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Inhibition of Gama-H2AX after Ionizing Radiation as a Biological Surrogate of Impaired Upstream DNA Damage Signaling and Radiosensitivity

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Author(s): Johnny Kao | Amir Lavaf | Chao-Hua Lan | Shibo Fu

Journal: Journal of Cancer Molecules
ISSN 1816-0735

Volume: 5;
Issue: 2;
Start page: 49;
Date: 2010;
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Keywords: H2AX | ATM | ATR | DNA-PKcs | radiosensitivity

ABSTRACT
AIM: Gama-H2AX is emerging as an important marker of ionizing radiation (IR)-induced double strand breaks. Based on genetic data, we evaluated inhibition of gama-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 gama-H2AX occurred in a dose-dependent 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 microM LY294002 (DNA-PKcs inhibitor), 8 Gy + 1 mM caffeine (ATM/ATR inhibitor) or 8 Gy + 20 microM wortmannin (DNA-PKcs, ATM and ATR inhibitor) resulted in a 55%, 51% and 88% reduction in relative fluorescence, respectively. Concurrent treatment with inhibitors of gama-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 gama-H2AX is redundantly phosphorylated by ATM, ATR and DNA-PKcs.
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