Increased γ-H2A.X Intensity in Response to Chronic Medium-Dose-Rate γ-Ray Irradiation

<div><h3>Background</h3><p>The molecular mechanisms of DNA repair following chronic medium-dose-rate (MDR) γ-ray-induced damage remain largely unknown.</p> <h3>Methodology/Principal Findings</h3><p>We used a cell function imager to quantitatively measure the fluorescence intensity of γ<em>-</em>H2A.X foci in MDR (0.015 Gy/h and 0.06 Gy/h) or high-dose-rate (HDR) (54 Gy/h) γ<em>-</em>ray irradiated embryonic fibroblasts derived from DNA-dependent protein kinase mutated mice (scid/scid mouse embryonic fibroblasts (scid/scid MEFs)). The obtained results are as follows: (1) Automatic measurement of the intensity of radiation-induced γ<em>-</em>H2A.X foci by the cell function imager provides more accurate results compared to manual counting of γ<em>-</em>H2A.X foci. (2) In high-dose-rate (HDR) irradiation, γ<em>-</em>H2A.X foci with high fluorescence intensity were observed at 1 h after irradiation in both scid/scid and wild-type MEFs. These foci were gradually reduced through de-phosphorylation at 24 h or 72 h after irradiation. Furthermore, the fluorescence intensity at 24 h increased to a significantly greater extent in scid/scid MEFs than in wild-type MEFs in the G₁ phase, although no significant difference was observed in G₂/M-phase MEFs, suggesting that DNA-PKcs might be associated with non-homologous-end-joining-dependent DNA repair in the G₁ phase following HDR γ<em>-</em>ray irradiation. (3) The intensity of γ<em>-</em>H2A.X foci for continuous MDR (0.06 Gy/h and 0.015 Gy/h) irradiation increased significantly and in a dose-dependent fashion. Furthermore, unlike HDR-irradiated scid/scid MEFs, the intensity of γ<em>-</em>H2A.X foci in MDR-irradiated scid/scid MEFs showed no significant increase in the G₁ phase at 24 h, indicating that DNA repair systems using proteins other than DNA-PKcs might induce cell functioning that are subjected to MDR γ<em>-</em>ray irradiation.</p> <h3>Conclusions</h3><p>Our results indicate that the mechanism of phosphorylation or de-phosphorylation of γ<em>-</em>H2A.X foci induced by chronic MDR γ<em>-</em>ray irradiation might be different from those induced by HDR γ<em>-</em>ray irradiation.</p>