EFFECT OF DNA-POLYMERASE INHIBITORS ON REPAIR OF GAMMA-RAY-INDUCED DNA-DAMAGE IN PROLIFERATING (INTACT VERSUS PERMEABLE) HUMAN FIBROBLASTS - EVIDENCE FOR DIFFERENCES IN THE MODES OF ACTION OF APHIDICOLIN AND 1-BETA-D-ARABINOFURANOSYLCYTOSINE

The mammalian DNA polymerase inhibitors aphidicolin and 1-beta-D-arabi nofuranosylcytosine (araC), when used in combination, inhibit the repa ir of DNA damage induced by gamma rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that s een with each inhibitor alone. Thus either aphidicolin modulates the r ate of intracellular accumulation of araC 5'-triphosphate (araCTP), th e presumed rate-limiting step in the genotoxic action of araC, or aphi dicolin and araC inhibit repair by different mechanisms. To explore th ese possibilities, we compared the effects of aphidicolin, araC, araCT P, and 2',3'-dideoxythymidine triphosphate (ddTTP) on repair of DNA da mage induced by Co-60 gamma radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in p ermeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 mu g/ml; 10 min) and incubated for 2 h with either chemical. The exte nt of repair inhibition by each drug was comparable in intact and perm eable cells, amounting to similar to 1.1 sites/10(8) daltons/2 h upon exposure to 150 Gy. The active metabolite of araC, araCTP, did not inh ibit repair in intact cells, but did so in permeable cells to an exten t within the range of that seen with araC or aphidicolin alone. The in cidence of DNA strand breaks accumulating in gamma-irradiated permeabl e cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was similar to 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrat ions of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populati ons, we have reason to be confident that within the short post-irradia tion period considered here, the observed drug-accumulated breaks trul y represent functional repair inhibition and not merely abortive patho logical responses. We thus conclude that (1) the accumulation of araCT P in intact cells is not limiting the ability of araC to inhibit DNA r epair; and (2) the mode of the inhibitory action of araC/araCTP on gam ma ray repair is different from that of aphidicolin. In contrast to th e observations with these chemicals, ddTTP (20 mu M), a potent inhibit or of DNA polymerase beta, did not produce any measurable effect on DN A repair in gamma-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair, These results strongly suggest that DNA polymerase beta plays no significant role in the repair of gamma radioproducts in human fibroblasts. The p ermeabilized cell system adopted here does not adversely influence the repair capability of human fibroblasts and may thus prove useful in s tudying the enzymology of DNA repair in proliferating cultures followi ng exposure to ionizing radiation and other genotoxic agents.