Using gamma-H2AX and H2AX quantitative ELISA for monitoring DNA damage induced by chemotherapeutic agents and irradiation exposure.

Abstract

2559 Background: Gamma-H2AX (γH2AX) is a biomarker for DNA double-strand breaks and programmed cell death, but variable relative amounts of H2AX in different samples causes ambiguity in the meaning of the γH2AX level unless it is related to total H2AX levels. We developed a 96-well plate-based ELISA for quantifying γH2AX and H2AX levels in crude extracts of tumor cells, CTCs and biopsy tissues and are validating it for applications in irradiation exposure monitoring and in pharmacodynamic evaluation of anti-cancer agents. Methods: The ELISA was used to analyze extracts of several NCI60 tumor cell lines that had been exposed to a variety of agents, including ionizing radiation, inhibitors of Top-1 (CPT, SN-38, Topotecan), PARP (ABT-888, AZD-2281, MK-4827) and ATR (VE-821, VE-822, AZD-6738, Compound 45, NU-6027), and their combinations. Combination regimens of CPT-11 with PARP inhibitors (ABT-888, AZD-2281, MK-4827) were further evaluated in vivo in the A375 xenograft mouse model. Patient samples obtained for research purposes were also examined by ELISA for feasibility and utility. Results: In vitro, dose-dependent increases in the ratio of γH2AX to H2AX were detected after escalating ionizing radiation exposure and concentration-dependent increases after Top-1 exposure. Treating with inhibitors of PARP or ATR alone did not significantly induce γH2AX. Combinations of Top-1 inhibitors with PARP or ATR inhibitors led to synergistic induction of DNA damage. Among five ATR inhibitors evaluated in combination with Top-1 inhibitors, VE-822 and AZD-6738 were observed to have the highest synergy for γH2AX induction, while NU-6027 showed none. Combinations of CPT-11 with ABT-888, AZD-2281 or MK-4827 showed synergistic induction of γH2AX in A375 xenografts in vivo. Additional testing of human specimens including PBMCs, bone marrow and tumor biopsies proved the assay’s clinical suitability and potential advantages. Conclusions: A newly developed quantitative ELISA for measuring both γH2AX and H2AX is ready for clinical validation for monitoring DNA damage induced by chemotherapeutic agents or irradiation exposure. Funded by NCI Contract No. HHSN261200800001E.