MacPhail, S. H., Banáth, J. P., Yu, Y. T., Chu, E. and Olive, P. L. Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells. Radiat. Res. 159, 759–767 (2003).
Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (γH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in γH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced γH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of γH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of γH2AX. With the exception of xrs5 cells, γH2AX level was approximately 3 times lower in unirradiated G1-phase cells than S- and G2-phase cells, and the slope of the G1-phase dose–response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G1-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5–10-fold higher levels of γH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of γH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G2-phase cells.