ATM activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis

Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G(1) cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981(P) and gamma H2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981(P) IF of G(1) cells, the ratio of IF of S to G(1) cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981(P) alone, or for ATM-S1981(P) and gamma H2AX IF combined, than for gamma H2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.