Contributed by Charles Heidelberger, October, 5, 1978. ABSTRACT. Chemical carcinogens ..... P. A., Benedict, W. F. & Ts'o, P. 0. P. (1977) Cancer Res. 37,.
Proc. Nati. Acad. Sci. USA Vol. 76, No. 2, pp. 930-934, February 1979
Chemical carcinogens produce mutations to ouabain resistance in transformable C3H/1OT1/2 Cl 8 mouse fibroblasts (transformation/screening system/ratio of transformation to mutation frequencies)
JOSEPH R. LANDOLPH AND CHARLES HEIDELBERGER* Los Angeles County-University of Southern California Comprehensive Cancer Center, Los Angeles, California 90033
Contributed by Charles Heidelberger, October, 5, 1978
ABSTRACT Chemical carcinogens induce mutations to ouabain resistance in the transformable mouse fibroblast cell line C3H/1OT1/2 Cl 8. The mutant phenotype is stable and heritable in the absence of selective agent, and dose-response curves for mutant frequency were obtained with N-methylN'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, benzo[apyrene, N-acetoxy-N-2-acetylaminofluorene, and the anti7,8-diol-9,10-epoxide of benzo[a]pyrene. The ratio of the malignant transformation frequency to the mutation frequency was 12 for benzo[a pyrene and 21 for -N-acetoxy-N-2-acetylaminofluorene. The development of the mutational assay reported here allows the use ofthis permanent cell line for comparison of mutation and transformation frequencies and as a screening system for xenobiotics that pose mutagenic or carcinogenic hazards to mammalian cells. It is well known that chemical carcinogens can induce malignant transformation in mammalian cell systems (1-9). However, it is not known with certainty if chemically induced transformation is a mutational event, and the precise molecular and cellular alterations that result in transformation have not been identified. The carcinogen-induced transformation frequencies reported (1-9) are generally of the order of 102 to 103 times greater than carcinogen-induced mutation frequencies measured in different mammalian cells (10-14), and these comparisons have recently been reviewed (15). When mutation and transformation were induced in the same cells by the same agent, ratios of transformation to mutation frequencies of 20 (16), 20-500 (17, 18), and 10 (19, 20) have been reported. However, some of these ratios of transformation to mutation frequencies could be artificially high due to the nontumorigenicity of some of the morphologic transformants in some systems. These ratios could be useful because they might help to define the number of genes that are targets for transformation. In addition to the theoretical utility of ratios of transformation to mutation frequencies obtained in the same cells, comparisons of mutagenesis and transformation are also potentially helpful to standardize the use of the mutagenic potential of agents as a predictor of their possible oncogenic potential (10-14, 2123). Consequently we have established and characterized a mutational assay for ouabain-resistant mutants in the C3H/LOT1/2 Cl 8 mouse fibroblast cell line (24), in which we have established a correspondence between morphologic and malignant transformation (7). Ouabain resistance (Ouar), which has been shown to be induced in many mammalian cell types by known mutagens (25-29), results from an altered sodium-potassium adenosinetriphosphatase (Na+,K+-ATPase) that no longer binds ouabain (Oua) or binds it less tightly than the wild-type enzyme
does (27). During the preparation of this manuscript, Chan and Little demonstrated mutagenesis to Ouar in these cells and showed that ultraviolet light produced a transformation/ouabain resistance ratio of 10 (20). MATERIALS AND METHODS Cell Culture Procedures. The C3H/1OT1/2 Cl 8 cell line has been described in detail elsewhere (24). Briefly, it is a hypotetraploid permanent fibroblast cell line derived from C3H mouse embryos, and has an extremely low level of spontaneous transformation. The cells are carried in basal Eagle's medium plus 10% fetal calf serum (GIBCO) and passaged as described (24). Fluorescent lights were not used in the laminar flow hoods in which this work was done. Cloning. To derive Ouar cell lines, clones were ring-isolated (30) from 100-mm plastic petri dishes (Corning) having five or fewer colonies per dish, and only one mutant colony was picked from each dish. Assays for Induction of Ouar. For these assays, 100-mm dishes were each seeded with 105 cells, and 10 dishes were used for each concentration of carcinogen tested. After 24 hr, carcinogen in acetone (0.5% final concentration in the medium, which caused no toxicity) was added directly to the dishes with swirling in the case of alkylating agents or ultimate forms of carcinogens. With hydrocarbons, the compounds were dissolved in medium plus serum and 0.5% acetone, and the original medium was replaced with carcinogen-containing medium. The cells were exposed to the compounds for 48 hr. For ultimate carcinogens with extremely short half-lives, this treatment provides a 48-hr expression time, whereas for hydrocarbons, which are continuously metabolized to ultimate forms during the exposure time, no exact expression time can be derived. Cells were then trypsinized, and 10-20 100-mm dishes per point were seeded with 1 X 105 cells in nonselective medium. Twenty-four hours later, the medium was aspirated, and selective medium containing 3 mM Oua was added to the cells. The Oua-containing medium was then changed to fresh 3 mM Oua on days 6 and 12 after addition of the original Oua, and the mutant colonies were fixed and stained after 16 days of exposure to Oua. Colonies greater than 2 mm in diameter were scored as Ouar. Oua-containing medium was prepared by addition of Oua (Sigma) to the medium, warming for 1-2 hr at 370C to dissolve the Oua, sterilizing the medium by filtration through 0.2 ,gm Gelman filters (Gelman), and then adding serum to 10% final concentration. Mutant frequencies were calculated either by the Poisson method, utilizing the fraction of dishes containing no colonies Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; BaP, benzo[a Ipyrene; MCA, 3-methylcholanthrene; N-AcO-AAF, N-acetoxy-N-2-acetylaminofluorene; Oua, ouabain; Ouar, ouabain resis-
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tant. * To whom reprint requests should be addressed.
Medical Sciences: Landolph and Heidelberger
(31, 32), or by the ratio of the observed number of mutant colonies to the number of surviving cells. Transformation and Toxicity Assays. Cytotoxicity and transformation assays were performed as per ref. 7; only type III foci were scored as transformed. The cytotoxicity and transformation assays were performed with cells of the same passage number, with the same medium and serum, and with the same 48-hr exposure to carcinogens as were the mutation assays. The medium was changed to medium lacking carcinogen in the cytotoxicity and transformation experiments when the mutation assay was reseeded for selection. Transformation frequencies were calculated by the ratio of type III foci (7) to the number of surviving cells, or by the Poisson method. At each concentration of carcinogen, 20-30 dishes were used in the transformation assays and 5 dishes in the cytotoxicity assays. Carcinogens. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG), benzo[a]pyrene (BaP), and 3-methylcholanthrene (MCA) were purchased from Sigma. The anti-7,8-diol-9,10epoxide of BaP was given to us by Kenneth Straub, Laboratory of Chemical Biodynamics, University of California, Berkeley, CA. N-Acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) was freshly prepared by Robert P. P. Fuchs, of the Institut de Biologie Moleculaire et Cellulaire, Strasbourg, France (on sabbatical in this laboratory). RESULTS Selection System for Ouabain Resistance. Exposure of C3H/1OT1/2 cells to 3 mM Oua for 16 days reduced the survival fraction to 10-5 (Fig. 1). Decreasing the K+ concentration in the medium increased the toxicity of Oua (Fig. 1), an effect previously seen in L cells (25) and in Chinese hamster V79 cells (unpublished observations). Other laboratories have selected Ouar mutants with 1 mM Oua (16, 25, 26, 29) and 3 mM Oua (25). Because the survival of C3H/1OT1/2 cells was too high (10-2) in 1 mM Oua, we used 16-day exposure to 3 mM Oua with medium changes on days 6 and 12 to select mutants.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 1. Effect of number of cells reseeded on the frequency of MNNG-induced Ouar mutants
Viable cells reseeded
Oua, change on day 6, 12-day exposure
MNNG-treated cells* 1.0 X 104 0.5 ± 0.3 4.0 X 104 3.0 i 0.0
5.0 ± 1.0 7.5 ± 0.5
Oua, no change, 16-day exposure
2.0 X 104
1.8 i 0.1
9.0 i 0.5
Oua, change on day 6, 16-day exposure
1.0 X 2.0 X 4.0 X 8.0 X
104 104 104 104
1.0 i: 0.7 2.3 ± 0.5 5.0 ± 2.0 10.0 ± 1.0
10.0 ± 7.0 12.0 ± 2.0 12.0 ± 1.0 12.5 I 0.5
Oua, changes on day 6 anddayl2, 16-day exposure
2.0 X 104 4.0X 104 8.0 X 104
1.5 ± 0.7 1.0±0.0 7.0 ± 0.6
8.0 ± 4.0 2.5 0.1 8.7 ± 0.1
Oua, changes on day 6 anddayl2, 16-day exposure
Acetone-treated cells 2.2 X 104 4.5 X 104 9.0 X 104 1.8 X 105
0 0 0 0