Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay

Phosphorylated H2AX (γ-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 γ-H2AX level. Furthermore, the assays commonly used for γ-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 γ-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 γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-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.磷酸化组蛋白H2AX（γ-H2AX）是检测DNA双链断裂（DSBs）的敏感标志物，但H2AX在不同细胞及组织类型中的表达存在显著差异，这使得对γ-H2AX水平的生物学意义解读颇具难度。此外，当前主流的γ-H2AX检测方法多基于显微镜成像，不仅操作流程繁琐，且样本通量较低。本研究报道了一种酶联免疫吸附测定（ELISA）方法，可同时定量检测磷酸化H2AX与总H2AX的绝对含量，通过计算γ-H2AX占总H2AX的百分比得到标准化数值，以此反映DNA损伤程度。我们验证了该方法可用于检测培养细胞及异种移植模型中由电离辐射或DNA损伤剂诱导产生的DNA双链断裂。此外，利用美国国家癌症研究所60株肿瘤细胞系（NCI-60）检测面板，我们证实了基础γ-H2AX比例与细胞突变水平之间存在显著相关性。这一拓展应用凸显了该检测方法可同时处理大量细胞提取物的优势，从而能够将检测结果与其他细胞特性进行关联分析。总体而言，γ-H2AX ELISA为DNA损伤的定量分析提供了全新的技术手段，有望助力我们更深入地解析肿瘤中的诱变通路，并为监测DNA损伤类抗癌药物的临床疗效提供极具价值的生物标志物。