Induction and repair of DNA single-strand breaks in EM9 mutant cho cells treated with hydrogen peroxide

Abstract

In this study we investigated the induction and rejoining of DNA single-strand breaks (SSBs) produced by H 2O 2 in the repair-deficient EM9 mutant Chinese hamster ovary (CHO) cell line. The effect of the poly(ADP-ribose)-transferase inhibitor 3-aminobenzamide (3-ABA) on SSB-rejoining and on cell killing was also evaluated. Results were compared with those obtained previously with the parent cell line (AA8). Cells were treated with H 2O 2 on ice for 1 h, after which they were either harvested or allowed to repair their damage at 37°C either in the presence or absence of 3-ABA (5 mM). The cells were then assayed either for survival using a colony-forming assay or for their level of DNA SSBs using alkaline elution. EM9 cells were somewhat more sensitive than AA8 cells to the cytotoxic effects of H 2O 2. However, because the repair mutant showed slightly lower levels of DNA SSBs than did its parental cell line, this sensitivity could not be explained on the basis of alterations in initial damage. The rejoining of the H 2O 2-induced DNA SSBs followed exponential kinetics in both cell lines; however, EM9 cells rejoined these breaks at a slower rate ( t 1 2 of 10 min) than did AA8 cells ( t 1 2 of 5 min). The increased sensitivity of the EM9 cells therefore appears to correlate with a reduced ability to remove these lesions from their DNA. As previously demonstrated for the AA8 cells, 3-ABA treatment resulted in both a retardation of the removal of H 2O 2-induced DNA SSBs and potentiation of cytotoxicity in the EM9 cells. However, the degree of these effects were similar for both AA8 and EM9 cells. These data provide further evidence that the cytotoxic effects of low concentrations of H 2O 2 are mediated by damage to DNA, and suggest that the rate at which DNA SSBs are rejoined is important for cell survival.