Phosphorylated Fraction Of H2ax As A Measurement For Dna Damage In Cancer Cells And Potential Applications Of A Novel Assay

Phosphorylated H2AX (gamma-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the gamma-H2AX level. Furthermore, the assays commonly used for gamma-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of gamma-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of gamma-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure gamma-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the gamma-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.