Abstract
Phosphorylated histone H2AX (γH2AX) has been thought to be a marker of DNA double-strand breaks (DSBs). We examined whether polyaromatic hydrocarbons (PAH), benzo[a]pyrene, 1-nitropyrene, 1,8-dinitropyrene, 3-nitrobenzanthrone and N-hydroxy-3-aminobenzanthrone, can phosphorylate H2AX in human HeLa cells by using immunofluorescence microscopy. These substances do not cause DSB directly but produce purine adducts in cellular DNA. After exposure of the cells to these chemicals with a concentration giving 10% cell survival followed by incubation for one hour, γH2AX foci appeared in the cell nuclei. All cells expressing γH2AX corresponded to those that incorporated bromodeoxyuridine after PAH treatment, indicating that PAH-induced H2AX phosphorylation correlated to S-phase entry of the cells. Cells exposed to ultraviolet light and camptothecin, which produce pyrimidine dimers and single-strand breaks (SSBs) in DNA, respectively, showed the same response. On the other hand, cells exposed to X-ray and etoposide, which produce DSBs, expressed γH2AX not only in S-phase cells. These results suggest that H2AX phosphorylation signals are transmitted from directly produced DSBs in all cell phases as well as from DNA strand stalling in the S-phase. H2AX phosphorylation by all substances used in this experiment is inhibited by wortmannin, implying that H2AX phosphorylation is regulated by ATM and/or ATR pathways.
