Modulation of X-irradiation-induced γ-H2AX focus stability in human blood lymphocytes: A technical note

Histones, essential building blocks of the nucleosome, undergo dynamic changes during the cell cycle. Phosphorylation of histone protein H2AX occurs at sites of DNA double-strand breaks (DSBs) and is a sensitive marker of DNA damage. Detection of phosphorylated H2AX, the γ-H2AX assay, has been widely used in experimental models, including human tissues and cell lines. The assay has been used in research on DNA repair, drug development, genotoxicity assessment, radiation biology, diagnostic radiology, and radiotherapy protocol optimization. Among these applications, the γ-H2AX assay has gained particular attention in radiation biodosimetry due to its rapid turnaround time. Despite its potential for radiation triage, the assay's utility in cases of delayed sample collection/processing remains limited, because quantitation is constrained by rapid dephosphorylation, catalyzed by phosphatases such as Protein Phosphatase 2 A (PP2A). Potential stabilizing agents, such as fostriecin, okadaic acid, calyculin A, and LiCl, have been suggested to preserve γ-H2AX by modulating phosphatase activity. We evaluated the possible stabilising effects of calyculin A and LiCl on γ-H2AX foci in irradiated human blood lymphocytes. Pre-treatment with either calyculin A (10 nM) or LiCl (20 mM) yielded higher levels of γ-H2AX foci, 0.5 h post-X-irradiation; however, the stability of the γ-H2AX foci declined, 24 h post-irradiation.