Histone H2AX phosphorylation after cell irradiation with UV-B: relationship to cell cycle phase and induction of apoptosis.

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (gammaH2AX) can be revealed immunocytochemically; the intensity of gammaH2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of gammaH2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m(2)-3.45 kJ/m(2)) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G(1), G(2) and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced gammaH2AX in G(1), G(2) and M was much higher in HeLa- than in HL-60- cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of gammaH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication, by aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of gammaH2AX. Furthermore, aphidicolin itself induced gammaH2AX in early-S phase cells. The induction of gammaH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of gammaH2AX in G(1), G(2) and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.