Spatial and temporal distribution of γH2AX fluorescence in human cell cultures following synchrotron-generated X-ray microbeams: lack of correlation between persistent γH2AX foci and apoptosis.

Formation of γH2AX foci (a marker of DNA double-strand breaks), rates of foci clearance and apoptosis were investigated in cultured normal human fibroblasts and p53 wild-type malignant glioma cells after exposure to high-dose synchrotron-generated microbeams. Doses up to 283 Gy were delivered using beam geometries that included a microbeam array (50 µm wide, 400 µm spacing), single microbeams (60-570 µm wide) and a broad beam (32 mm wide). The two cell types exhibited similar trends with respect to the initial formation and time-dependent clearance of γH2AX foci after irradiation. High levels of γH2AX foci persisted as late as 72 h post-irradiation in the majority of cells within cultures of both cell types. Levels of persistent foci after irradiation via the 570 µm microbeam or broad beam were higher when compared with those observed after exposure to the 60 µm microbeam or microbeam array. Despite persistence of γH2AX foci, these irradiation conditions triggered apoptosis in only a small proportion (<5%) of cells within cultures of both cell types. These results contribute to the understanding of the fundamental biological consequences of high-dose microbeam irradiations, and implicate the importance of non-apoptotic responses such as p53-mediated growth arrest (premature senescence).