Effect of DNA polymerase inhibitors on repair of γ ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-β- d-arabinofuranosylcytosine

Abstract

The mammalian DNA polymerase inhibitors aphidicolin and 1-β- d-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by γ rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that seen with each inhibitor alone. Thus either modulates the rate of intracellular accumulation of araC 5′-triphosphate (araCTP), the presumed rate-limiting step in genotoxic action of araC, or aphidicolin and araC inhibit repair by different mechanisms. To explore these possibilities, we compared the effects of aphidicolin, araC, araCTP, and 2′,3′-dideoxythymidine triphosphate (ddTTP) on repair of DNA damage induced by 60Co γ radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in permeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 μg/ml; 10 min) and incubated for 2 h with either chemical. The extent of repair inhibition by each drug was comparable in intact and permeable cells, amounting to ∼ 1.1 sites/10 8 daltons/2 h upon exposure to upon Gy. The active metabolite of araC, araCTP, did not inhibit repair in intact cells, but did so in permeable cells to an extent within the range of that seen with araC or aphidicolin alone. The incidence of DNA strand breaks accumulating in γ-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was ∼ 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrations of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populations, we have reason to be confident that within the short post-irradiation period considered here, the observed drug-accumulated breaks truly represent functional repair inhibition adn not merely abortive pathological responses. We thus conclude that (1) the accumulation of araCTP in intact cells is not limiting the ability of araC to inhibit DNA repair; and (2) the mode of the inhibitory action of araC/araCTP on γ ray repair is different from that of aphidicolin. In constrast to the observations with these chemicals, ddTTP (20 μM), a potent inhibitor of DNA polymerase β, did not produce any measurable effect on DNA repair in γ-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair. These results strongly suggest that DNA polymerase β plays no significant role in the repair of γ radioproducts in human fibroblasts. The permeabilized cell system adopted here does not adversely influence the repair capability of human fibroblasts and may thus prove useful in studying the enzymology of DNA repair in proliferating cultures following exposure to ionizing radiation and other genotoxic agents.