Results from in vivo and in vitro studies showing that antioxidants may act as anticarcinogens support the role of active oxygen in carcinogenesis and provide impetus for exploring the functions of dietary antioxidants in cancer prevention by using in vitro models. We examined the single and combined effects of selenium, a component of glutathione peroxidase, and vitamin E, a known antioxidant, on cell transformation induced in C3H/1OT-1/2 cells by x-rays, benzo[alpyrene, or tryptophan pyrolysate and on the levels of cellular scavenging systems and peroxide destruction. Incubation of C3H/1OT-1/2 cells with 2.5 ,IM Na2SeO3 (selenium) or with 7 ,IM a-tocopherol succinate (vitamin E) 24 hr prior to exposure to x-rays or the chemical carcinogens resulted in an inhibition of transformation by each of the antioxidants with an additive-inhibitory action when the two nutrients were combined. Cellular pretreatment with selenium resulted in increased levels of cellular glutathione peroxidase, catalase, and nonprotein thiols (glutathione) and in an enhanced destruction of peroxide. Cells pretreated with vitamin E did not show these biochemical effects, and the combined pretreatment with vitamin E and selenium did not augment the effect of selenium on these parameters. The results support our earlier studies showing that free radical-mediated events play a role in radiation and chemically induced transformation. They indicate that selenium and vitamin E act alone and in additive fashion as radioprotecting and chemopreventing agents. The results further suggest that selenium confers protection in part by inducing or activating cellular free-radical scavenging systems and by enhancing peroxide breakdown while vitamin E appears to confer its protection by an alternate complementary mechanism.
Malignant transformation in vitro of hamster embryo cells and mouse C3H lOT'/2 cells by x-rays, ultraviolet light, and chemical carcinogens was inhibited by benzamide and by 3-aminobenzamide at concentrations that are specific for inhibition of poly(ADP-ribose) formation. These compounds slow the ligation stage of repair of x-ray and alkylation damage but not of ultraviolet light damage. At high concentrations they also inhibited de novo synthesis of DNA purines and DNA methylation by S-adenosylmethionine. The suppression of transformation by the benzamides is in striking contrast to their reported effectiveness in enhancing sister chromatid exchange, mutagenesis, and killing in cells exposed to alkylating agents. Our results suggest that mechanisms regulating malignant transformation are different from those regulating DNA repair, sister chromatid exchange, and mutagenesis and may be associated with changes in gene regulation and expression caused by alterations in poly(ADP-ribosyl)ation.Malignant transformation of cells in vitro by radiation or chemical carcinogens is a reproducible quantitative system for studying the mechanisms of carcinogenesis (1-7). Although fixation of transformation and mutagenesis involves DNA metabolism and cell replication (2, 4-6), the numbers and kinds of genes involved are not known. Poly(ADP-ribose) is an important cellular regulatory molecule (8, 9) and its synthesis can be inhibited by 3-aminobenzamide (3ABzA) and benzamide (BzA). We therefore investigated the effects of these inhibitors on transformation by a variety of DNAdamaging agents with careful comparison to their effects on DNA repair and nucleotide precursor pathways over a range of concentrations. These agents, at concentrations below those at which side effects were evident, inhibited malignant transformation in vitro, in contrast to their reported enhancement of sister chromatid exchange (10-12) and mutagenesis (13) Mouse embryo fibroblast cells C3H 10T1/2 (clone 8), originally obtained from C. Heidelberger (University of Southern California, Los Angeles), were treated as described (3,7). Stock cultures were maintained at 370C, aerated with 5% CO2 in air, in Eagle's basal medium containing 10% heatinactivated fetal calf serum, penicillin (50 units/ml), and streptomycin (50 tkg/ml).At 24 hr after plating, cells were irradiated with a Siemens 300-kVp constant-potential generator with an additional filter of 0.2 mm of Cu at room temperature with 3 or 4 Gy of xrays at a dose rate of 0.322 Gy/min and incubated at 37°C in 5% C02/95% air with weekly changes of medium. After a 10-day incubation period for the hamster cells (1, 2) and 6 weeks for the lOT1/2 cells (2, 3, 7), cultures were fixed and stained with Giemsa stain. Assays for cell survival, cloning efficiency, and transformation were carried out as described previously for the hamster cells (1-4) and for the C3H 10T1/2 cells (2, 3, 7). For experiments with UV light, C3H 10T1/2 cells were exposed to 10-13 J/m2 (254 nm, dose rate of 0.15-1.3 J/m2_sec).For ex...
DNAs from hamster embryo cells and mouse C3H/lOT1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and
The direct oncogenic potential of X rays has been demonstrated by the in vitro neoplastic transformation of mammalian cells in culture, a technique which permits the study of oncogenesis in the absence of host-specific effects. Although several agents are known to modulate in vitro neoplastic transformation by X rays, little is known of the effects of hormones. We now describe experiments which show that the presence of thyroid hormone is necessary for in vitro neoplastic transformation by X rays in cells of an established mouse fibroblast culture (C3H/10T1/2) and in early-passage diploid hamster embryo cells.
Incubation of primary cultures of hamster embryo cells (HEC) or mouse fibroblasts (C3H/IOTI/2 cells) in media depleted of thyroid hormones does not alter cell growth or survival but renders the cells resistant to neoplastic transformation by benzo [a]pyrene (B[a]P) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), carcinogens which yield transformation rates of 10-4_10-2 in media supplemented with triiodothyronine (T3). In C3H/10T'/2 cells, the times of addition or removal of the hormone indicate that T3 exerts maximum effect when added 12 hr prior to treatment with B[a]P and that the progression of transformation from the time of initiation by the carcinogen to full expression and the appearance of transformed foci was independent of the presence or absence of the hormone in the medium. Dependence of transformation on T3 concentration in the medium was observed over the physiological range of 1 pM to 100 nM in C3H/1OT1/2 cells treated with B[a]P. These results were similar to our previous findings on the T3 dose-related induction of radiogenic transformation and of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the potential involvement of protein synthesis in T3 action is the suppression of T3-and B[a]P-dependent transformation by cycloheximide at concentrations that inhibit protein synthesis by ==50% in the C3H/1OT'12 cells. We suggest that thyroid hormone induces the synthesis of a host protein that plays a key role in neoplastic transformation by direct-acting chemical carcinogens and by those requiring metabolic activation. In our previous studies, similar T3-dependent mechanisms were implicated in radiogenic transformations.Our earlier studies, with primary cultures of hamster embryo cells (HEC) and mouse embryo fibroblasts (C3H/LOTI/2), an established line, indicated that thyroid hormones are essential factors in the induction of neoplastic transformation by x-irradiation (1, 2). Removal of triiodothyronine (T3) and thyroxine (T4) from serum-supplemented media eliminated radiogenic transformation in both cellular systems without modifying the rates of cell survival or cell growth (1, 2). The addition of T3 (1 pM to 100 nM) to the thyroid hormone-depleted medium reestablished transformation at frequencies that were T3 dose dependent. The T3 concentration dependence of the transformation response to x-rays was the same as the concentration dependence of the Na+,K+-ATPase response (2).In these earlier studies (2), the maximal transformation frequency was observed when T3 was added to the medium 12 hr prior to x-ray treatment. In contrast, when T3 was added to the hormone-depleted cells 24-48 hr after exposure to x-rays, no transformation was observed. These findings strongly suggested that thyroid hormones serve as crucial factors in the initiation of cellular neoplastic transformation by x-irradiation.The present experiments were undertaken to assess the role of thyroid hormone in neoplastic transformation induced by spec...
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