Temporal and spatial patterns of γH2AX signaling in different human cells after exposure to X-rays and UV-C light

Abstract The phosphorylation of H2AX at serine 139 (γH2AX) is a marker of DNA damage response, well characterized after ionizing radiation (IR) exposure. In this work we compared γH2AX activation in different human cell lines exposed to X-rays, up to 5 Gy, or UV-C light (below ionization threshold), up to 40 J/m 2 . We selected HeLa, Caco-2 and HaCaT cells as models with different origins and radiosensitivity, conventionally screened with the clonogenic assay. We integrated data from fluorescence microscopy and flow cytometry. Results highlight that γH2AX spatial pattern is determined by the genotoxic agent: X-rays induce discrete, countable foci (associated with DNA double-strand breaks), while UV-C leads to pan-nuclear signal distributions. Instead, dose-response curves are strongly dependent on the cell-line, showing linear or saturating trends, not attributable to the readout technique. After 5 Gy X-rays, γH2AX signal peaks within 1 hour. The residual intensity at 24 hours is compatible with the control for all cell lines; however, cells show different clonogenic survival fractions at 7 days. The kinetics of γH2AX positive cells following 20 J/m 2 UV-C exposure is completely different, with a slower accumulation and persistent signaling from viable cells (not restricted to the S-phase) up to 24 hours, but with a loss of clonogenic potential in the long-term. These findings emphasize that γH2AX marker may be considered as a functional sensor of DNA damage response rather than a marker of specific DNA lesions, and that both radiation quality and cellular context must be considered for its correct interpretation.