Background: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted ␥H2AX. The intensity of immunofluorescence (IF) of ␥H2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the druginduced (DI) IF of ␥H2AX, and in particular to distinguish it from AA ␥H2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. Methods: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA crosslinking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of ␥H2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of ␥H2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. Results: In cells treated with TPT or MTX, the increase in DI-␥H2AX IF peaked at 1.5 or 2 h, and was maximal in Sor G 1 -phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the ␥H2AX IF was less intense, peaked later (3 h) and showed no cell cyclephase specificity. In the presence of phosphatase inhibitor

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