Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage

DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP ) are activated by DNA strand breaks and participate in DNA repair. We inve stigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU102 5), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) an d single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells fol lowing exposure to ionizing radiation (IR) or temozolomide. WM (20 mu M) or NU1025 (300 mu M) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 +/- 0.6 and 1.7 +/- 0.2, res pectively. When used in combination, a DEF10 of 7.8 +/- 1.5 was obtained. W M or NU1025 potentiated the cytotoxicity of temozolomide, and an additive e ffect on the DEF10 value was obtained with the combined inhibitors. Using t he same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution, Cells exposed to IR were post-incubated for 30 min to allow repair to occur . WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 +/- 0.04 and 1.20 +/- 0.05, respectively, compared with 1.01 +/- 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025, SSB levels were increased to 1.27 +/- 0.05 with NU1025 compared with IR alone, 1.02 +/- 0. 04, The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 mu M WM and 300 mu M NU1025, DSB repair kineti cs were studied. Both inhibitors increased net DSB levels over a 3 h time p eriod; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR, The combined use of DNA repair inhibitors may have therapeutic potential.