Inhibition of malignant transformation in vitro by inhibitors of poly(ADP-ribose) synthesis.

Abstract

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...

Eukaryotic nuclear ADP-ribosylation reactions

“…This finding has been extended by Borek et al (1984) (1) Cells are reported to possess both ADPribose dependent and -independent DNA repair pathways (Bohr & Klenow, 1981;Zwelling et al, 1982) and the independent pathway is suggested to be more rapid and error-prone than the dependent pathway. This may not be the case, however, in the putative independent pathway in all cell types.…”

“…Borek et al (1984) have only recently shown that benzamide and 3-aminobenzamide inhibit both purine synthesis and methylation mediated by S-adenosylmethionine. The effects of these inhibitors on differentiation in intact cells may be due to glucose starvation, since Grunfeld & Shigenaga (1984) found that nicotinamide, benzamide and bromodeoxyuridine inhibit deoxyglucose uptake in differentiated 3T3-L1 fat cells, OK opossum kidney cells and UMR bone cells.…”

“…Borek et al (1984) describe malignant transformation induced by various agents as a multistage process initiated by the induction of DNA damage and fixation of this damage by DNA and cellular replication. We know that ADPRT inhibitors potentiate the cytotoxic effects of N-methyl-Nnitrosourea, dimethyl sulphate, y-irradiation, BCNU, and bleomycin.…”

Chemical and Molecular Biological Aspects of Alkylhydrazine-Induced Carcinogenesis in Human Cells in Vitro. Revised

“…Alkylation of oxygen atoms of purines and pyrimidines of DNA has been suggested to be critical to mutagenesis and carcinogenesis (40) and a positive correlation has been found between tumorigenesis and the formation and persistence of the promutagenic lesion 06 alkylguanine in target tissues (12,(23)(24)(25)31,33 Ci/mmole) were supplied by New England Nuclear Corporation, (Boston, MA). Table 1).…”

Tumor promoter phorbol-12-myristate-13-acetate induces poly(ADP)-ribosylation in fibroblasts.

“…Poly(ADP)-ribosylation (ADPR) of chromosomal proteins may represent a mechanism of regulation of gene expression by active oxygen because it is intimately related to the redox state of the cell, DNA strand breakage and chromatin conformation (Hollenberg and Ghani, 1982;Uchigata et al, 1982;Purnell et al, 1980;Mandel et al, 1982;Poirier et al, 1985). Evidence for a role of ADPR in DNA repair (Jacobson et al, 1983;Wielckens et al, 1983;Durkacz et al, 1980), cell differentiation (Althaus et al, 1982;Johnstone and Williams, 1982;Farzaneh et al, 1982) and malignant transformation (Kun et al, 1983;Borek et al, 1984) has been obtained. We now report that the potent mouse skin promoter phorbol-12-myristate-13-acetate (PMA), which produces a prooxidant state in several cell types (see Cerutti, 1985), increased poly(ADPR) levels via the intermediacy of active oxygen in mouse embryo fibroblasts C3H1OT1/2 and human fibroblasts 3229 (Singh et al, 1985a).…”