Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate.

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/10T1/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.

Weak promotion of C3H/10T1/2 cell transformation by repeated treatments with formaldehyde.

C3H/10T 1/2 cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine and then repeatedly exposed to formaldehyde (0.1 to 2.0 micrograms/ml). Exposure of N-methyl-N'-nitro-N-nitrosoguanidine-initiated cultures to formaldehyde concentrations of 0.5 or 1.0 micrograms/ml in a variety of treatment regimens resulted in focus formation in up to 9% of the treated dishes. Transformed foci were observed in 2% or less of the cultures treated with N-methyl-N'-nitro-N-nitrosoguanidine or formaldehyde alone. Formaldehyde thus appears to be only a weak tumor promoter for C3H/10T 1/2 cell transformation.

Factors influencing the promotion of transformation in chemically-initiated C3H/10T1/2 Cl 8 mouse embryo fibroblasts.

Treatment of low density asynchronous cultures of C3H/10T1/2 Cl 8 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiates the process of transformation and produces significant numbers of transformed foci only when treated cultures are subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell culture variables which influence the outcome of this initiation and promotion system were studied. A TPA concentration of 0.25 micrograms/ml was found to be optimal for the promotion of focus production and the presence of TPA was required both during logarithmic growth and throughout confluence. The lot of fetal calf serum used to cultivate the cells also played a determining role in focus production. Of nine serum lots purchased from four different suppliers, only two were suited for initiation and promotion studies with MNNG and TPA. In contrast, seven of these lots were adequate for transformation studies with 3-methylcholanthrene. Factors which adversely influenced focus production included the use of fungizone or the use of high passage stock cultures. These studies demonstrate that cell culture variables which influence promotion in these cells can be controlled and that this system can be successfully used in studies of the cellular mechanism of in vitro promotion and for the detection of genotoxic substances.

Initiation of C3H/10T1/2 cell transformation by N-methyl-N'-nitro-N-nitrosoguanidine and aflatoxin B1.

The utility of C3/H/10T1/2 mouse embryo fibroblasts for the detection of carcinogenic substances has been limited by their apparent insensitivity to the oncogenic effects of direct-acting alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and procarcinogens such as aflatoxin B1 (AFB1). Because the process of C3H/10T1/2 transformation can be observed to proceed through discrete stages of initiation and promotion, we have considered the possibility that MNNG and AfB1 may only initiate C3H/10T1/2 transformation. Treatment of asynchronous C3H/10T1/2 cells with MNNG or AfB1 alone generally produced few transformed foci. If MNNG or AfB1 treatment was followed by the exposure of cells to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), numerous transformed foci were produced. Phorbol did not enhance transformation by either substance. MNNG and AfB1 thus appear to be initiating agents for transformation. TPA also enhanced the transformation of C3H/10T1/2 cells by low doses of 3-methylcholanthrene (3-MCA), but transformation by high concentrations of 3-MCA was inhibited by the presence of TPA. These studied suggest that the sensitivity of the C3H/10T1/2 transformation system to potential carcinogens can be dramatically heightened if the bioassay is conducted in the presence and absence of TPA.

Responses of Fanconi anemia fibroblasts to adenine and purine analogues.

Fanconi anemia (FA) fibroblasts are known to be exceptionally sensitive to the cytotoxic action of mitomycin C (MMC). The survival of FA cells was enhanced significantly when 0.5 mM caffeine or 0.5 mM adenine was added for 72 h after the cells were exposed to MMC. In other experiments in which MMC was not used, FA fibroblasts were shown to be significantly more sensitive than control cells to 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and 6-azauridine (6-AU). These observations offer a new approach to defining the basic biochemical defect in FA.