title: 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.
abstract: The mammalian DNA polymerase inhibitors aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair 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 seen with each inhibitor alone. Thus either aphidicolin modulates the rate of intracellular accumulation of araC 5'-triphosphate (araCTP), the presumed rate-limiting step in the 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 gamma 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 micrograms/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 approximately 1.1 sites/10(8) daltons/2 h upon exposure to 150 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 gamma-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was approximately 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 and 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 gamma ray repair is different from that of aphidicolin. In contrast to the observations with these chemicals, ddTTP (20 microM), a potent inhibitor of DNA polymerase beta, did not produce any measurable effect on DNA 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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Molecular Oncology Program, Cross Cancer Institute, Edmonton, Alberta, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Mirzayans
authors.forename: L
authors.initials: L
authors.lastname: Enns
authors.forename: S
authors.initials: S
authors.lastname: Cubitt
authors.forename: K
authors.initials: K
authors.lastname: Karimian
authors.forename: B
authors.initials: B
authors.lastname: Radatus
authors.forename: M C
authors.initials: MC
authors.lastname: Paterson
mesh_terms: D016590:Aphidicolin / Q000494:pharmacology*; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D002648:Child; D003561:Cytarabine / Q000494:pharmacology*; D004260:DNA Repair / Q000187:drug effects*; D004338:Drug Combinations; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D012503:Saponins / Q000494:pharmacology
chemical_list: D004338:Drug Combinations; D019384:Nucleic Acid Synthesis Inhibitors; D012503:Saponins; D003561:Cytarabine; D001085:Arabinofuranosylcytosine Triphosphate; D016590:Aphidicolin