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Relationship Between Somatic Mutation and Neoplastic 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
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