- 期刊
Inhibition of γ-H2AX after Ionizing Radiation as a Biological Surrogate of Impaired Upstream DNA Damage Signaling and Radiosensitivity
並列摘要
AIM: γ-H2AX is emerging as an important marker of ionizing radiation (IR)-induced double strand breaks. Based on genetic data, we evaluated inhibition of γ-H2AX as a reporter of aberrant DNA repair and radiosensitivity.METHODS: PC-3, DU145 and LNCaP prostate cancer cells were treated with radiation and small molecule inhibitors of ATM/ATR, DNA-PKcs or both. Cells were analyzed using fluorescence activated cell sorting with doublets and cell debris gated out. Mean fluorescence was calculated, adjusting for DNA content.RESULTS: Within 20 min after IR, induction of γ-H2AX occurred in a dosedependent fashion for IR doses from 0 to 8 Gy in all three cell lines. Compared to 8 Gy alone, cells treated by 8 Gy + 50 μM LY294002 (DNA-PKcs inhibitor), 8 Gy + 1 mM caffeine (ATM/ATR inhibitor) or 8 Gy + 20 μM wortmannin (DNA-PKcs, ATM and ATR inhibitor) resulted in a 55%, 51% and 88% reduction in relative fluorescence, respectively. Concurrent treatment with inhibitors of γ-H2AX resulted in significantly increased tumor cell radiosensitivity.CONCLUSION: This streamlined flow cytometry assay is a robust and biologically useful approach to elucidating response to DNA damage after IR and drug therapy. These data support the notion that γ-H2AX is redundantly phosphorylated by ATM, ATR and DNA-PKcs.
延伸閱讀
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