ATM Phosphorylation Kinetics as a Biological Reporter of Cellular Radiosensitivity

AIM: Exposure to ionizing radiation (IR) causes DNA strand break formation, result-ing in the rapid activation and autophosphorylation of ATM on Ser-1981. We hypothe-size 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 poly-clonal anti-phospho-H2AX or monoclonal anti-phospho-ATM phospho-specific anti-bodies. 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 deter-mine the relative radiosensitivity of PC-3, DU-145, SCC25, and SQ-20B cells. RESULTS: Within 10 min after IR, discrete foci of ATM autophosphorylation ap-peared at putative sites of DNA strand breaks. The number and relative brightness of γ-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 γ-H2AX foci, which was most evident at 30 min after IR. CONCLUSION: Our preliminary data suggest that persistence of ATM autophos-phorylation 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