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ATM Phosphorylation Kinetics as a Biological Reporter of Cellular Radiosensitivity

Author(s): Amir Lavaf | Shibo Fu | Sheila Peters | Yen-Nien Hou | Barry S. Rosenstein | Johnny Kao

Journal: Journal of Cancer Molecules
ISSN 1816-0735

Volume: 5;
Issue: 1;
Start page: 9;
Date: 2009;
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Keywords: ATM | ionizing radiation | gamma-H2AX | radiosensitivity

AIM: Exposure to ionizing radiation (IR) causes DNA strand break formation, resulting in the rapid activation and autophosphorylation of ATM on Ser-1981. We hypothesize that quantitative analysis of ATM autophosphorylation may serve as a reporter of DNA damage induction and repair after IR in two ATM wild-type prostate cancer cell lines PC-3 and DU-145 and two oral squamous cell carcinoma cell lines SCC25 and SQ-20B. METHODS: Immunocytochemistry was performed on glass coverslips using polyclonal anti-phospho-H2AX or monoclonal anti-phospho-ATM phospho-specific antibodies. Cells were treated with 0 to 4 Gy of IR, incubated, fixed, and stained. Stained cells were analyzed using epifluorescence microscopy and discrete foci of brightness in each nucleus were counted. In vitro clonogenic assays were performed to determine the relative radiosensitivity of PC-3, DU-145, SCC25, and SQ-20B cells.RESULTS: Within 10 min after IR, discrete foci of ATM autophosphorylation appeared at putative sites of DNA strand breaks. The number and relative brightness of gamma-H2AX and ATM autophosphorylation foci increased linearly with dose at 30 min after clinically relevant IR doses in these cell lines. The relatively radiosensitive PC-3 and SCC-25 cells retained a greater percentage of ATM autophosphorylation and gamma-H2AX foci, which was most evident at 30 min after IR. CONCLUSION: Our preliminary data suggest that persistence of ATM autophosphorylation is a promising reporter of in vitro radiosensitivity. Further research is necessary to validate this finding and to develop additional applications for ATM autophosphorylation in radiation research.
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