Predictive Effect Of γH2AX Expression On The Radiosensitivity Of Glioma

Objective:Glioma is a common intracranial tumors,which is intracranial primary malignant tumor. Glioma grows aggressively which is difficult to be completely completely excised by surgery. Postoperative radiotherapy is an important treatment of glioblastoma. However, the therapeutic effect of radiotherapy is not good enough because of the radiation resistance of glioblastoma.In recent years, a new biomarker, the phosphorylated histone H2AX, has become a powerful tool to monitor DNA DSBs in translational cancer research. In the present article,we observe the expression changes of yH2AX in high grade glioma cell and to investigate the relationship between the expression of yH2AX and the radiosensitivity of high grade glioma cells in vitro.Methods:1.The radiosensitivity of glioma (U87、U251、 LN229) cells were evaluated by using cell clonogenic assays.The cells were then irradiated using USA Varian walian600linear accelerator(source skin distance was100cm. dose rate was300cGy/min,accelerator6MV)to deliver the indicated doses(0,2,4,6,8and10Gy)at room temperature. After incubation for14days to allow the formation of macroscopic colonies, the plates were fixed with methanol and stained with Giemsa. Colonies containing at least50cells in size were counted. The fraction surviving a given X-ray dose was calculated based on the survival of irradiated cell.Survival data after a radiation dose were fit by analysis of single factor variance.2.The levels of yH2AX of glioma U251, U87and LN229cell lines were detected by western blotting. According to incubation time after the X-ray irradiation for each cell line, The experiments are randomly divided into9groups(0h group,0.5h irradiation group,1H irradiation group,2h irradiation group,6h irradiation group,12h irradiation group,24h irradiation group,36h irradiation group and48h irradiation group. Using of cells in the exponential growth phase of the plant, the cells were then irradiated using USA Varian walian600linear accelerator and incubated. Sample protein lysates in sample buffer from each tissue were loaded within each well. Gels were run for2hours for maximum separation. Wet transfer was performed for45mins at constant current using PVDF membrane presoaked in methanol. The membrane was blocked in BSA blocking buffer. The membrane was then washed in TBST×1for15min each. The membranes were then incubated overnight with primary antibodies directed at β-actin and γH2AX. Subsequently, the membranes were washed in0.2%TBST×3for15min each. The membrane was then incubated with secondary antibody for1h. Chemiluminescent detection was then used to detect expression of γH2AX, respectively.3. Correlation between γH2AX expression and radiosensitivity of glioma cells was calculated using statistical analysis.The value difference of protein in6h group and0.5h group instead of expression of the relative decay rate of γH2AX.And the value difference of the objective protein peak and initial value difference represents the elevated degree of γH2AX protein. Then we can analyse the relationship between radiotherapy sensitization ratio and the elevated degree of γH2AX protein,as well as between radiotherapy sensitization ratio and residual amount expression of the relative decay rate of γH2AX.Results:1. For glioma U251、U87and LN229cells lines,the clone forming rate were gradually decreased with the increase radiation dose,and the radiotherapy sensitization ratio of U87cells was slightly higher compared with LN229、U251cells by analysis of single factor variance(P=0.000).In the western blotting assay,the kinetics of the expression of γH2AX protein after irradiation was festured by increase and decay.The γH2AX expression of U87cells after irradiation reached the peak value much later by analysis of variance of randomized block design(P=0.000、0.000、0.001).There was positive correlation between radiotherapy sensitization ratio and the elevated degree of γH2AX protein,as well as between radiotherapy sensitization ratio and residual amount expression of the relative decay rate of γH2AX.(r=0.733, p=0.025;r=0.672,P=0.047).Conclusions:1.The SF2values of1.31±0.04(U87),1.14±0.02(LN229) and1.00±0.00(U251)found here reveal that U87cells is the radiosensitive cells,the U251is the radioresistant cells 2. Comparison of the three glioma cell lines also revealed that the radioresistant U251exhibited the maximum γH2AX foci expression1h after irradiation whereas its radiosensitive counterpart (U87cells) reached the peak value much later3. Both the maximum amout of the induced histone γH2AX and the relative rate of decay was slightly higher in radiosensitivity (U87) cells compared with the radioresistant(U251)cells.4.The phosphorylated histone γH2AX is expected to become a powerful tool to monitor DNA DSBs and to predict the radiosensitivity in glioma radiotherapy.