DNA damage detected with gammaH2AX in endometrioid adenocarcinoma cell lines.

Phosphorylation of histone H2AX (gammaH2AX) is a sensitive marker of DNA damage, particularly induction of DNA double-strand breaks. Using multiparameter cytometry we explored the effects of doxorubicin (DOX), cisplatin (CDDP) and 5-fluorouracil (5-FU) on four types of endometrioid adenocarcinoma cell lines (HEC-1A, HEC-1B, Ishikawa, KLE) correlating the drug-induced increases in phosphorylated H2AX (gammaH2AX) with cell cycle phase, induction of apoptosis and induction of cell senescence, the latter detected by analysis of beta-galactosidase. The study revealed significant differences among the cell lines in the effects of DNA damage vis-a-vis cell cycle phase specificity, induction of apoptosis or senescence following drug treatment. DOX treatment showed little cell cycle specificity in terms of induction of gammaH2AX, and its mechanism, which is similar to another anthracycline DNA topoisomerase II inhibitor mitoxantrone, may involve oxidative DNA damage modulated by other factors. Treatment with CDDP and 5-FU led to phosphorylation of H2AX preferentially in S-phase cells, consistent with the induction of replication stress. The response of Ishikawa cells expressing wt p53 was different compared to other cell lines. The data suggest that the treatment of endometrioid adenocarcinoma with these drugs may have to be customized to individual patients. The flow cytometric bivariate analysis of gammaH2AX and DNA content is a useful technique for better understanding the effects of antitumor agents and may contribute to customized patient treatments.