International Journal of Radiation Oncology*Biology*Physics
ICTR 2003: Translational research and pre-clinical srategy studyPhosphorylation of histone H2AX as a measure of radiosensitivity☆
Introduction
Development of methods to predict tumor and normal cell response to radiation represents an important approach to improving response to radiation therapy. Although the basis for differences in response is undoubtedly multifactorial, identification and validation of functional assays that integrate these responses is essential. Of particular importance is the measurement of repair of radiation-induced double-strand breaks, because these lesions, if unrepaired, lead to cell death. Recently, it has been shown that histone H2AX becomes phosphorylated immediately after irradiation and is believed to recruit DNA repair factors to sites of DNA double-strand breaks 1, 2. Visible foci containing thousands of molecules of γH2AX are found at each double-strand break, making it possible to detect a single break within a nucleus. Using a flow cytometry method, we have been able to detect double-strand breaks produced by doses as low as 10–20 cGy, almost 2 orders of magnitude more sensitive than traditional methods used to detect these lesions 3, 4. Although image analysis of γH2AX foci is a more sensitive method for detecting individual double-strand breaks (5), flow cytometry provides an accurate way of measuring the rate of formation and loss of γH2AX in heterogeneous populations of cells.
Because γH2AX is associated with double-strand break repair, we reasoned that the kinetics of formation or loss of these foci might be related to the efficiency of repair. Although the number of γH2AX foci formed after irradiation is consistent with the number of double-strand breaks, the kinetics of foci development and loss differ markedly from those of double-strand break rejoining. Loss of γH2AX may therefore be indicative of factors in addition to strand break rejoining, such as “repair” of higher order chromatin organization. Recently, we examined the kinetics of γH2AX after irradiation of 10 cell lines. The rate of loss of γH2AX was associated with a greater ability to survive exposure to radiation, as measured by the clonogenic fraction after 2 Gy (4). We have extended this study of γH2AX kinetics to examining the response of cells from transplanted tumors and normal tissues of mice. Because hypoxia is a known prognostic factor that reduces the number of radiation-induced double-strand breaks by a factor of about 3, we explored the possibility of using γH2AX expression to identify hypoxic cells in murine tumors.
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Cell lines
SiHa human cervical carcinoma cells, obtained from American Type Culture Collection, were maintained with twice-weekly subculture in minimal essential medium containing 10% fetal bovine serum. Cells at ambient temperature were exposed to 250-kV X-rays at a dose rate of 5.8 Gy/min. For some experiments that required equilibrating cells with different oxygen mixtures, SiHa cells were placed in glass spinner culture flasks at a density of 2 × 105 cells/mL and were continuously gassed with various
Results
Previous experiments established that maximal expression of γH2AX occurred within 1 h after irradiation of several cultured cell lines (4), and similar kinetics for development were observed here for SiHa xenograft cells (Fig. 1a). Levels in SiHa cells subsequently declined with a half-time of approximately 5 h. The overall pattern of expression was similar for different mouse normal tissues, although the relative γH2AX intensity was higher, because the background level of expression was quite
Discussion
There have been efforts in the past to use double-strand break rejoining kinetics or residual damage to identify radioresistant tumor cells. However, a clear consensus concerning the predictive ability of double-strand break repair kinetics has not emerged 7, 8, 9, 10, 11, 12, 13. There is general agreement, however, that repair fidelity is a critical factor and that this cannot be measured using conventional double-strand break rejoining methods. At this point, it is not known whether loss of
Acknowledgements
The technical assistance of Laura Sinnott and Denise McDougal is gratefully acknowledged.
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Supported by Grant No. 12069, awarded by the National Cancer Institute of Canada, with funds provided by the Canadian Cancer Society.