltransferase (HPRT) functions of these cells. Syrian hamster may represent different stages in this progressive transforma- tion. The results are discussed in this context and the role of embryo cells also have been utilized for quantitative studies of mutagenesis in the transition between various stages is con- in vitro transformation by chemical carcinogens (9, 10). In these sidered. Neoplastic transformation may be initiated by a mu- studies, transformation has been measured by the frequency tational change, but it cannot be described completely by a of cells which either yield morphologically transformed colonies single gene mutational event involving a dominant, codominant, (9, 10) or are capable of anchorage-independent growth (11). or X-linked recessive locus. Neoplastic transformation induced transformation an by chemical carcinogens is more complex than a single gene Morphological is early alteration of Syrian mutational process. Thus, this comparative study does not give hamster cells after exposure to chemical carcinogens (7, 9, 10), experimental support to predictions of the carcinogenic po- whereas anchorage-independent growth correlates very well tential of chemicals based on a simple extrapolation of the re- with the ability of the cells to produce tumors in vivo (12, 13). sults obtained from conventional somatic mutation assays. In this report, these two phenotypic alterations associated with neoplastic transformation are compared with known somatic Since proposed by Boveri in 1914 (1), somatic mutation as a mutations in terms of observed frequency and time for detec- basis for the heritable alteration in malignant cells has been a tion after carcinogen treatment. Both morphological trans- popular hypothesis (2, 3). This hypothesis provides, at least in formation and anchorage-independent growth have features part, the rationale for the use of mutagenesis tests for the de- distinct from conventionally studied somatic mutations. While tection of biohazardous chemicals. The relationship, however, such mutations between somatic mutation and neoplastic transformation is can be characterized by a single-step mutation unclear. An examination of this relationship requires that each process, neoplastic transformation cannot be described ade- process be quantitated and that the mechanism of each process quately in such terms. Neoplastic development in vivo (14, 15) be defined. The process of somatic mutation can be studied and in vitro (5, 6) has been described as a multistep progressive reliably by examining various heritable phenotypic alterations process. Although such a multistep process might be initiated of mammalian cells, particularly resistance to certain drugs. by a mutational change, it cannot be completely described by Additionally, the basis of somatic mutation can be defined at a single gene mutational event involving a dominant, codom- the molecular level in biochemical terms (4). Neoplastic inant, or X-linked recessive locus, because secondary changes transformation, in contrast, is less well understood, a fact par- must occur. The relationship between mutagenesis and carci- tially attributable to the lack of a definitive phenotypic alter- nogenesis of Syrian hamster embryo cells in culture is therefore ation characteristic of malignancy (5). Although tumor for- discussed with reference to the progressive multistep nature of mation in vivo serves to define neoplastic transformation of cells neoplastic transformation. in vitro, tumorigenicity is a multifaceted phenomenon which MATERIALS AND METHODS is difficult to analyze at the molecular or cellular level. Ac- Cells. Syrian hamster embryo cell cultures were established cordingly, several other in vitro phenotypic characteristics Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase; The costs of publication of this article were defrayed in part by the MNNG, N-methyl-N'-nitro-N-nitrosoguanidine. payment of page charges. This article must therefore be hereby marked * Present address: National Institutes of Health, National Institute of "advertisement" in accordance with 18 U. S. C §1734 solely to indicate Environmental Health Sciences, P.O. Box 12233, Research Triangle this fact. Park, NC 27709. 3297 Downloaded by guest on October 2, 2021 8298 Cell Biology: Barrett and Ts'o Proc. Natl. Acad. Sci. USA 75 (1978)

from 13-day gestation fetuses collected aseptically by Caesarian sphere for 28 days, after which time the number of colonies was section from inbred Syrian hamsters, strain LSH/ssLAK determined. Cloning efficiency in soft agar was expressed as (Lakeview Hamster Colony, Newfield, NJ), or outbred hamsters the percentage of plated cells that formed visible colonies from Engle Laboratories (Farmersburg, IN). Pools of primary containing over 25 cells. The population doublings were de- cultures from littermates were stored in liquid nitrogen. Sec- termined from the number of cells obtained at confluency, ondary cultures were initiated from the frozen stocks, and all when a subculture was established. experiments were performed with tertiary or later cultures. All In the direct transformation assay, colonies formed eight days cultures were routinely tested by Microbiological Associates and after treatment were examined for morphological transfor- found free of mycoplasma contamination. mation. (These plates were established and treated in parallel Media and Growth Conditions. The cells were grown in with those used for mutation assay.) IBR modified Dulbecco's Eagle's reinforced medium (Biolabs, Northbrook, IL) supplemented with 0.22% NaHCO0 (wt/vol) RESULTS and 10% Rehatuin filter-sterilized fetal bovine serum (Reheis The Detection Time of Morphological Transformation and Chemical Company, Kankakee, IL) without antimicrobial Anchorage-Independent Growth in Syrian Hamster Embryo to ac- agents. Cells were transferred by gentle trypsinization with Cells after Exposure Benzo[alpyrene. The temporal 0.1% trypsin solution (1:250, GIBCO, Grand Island, NY) for 5 quisition of various phenotypic transformations of Syrian min at 370. hamster embryo cells after exposure to carcinogen or mutagen Mutation Assays. Details of these methods have been pub- has been under active investigation in our laboratory (6, 7). lished (8). For the respreading assay, tertiary passage Syrian Table 1 shows the temporal relationship between the appear- hamster embryo cells were inoculated at a density of 5 X 105 ance of morphologically transformed colonies and the ap- cells in a 75-cm2 flask. After 15 hr, these cells were treated with pearance of colonies capable of growth in soft agar (anchor- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 1 MiM or age-independent growth). As shown, morphologically trans- 5 MM for 2 hr, or they were treated with benzo[ajpyrene at 1 formed colonies appeared in the population 8 days after Ag/ml or 10 Mug/ml for 24 hr. After the exposure time, the cul- treatment. Anchorage-independent growth was measured at tures treated with MNNG were washed twice with phos- every passage after treatment with benzofaIpyrene by testing phate-buffered saline and the flasks treated with benzo[a]- 106 cells from each culture for growth in agar. Anchorage- pyrene were washed five times with complete medium (con- independent growth was not observed until 6-15 passages after taining 5% serum). The cultures were then grown to confluency, treatment, depending on individual experiments. The detection subcultured at a split ratio of 1:10, and again grown to con- time of this transformation thus required 32-75 population fluency. Untreated cultures normally attained confluency in doublings. When initially detected at 6-9 post-treatment pas- 7 days, whereas cultures treated with 5 MtM MNNG or 10 ,g/inl sages, the frequency of this alteration was approximately 10-6 benzo[a]pyrene required a longer time period. to 10-5 and increased slightly at later passages. Other studies At each passage, 105 cells were plated in each of 10 100-mm involving isolated clones indicated that the long delay in ex- petri plates. After 15 hr, selective medium containing either pression of this transformed phenotype is not due to selection 8-azaguanine at 40 ug/ml, 6-thioguanine at 2 ug/ml, or ouabain of a few transformed cells present early after treatment (6). at 1.25 mM was added to the plates. After 1 10-20 days incu- Quantitative Comparisons among Somatic Mutation, bation, with changes of the selective medium every 3-4 days, Morphological Transformation, and Anchorage-Independent the colonies either were scored after fixation and staining or Growth. The effects of variation of the following parameters were isolated. For the direct assay of mutation, tertiary passage Syrian hamster embryo cells (105) were plated overnight in 100-mm Table 1. The temporal relationship between the appearance of petri plates, then treated with MNNG, benzo[a]pyrene, or morphologically transformed colonies and the appearance of solvent only for the specified period of time and washed as colonies grown in soft agar in Syrian hamster embryo cells exposed described. The cells were resupplied with complete medium to benzo[alpyrene* in the absence of selective agent and incubated for a recov- Post- % morphologically Growth in Total population ery/expression period of 1-3 days. After this period, 8-aza- treatment transformed soft agar, doublings at guanine at 40 Mg/ml was added to the medium and clones were passaget coloniest colonies/106 cells subculture§ grown in this selective medium for 3-4 weeks, with selective medium changes every 3-4 days. 0 1.1 0 Transformation Assay. The frequencies of morphological 1 1.1 0 3.8 and anchorage-independent transformants within the treated 2 0.75 0 7.9 3 0.5 0 13.5 cultures were determined concomitantly with the somatic 4 2.0 0 19.2 mutation For the respreading assay, cells at each frequencies. 5 1.7 0 27.4 were at for eight days passage plated low density and incubated 6 2.7 3 32.4 were to to form colonies. These colonies enumerated determine 7 7.2 16 37.0 cytotoxity and also scored for morphological transformation 8 11.8 18 47.7 with a stereodissecting microscope, using established criteria 9 25.0 41 46.9 (9, 10). At each passage, 200-600 colonies were examined from 12 >90 49 55.0 each culture. Additionally, 106 cells from each culture were tested at each passage for colony formation in semisolid agar * Cells were exposed to 1 jtg of benzo[alpyrene per ml for 24 hr, grown by using procedures described by Macpherson and Montagnier in mass culture, and assayed as described for each transformed when subcultured at various passages after treatment. as (17). Suspensions phenotype (16), modified by Kakunaga and Kamahora t The cells were subcultured every 6-9 days. of 105 cells in 4 ml of 0.3% Difco agar in complete medium Number of colonies judged morphologically transformed by the supplemented with 0.1% bactopeptone were plated in 60-mm criteria described per total number of colonies X 100%. dishes over a basal layer of 0.6% agar in complete medium. All § Number of population doublings calculated from number of initial plates were incubated at 370 in a 5% C02-humidified atmo- cells per culture and number of cells obtained at time ofsubculture. 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on the frequencies of somatic mutation and morphological Table 3. Comparison of morphological transformation and transformation were examined: (i) The genetic loci for the somatic mutation by direct assay somatic mutations-i.e., ATPase mutants vs. HPRT mutants; Morphological Apparent (ii) the selective agent for mutations of HPRT-i.e., 6-thio- transformation mutation guanine vs. 8-azaguanine; (iii) the method of assay-i.e., re- Treatment frequency frequency spreading vs. direct; (iv) the animal source for the cell prepa- rations; (v) the type of carcinogen; and (vi) the dose of carcin- 1 MM MNNG 8 x 0-3 8.5 X 10-5 ogen. The data for these variables are summarized in Tables (2 hr) (94) 2 and 3. 5 AM MNNG 1.9 X 10-2 2.5 X 10-4 For the mutation studies the conditions employed have been (2 hr) (76) B[a]P* at 1 Ag/ml 1.1 X 10-2 2 X10-4 shown to select for mutants that are stable in the absence of the (24 hr) (55) selective agent and have a low reversion frequency. The AGr B[aJP at 10 Mg/in 3.0 x 10-2 5 X 10-4 mutants are crossresistant to 6-thioguanine, and mutants se- (24 hr) (60) lected with either purine analog have a low level of HPRT ac- tivity. The ouabain resistant mutants have Na+/K+ ATPase Cells were allowed to attach overnight and then exposed to car- activity with an altered sensitivity to ouabain (8). cinogen at specified concentrations. After specified exposure time, the carcinogen was removed by extensive washing and the cells were The mutation frequencies in Tables 2 and 3 were determined either allowed to form colonies for 8 days for determination of mor- at an optimal expression time (6-8 population doublings) after phological transformation or allowed to grow for 48 hr followed by carcinogen exposure. The frequencies were also corrected for addition of8-azaguanine at 40 Mg/ml for selection ofAGr mutants as cytotoxicity and for the recovery efficiency of the mutant cells described (8). The mutation frequencies are corrected for cytotoxicity during the selective assay, which was 18-20%, as determined and recovery efficiency of 20% for the resistant colonies. The by a reconstitution experiment (8). frequencies are expressed as number of transformed or resistant colonies per surviving cell. The ratio of morphological transformation Similar frequencies of induced mutations of HPRT were to mutation is given in the parentheses. observed with either 6-thioguanine or 8-azaguanine selection. * Benzo[a]pyrene. Isolated mutant colonies were also qualitatively similar re- gardless of selective agent. A small variation (1.3-4 fold) in the induced frequencies of ATPase vs. HPRT mutants was ob- a comparative evaluation of the frequencies determined by this served. The direct assay resulted in a higher frequency of method. mutants than the respreading assay, which may reflect the In contrast to the somatic mutation frequencies, it was noted uncertainty of quantitating somatic mutation by this method that different embryo preparations from the same as well as (18). However, this experimental approach is used frequently different suppliers provided different induced frequencies of for quantitating morphological transformation (9, 10); there- morphological transformation. As shown in Table 2, two sep- fore, we have included the data for somatic mutation to allow arate preparations, after treatment with carcinogens, differed by nearly 5-fold with respect to induced morphological trans- Table 2. Comparison of morphological transformation and formation frequencies, although the mutation frequencies and somatic mutation by respreading assay cytotoxicity differed by less than ±50% of the reported fre- quency (which represents the standard error in these deter- Morphological minations). This variation in transformation-response of dif- transformation Mutation ferent embryo preparations confirms the results of Pienta et al. frequency frequency (19). (X 103) (X 105) The frequency of both morphological transformation and Treatment SHE*-1 SHE-2 ATPase HPRT somatic mutation increased with increasing dose of carcinogen. 1 AM MNNG <1 2 2.1 0.7-1.6 Doses of benzo[a]pyrene and MNNG that provided comparable (2 hr) (<47-96) (63-285) cytotoxicities were chosen. Regardless of which carcinogen was 5 JM MNNG 8 10 32 10-11 applied, the induced frequency of mutation or morphological (2 hr) (25-31) (73-100) transformation was the same at a given cvtotoxicitv. The B[a]Pt at 1,ug/ml 1 2.7 2.1 0.79 cytotoxicity (percent r efficiency) was (24 hr) (48-129) (126-342) 31-50% for 1-5 ,M MNNG and 28-50% for benzo[a]pyrene B[a]P at 10 ,g/ml 4 20 16 3.7-6 at 1-10.ug/ml. (24 hr) (25-125) (67-540) Because the frequencies of both induced morphological Cells were exposed to carcinogen at specified dose and then allowed transformation and somatic mutation were measured con- to grow for two passages after treatment. At the second passage, the comitantly in cultures of Syrian hamster embryo cells treated cells were plated at 5 X 103 cells/plate and allowed to form colonies with benzo[a]pyrene or MNNG, these two frequencies were for 8 days to determine morphological transformation. Concomitantly, compared directly. It should be noted that the frequency of cells were plated at 105 cells per plate and grown for 10-14 days in morphological transformation varied in response to the vari- medium containing 1.25 mM ouabain, 8-azaguanine at 40 Mg/ml, or 6-thioguanine at 2 Mg/ml as described. The morphological transfor- ables mentioned above. Nevertheless, the frequency of mor- mation frequency (transformed colonies per surviving cell) was phological transformation always exceeded significantly the measured for two preparations of Syrian hamster embryo cells and frequency of somatic mutation. The ratio of the morphological found to vary as indicated. The mutation frequencies of the two cell transformation frequency within a given culture varied by preparations were essentially identical and are averaged for the 25-540, depending upon the conditions and cells employed. ATPase locus (Ouar) and the HPRT locus (AGr and TGr, which are An average ratio of approximately 100 existed for most con- listed separately when different). The mutation frequencies are cor- ditions. A rected for cytotoxicity and mutant recovery as described (8). The similar ratio existed for the spontaneous frequencies ratios of morphological transformation to mutation are given in the (Table 4). However, it is difficult to determine accurately the parentheses. spontaneous frequency of morphological transformation, be- Syrian hamster embryo. cause a selective assay is not employed and only small numbers t Benzo[ajpyrene. of colonies are observed. Six spontaneous morphologically Downloaded by guest on October 2, 2021 3300 Cell Biology: Barrett and Ts'o Proc. Nati. Acad. Sci. USA 75 (1978) Table 4. Comparison of phenotypic changes of Syrian hamster ventionally studied somatic mutations (20-22), the frequency embryo cells of anchorage-independent transformation is less than 10-5- 10-ff After this expression time, somatic mutations at the HPRT Morpho- and Na+/K+ ATPase loci were approximately 10-4. Therefore, logical Anchorage anchorage-independent growth occurs either at a much lower Somatic transforma- independent frequency or only after a much longer time than somatic mutation tion growth* mutations. Observed frequency <10-6 10-4t <1.4 X 10-8 Because the sensitivity level of the soft agar assay is 10-5- (spontaneous) 10-6(6), if the frequency of this transformation was <10-6 at Observed frequency 10-_1O-4 10-3-10-2 10-5-10-6 an earlier passage, it would not be detected. However, certain (carcinogen treated) initial alterations induced by carcinogen had to occur.at a fre- Expression time or 6-8 <8 32-75 quency >10-6, since approximately 50% of all treated cultures detection time* containing 5 X 105 cells ultimately transformed (6). Cells from * As measured by colony formation in soft agar. colonies isolated 13 population doublings after benzo[ajpyrene t Six spontaneous morphologically transformed colonies were ob- treatment do not exhibit anchorage-independent growth ini- served per -62,000 control colonies examined. tially, but develop this potential after approximately 50 pop- Population doublings. ulation doublings (6). When half of the cells of the isolated colonies were assayed after growth to a population of 106 cells transformed colonies were observed per approximately 62,000 (approximately 23 population doublings after treatment), an- control colonies examined. Table 4 also lists the frequency of chorage-independent transformation was not observed at that anchorage independent growth in the treated and untreated time, but was observed in the culture after further growth. populations of Syrian hamster cells in culture. Therefore, the expression of anchorage-independent growth requires many cell divisions after carcinogen treatment, at least) DISCUSSION 23 population doublings, and most likely 32-75 population One of the difficulties in delineating the relationship between doublings. somatic mutation and neoplastic transformation has been the Thus, both phenotypic transformations-i.e., changes in lack of a system suitable for studying both mutagenesis and morphology and anchorage-independent growth in agar-have carcinogenesis concomitantly in vitro. Syrian hamster embryo characteristics distinct from conventionally studied somatic cells in culture provide such an in nitro system. With these cells mutations. This difference between somatic mutations and ( we have studied quantitatively somatic. mutations at the phenotypic transformations indicates a fundamental difference i Na+/K+ ATPase and HPRT loci. Also, we and others have in the nature of these two processes. The basis for this difference evaluated in vitro transformation quantitatively by measuring may be understood by consideration of the progressive nature two changes, morphological transformation and growth in soft of neoplastic transformation. agar (7, 9-11). A critical comparison can thus be made con- Evidence exists that tumor development in vivo is a com- cerning the induction of both mutation and transformation in plicated process involving multiple steps through qualitatively this cell system. different stages (14, 15). We have presented evidence that As shown in Table 4, neither morphological transformation neoplastic transformation in vitro, like neoplastic development nor the transformation to anchorage independence resembled in vivo, is a progressive process (6). Most mutational processes somatic mutations such as those reported here. Morphological are described by two states, wild type and mutant, with the transformation occurs within the time required for the for- transition between these states comprising a single step (4). mation of a colony (less than eight doublings), which is similar Although the multistep, progressive process of neoplastic to the time required for the optimal expression of somatic transformation might be initiated by a mutational change, it mutations (20-22). The frequency of this transformation, cannot be described completely by a single-gene mutational however, is 25-540 times higher than that determined at the event involving a dominant, codominant, or X-linked recessive HPRT and Na+/K+ ATPase loci (Tables 3 and 4). Huberman locus, because secondary changes must occur. To describe this et al. (23) also calculated the ratio of induced morphological process fully, one must define the number of stages involved, transformants to presumptive Na+/K+ ATPase mutants and as well as the mechanism of the transition between each stage, reported a value of approximately 20. Our results with one whether mutational or not. This definition will thus require embryo preparation agree quite well with this ratio; however, experimental identification and characterization of the various when other mutations (HPRT+ - HPRT-) and cell prepara- preneoplastic stages involved. tions were used, we observed higher ratios for this comparison. The obvious question is whether these multiple stages in Our results indicate that the lower limit for this ratio of mor- neoplastic transformation can be attributed to successive phological transformation frequency/somatic mutation fre- somatic mutations or perhaps to an interplay of genetic and quency may be 20-25, while the average value is approximately epigenetic factors. The phenotypic transformations of cells after 100. Thus, when a culture receives a dose of carcinogen or carcinogen treatment may represent different stages in the mutagen, morphological transformation occurs with a fre- transformation process and therefore can be discussed in this quency at least 20-fold and most often 100-fold higher than that context. of somatic mutation. dR d '*rn$Q (0ue" . Morphological transformation of Syrian hamster embryo cells Anchorage-independent growth is a cellular characteristic is the first observable change after carcinogen treatment, correlated closely with tumorigenicity (12, 13), suggesting that suggesting that this alteration may represent the "initiation" it may be a more reliable marker for neoplastic transformation of transformation. The high frequency of this transformation (11). The frequency at which this alteration is detected in car- may be indicative of an abnormally larg target size (23), mu- cinogen-treated cultures is 10-6-10-5; however, the earliest tational "hot spots," or, alternatively, a nonmutatialteration time at which this transformation can be detected is 32-75 in gene expression. It should be noted, however, that when population doublings after treatment. After an expression time initially detected, the morphologically transformed cells are of 6-13 population doublings, which is optimal for most con- not tumorigenic and cannot grow in agar (6). The number of Downloaded by guest on October 2, 2021 Cell Biology: Barrett and Ts'o Proc. Nati. Acad. Sci. USA 75 (1978) 3301 morphologically transformed colonies which progress toAgo diploid Syrian hamster embryo cell and the ultimate tumori- lignancy is uncertain, although our initial examination of this genic cell are needed. Recognition of carcinogenesis as a mul- problem indicates that less than 10% of the colonies can be tistep, progressive process emphasizes the complexity of this successfully isolated and demonstrated to have neoplastic po- problem, yet hopefully provides further insight into the sig- tential. The morphological transformation frequency may not nificance of phenotypic transformations in neoplastic devel- represent the "true" frequency of neoplastic transformation. opment and the role of mutagenesis in the various stages of A further understanding of this problem must await determi- carcinogenesis. It should be noted that somatic mutation assays, nation of the molecular and cellular basis of morphological such as those on the HPRT locus and on the Na+/K+ ATPase transformation. locus, have been widely adopted as assay systems for the bio- The ability of cells to grow in soft agar may represent a late hazards of chemicals. Until more is known about the relation- stage in neoplastic development, because it appears to correlate ship between somatic mutation and neoplastic transformation, very well with the tumorigenicity of Syrian hamster cells in- it may not be experimentally justifiable to predict the carci- duced by benzo[a]pyrene. Our data are consistent with this nogenic potential of compounds based on a simple extrapolation phenotypic transformation occurring by a multistep process, of the results from these somatic mutation assays. at least as a two-step process. If the initial carcinogen treatment resulted in a putative preneoplastic population, cells capable of could have after 1. Boveri, T. H. (1914) Zur Frage der Entstehung maligner Tu- anchorage-independent growth developed moren E. a second mutation that occurred spontaneously during growth (Fischer, Jena, Germany). in culture. The detection time 2. Miller, E. C. & Miller, J. A. (1971) in Chemical Mutagens, ed. (32-75 population doublings) A. Vol. and of this alteration are Hollaender, (Plenum, NY), 1, pp. 83-119. frequency (i0-5-10-6) phenotypic 3. McCann, J. & Ames, B. N. (1976) Proc. Natl. Acad. Sci. USA 73, consistent with the predicted values for such a process based 950-954. on the spontaneous mutation rate of Syrian hamster cells (1.2 4. Siminovitch, L. (1976)XCell 7, 1-11. X 10-8 mutations per cell/generation, Barrett, J. C., Crawford, 5. Barrett, J. C.. & Ts'o, P.-O. P. (1978) in Polycyclic Hydrocarbons B. D., Braiterman, L., and Ts'o, P. 0. P., unpublished data). and Cancer: Chemistry, Molecular Biology, and Environment, This hypothesis is supported further by experiments with eds. Ts'o, P. O. P. & Gelboin, H. V. (Academic, NY), Vol. 2, pp. baby hamster kidney cells, if it is assumed that these cells rep- 235-267. resent a putative preneoplastic population. Bouck and 6. Barrett, J. C. & Ts'o, P. 0. P. (1978) Proc. Natl. Acad. Sci. USA DiMayorca (11) have presented some evidence that the tran- 75, in press. sition of baby hamster kidney cells from the anchorage-de- 7. Barrett, J. C., Crawford, B. D., Grady, D. L., Hester, L. D., Jones, pendent to the anchorage-independent growth state occurs as P. A., Benedict, W. F. & Ts'o, P. 0. P. (1977) Cancer Res. 37, a one-step mutational event. Ishii et al. (24) also have studied 3815-3823. the quantitative transformation of baby hamster. kidney cells 8. Barrett, J. C., Bias, N. E. & Ts'o, P. 0. P. (1978) Mutation Res., by chemical carcinogens and UV irradiation, using growth in 50, 121-136. soft agar and tumorigenicity as assays. They concluded also that 9. Berwald, Y. & Sachs, L. (1965) J. Nati. Cancer Inst. 35, 641- this transformation occurs with a very short expression time and 661. with a frequency consistent with accepted single-locus somatic 10. DiPaolo, J. A., Donovan, P. & Nelson, R. L. (1969) J. Natl. Cancer mutations. Baby hamster kidney cells are an established, an- Inst. 42, 867-874. euploid cell line of Syrian hamster origin which often forms 11. Bouck, N. & diMayorca, G. (1976) Nature 264,722-727. tumors when injected in vivo in sufficient numbers (25, 26). In 12. Freedman, V. H. & Shin, S.-I. (1974) Cell 3,355. normal hamster 13. Shin, S.-I., Freedman, V. H., Risser, R. & Pollack, R. (1975) Proc. contrast, diploid Syrian embryo cells are Natl. Acad. Sci. USA 72,4435-4439. characterized by their lack of tumorigenicity. No tumors are 14. Foulds, L. (1969) Neoplastic Development (Academic, London) observed when 109 (27) cells are injected into hamsters. These Vol. 1. observations are consistent with the hypothesis that normal 15. Foulds, L. (1975) Neoplastic Development (Academic, London) hamster embryo cells require two or more alterations (somatic Vol. 2. mutations?) to gain tumorigenic capability, whereas baby 16. Macpherson, I. & Montagnier, L. (1964) Virology 23, 291- hamster kidney cells represent a preneoplastic population that 294. requires only a single mutational event to exhibit tumorigeni- 17. Kakunaga, T. & Kamahora, J. (1968) Biken J. 11, 313-332. city. Recent experiments of Spandidos and Siminovitch (28) 18. Myhr, B. C. & DiPaolo, J. A. (1975) Genetics 80, 157-169. strongly support these conclusions. They studied the transfer 19. Pienta, R. J., Poiley, J. A. & Lebhuz, W. B. (1977) Int. J. Cancer of anchorage independence in hamster cells by isolated meta- 19,642-655. phase chromosomes of Chinese hamster ovary cells. By this 20. Arlett, C. F. & Harcourt, S. A. (1972) Mutation Res. 16, 301- technique, transfer to baby hamster kidney cells of the ability 306. to grow in agar occurred with a frequency similar to the transfer 21. Penman, B. W. & Thilly, W. G. (1976) Somat. Cell Genet., 2, of single-gene markers. In contrast, when similar experiments 325-30. were performed with primary hamster cells as recipients, no 22. Van Zeeland, A. A. & Simons, J. W. I. M. (1976) Mutation Res. transferents to the ability to grow in agar were observed. They 35, 129-138. concluded that at least two events are involved in the trans- 23. Huberman, E., Mager, R. & Sachs, L. (1976) Nature 264 360- 361. formation to agar-growth ability. Furthermore, they were able 24. Y., Elliott, J. A., Mishra, N. K. to transfer this property to a morphologically transformed Ishii, & Lieberman, M. W. (1977) Cancer Res. 37, 2023-2029. hamster cell line that had senescence and to escaped cells that 25. Stoker, M. & MacPherson, I. (1964) Nature 203, were transformed in vitro by benzo[a]pyrene but had not 1355-1357. to the 26. Defendi, V., Lehman, J. & Kraemer, P. (1963) Virology, 19, progressed ability to grow in agar. These results strongly 592-598. support a multistep process for neoplastic transformation in 27. Huberman, E. & Sachs, L. (1966) Proc. Natl. Acad. Sci. USA 56, vitro as presented by us in this report and elsewhere (5, 6). 1123-1129. Further studies to define the stages between the normal, 28. Spandidos, D. A. & Siminovitch, L. (1977) Cell. 12,675-682. Downloaded by guest on October 2, 2021