Dose-response And Time-effect Between Ionizing Radiation(IR) And Histone H2AX Phosphorylation(γH2AX) Of Tumor Cell

Recently, a new DNA damage repair-related protein, the phosphorylated histoneH2AX (γH2AX) has been entering the vision of researchers. Mechanistically, ATM,ATR and DNA-PK, members of the PI3 kinase family, phosphorylated the of H2AXat the DSBs points. ThenγH2AX recuited other DNA damage repair factors to DSBsand provide a place for DNA damage repair. When DSBs were introduced intospecific partial nuclear volumes of cells by pulsed microbeam laser,γH2AX foci onlyform at these sites. The number ofγH2AX foci formed in a cell appears to be directlyrelated to the number of DNA double stranded breaks. Linear dose-responserelationships can be generated by counting individual foci or by measuringγH2AXintensity using flow cytometry. Besides, the half loss timeÏ„_1/2 ofγH2AX afterirradiation was shown to correlate with radiation sensitvity of human tumor cells andnormal cells(R²ï¼ž0.7). Thus,γH2AX analysis was a sensitive method to detect DSBs,and had potiential to be a new surrogate marker for radiosensitivity prediction of cells.This study contained two main parts. In the first part, severalγH2AX detectionmethods were applied to generate dose-response relationships, which then werecompared for differentiation. In the second part,γH2AX time-effect after irradiationwere studied, curves were fit to data and some parameters related toγH2AXtime-course were calculated.Based on the above studies, the following conclusion should be addressed:1. Ethanol-dual stained was a suitableγH2AX detection method, its slopes ofdose-response line measured by flow cytometer, and correlation was best inall detection methods.(ethanol-dual stained＞PFA-single stained＞ethanolsingle stained);2. Saturation phenomenon occurred at high dose groups when usingmicroscope.;3. TheγH2AX linear dose-response relationship was confirmed by neutral comet assay.4. The kinetics ofγH2AX after irradiation was featured by rapid increase andslow decay. The peak ofγH2AX level was reached at 1 hour post-irradiation.The best fit model of long time decay(1～20 hours) data was one orderexponential curve.The post-irraidation half loss time ofγH2AX was dose-dependent of ionizing radiation.In sum, our study provided more evidence for the standardization ofγH2AXdetection, and relationship betweenγH2AX post-irradiation time-effect andradiosensitivity of cells. However, the foundings of this study should be confirmedand extended by broad cell lines, and animal experiments, especially the lymphocyteswhich are easily obtained from clinics. Also, comparative study onγH2AX and cometassay measuring radiation dose-/time-effect should be involved in our future study todisclose more details ofγH2AX function of on DNA damage repair.