Cell cycle-dependent intervention by benzamide of carcinogen-induced neoplastic transformation and in vitro poly(ADP-ribosyl)ation of nuclear proteins in human fibroblasts.

Abstract

Human fibroblasts were subjected to nutritionally induced GI block, followed by release and subsequent entry into S phase, and exposed to nontoxic concentrations of carcinogens in early S phase. Cell transformation occurred as determined by early morphologic cell alterations, anchorage-independent colony formation, cell invasiveness, and augmentation of Ab 376 human malignancy-specific cell-surface antigenic determinant. Methylazoxymethanol acetate was the most potent transforming agent at doses that were negative in toxicity tests. Benzamide (10 FM intracellular concentration), a specific inhibitor of poly(ADPribose) polymerase, prevented transformation in a cell cycle-specific manner, maximal prevention coinciding with early S phase, also characteristic of maximal susceptibility to transformation. Neither an interference of carcinogen deoxyguanosine nucleoside adduct formation nor a chemical reaction between benzamide and carcinogens was detected. Methylazoxymethanol acetate at transforming but nontoxic dose partially inhibited poly(ADP-ribosyl)ation to about the same extent as benzamide. However, simultaneous exposure of cells to both agents in early S phase, resulting in the prevention of transformation, augmented poly(ADPribosyl)ation above the controls. Enzymatic activities ran parallel with the formation of DNA-associating polymer-nonhistone protein adducts that are assumed to regulate the physiological function of chromatin at the structural level.A significant increase in poly(ADP-ribosyl)ation of nonhistone proteins was found in an early precancerous state of hamster liver (1) and in regenerating rat liver (2), suggesting that cellular regeneration may be a common reason for the accelerated enzymatic rates. Cellular regeneration is known to augment the number of cells in S phase and this has been correlated with the facilitation of neoplastic transformation in organs (3) and in cultured C3H/101/3 CL8 mouse fibroblasts (4). Poly(ADP-ribosyl)ation also exhibits a cell cycle-dependent oscillation, a maximum coinciding with S phase after a release of G1 block (5). These observations tend to suggest an as yet undefined connection between (poly ADP-ribosyl)ation, cell cycle, and neoplastic transformation.Ontogenic development (6), the effect of developmental hormones (7-9), and differentiation (10) also coincide with changes in rates of poly(ADP-ribosyl)ation of mainly nonhistone proteins (11, 12) that are thought to regulate selective gene expression (13). Identification of poly(ADP-ribose) as a unique nucleic acid (14, 15) that is covalently bound to presumably DNA-associated proteins may provide a molecular model of chromatin regulation. Ionic environment-dependent helical polymer chains of poly(ADP-ribose) (15) may act as crosslinking agents between regulatory proteins and, depending on their nature and localization, could alter chromatin conformations, expressed as karyological changes that accompany the cell cycle in normal and malignant cells (16,17). The poly(ADP-ribosyl)ation-dependent var...

Eukaryotic nuclear ADP-ribosylation reactions

“…In contrast to these reports Kun et al (1983) found that ADPRT inhibitors block neoplastic transformation in human fibroblasts subjected to transforming but non-toxic doses of carcinogens. 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.…”

“…(2) Contrasting results may be due simply to the different concentrations of ADPRT inhibitor used (Kun et al, 1983 (Sims et al, 1983). Finally, it should be noted that there is no evidence that transformation results from inaccurate repair of strand breaks for any DNA-damaging agent.…”

Regulation of malignant phenotype and bioenergetics by a π-electron donor-inducible mitochondrial MgATPase

“…PARP -/-fibroblasts and their PARP + counterparts, plated at 0.5x10 6 cells/plate, grew at the same rate. However, exposure (3 times weekly for 4 h during a period of 3 weeks) to the chemical carcinogens methylnitronitrosoguanidine (17 μM) or benzo(a)pyrene (2.6 μM), each dissolved in DMSO, converted PARP -/-fibroblasts to a malignant phenotype as assayed by colony formation (13). This carcinogeninduced malignant phenotype could not be reverted to the non-malignant cell type by incubation with 100 μM AI.…”

Inhibition of poly(ADP‐ribosyl)ation does not prevent lymphocyte entry into the cell cycle

“…As pointed out by Cleaver et al [18], little is known about the effective concentrations of ADPRT inhibitors in intact ceils as opposed to permeable ones. Kun et al [28] observed that, in intact fibroblasts, 1 mM 3ABA in the culture medium resulted in a concentration of 10/zM inside the cell, which apparently increased, rather than inhibited, ADPRT activity. Furthermore, very little is known about the rate at which benzamides and nicotinamide are metabolized in intact cells, i.e.…”