Cluster Size Analysis for γH2AX Assay: A Novel Tool for DNA Damage Quantification in Cells with Diverse Radiosensitivity
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Introduction :DNA double-strand breaks (DSBs) are considered the most lethal of all DNA lesions and are responsible for the bulk of cytotoxic effects induced by ionizing radiation (IR) and cancer chemotherapeutics.Objectives:This study focuses on radiation-induced DNA damage in glioblastoma cell lines characterized by disparate radiosensitivity, where divergent degrees of complex damage induction were observed.Methods:Fluorescence microscopy evaluation showed an expected linear increase in the relative foci number, localization, and signal intensities in cells with absorbed doses up to 6 Gy, albeit with repeatable pattern variation among the different cell lines. The cluster size parameter was introduced as a novel quantification approach for γH2AX assay analysis when considerable discrepancies in cell sensitivities are persistent.Results:This approach provides a robust tool to differentiate dose-response shape results, allowing for the distinction of seemingly similar responses. Conclusion: The method, addressing each cell line's unique cluster size distribution, is thus recommended for granular analysis of γH2AX foci in multiple cell lines of similar origin, demonstrating its impact on therapeutic responses in heterogeneous tumors.Graphical Abstract:
