ABSTRACT
Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P < 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] %,[7.84 ± 0.68] %,[8.40 ± 0.50] % and [14.03 ± 1.03] %) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] %,F =35.879,P < 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P < 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P < 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P <0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.
ABSTRACT
Objective:To study the feasibility and reliability of using phosphorylated H2AX(?H2AX)as a predictor for sensitivity of hepatic carcinoma cell HepG2.215 to chemotherapy agents: etoposide, doxorubicin, mitomycin, and cisplatin. Methods: HepG2.215 cells were exposed to etoposide, doxorubicin, mitomycin or cisplatin of 1, 2, 4 and 20 concentration index (CI). Untreated HepG2.215 cells were taken as control. The proportion of HepG2.215 cells expressing ?H2AX was measured by flow cytometry, the number of ?H2AX foci in HepG2.215 cells was measured by immunocytochemistry, and cell proliferation was measured by MTT. The correlation between the number of ?H2AX foci and the percentage of HepG2.215 cells expressing ?H2AX in HepG2.215 cells was analyzed; the correlation of CI with the percentage of HepG2.215 cells expressing ?H2AX or ?H2AX foci and inhibitory rate of cell proliferation was analyzed; and the correlation of inhibitory rate of cell praliferation with the percentage of HepG2.215 cells expressing ?H2AX or ?H2AX foci was also analyzed. Results: There was a positive correlation between the number of ?H2AX foci and the percentage of HepG2.215 cells expressing ?H2AX in HepG2.215 cells after treatment with the above 4 agents (all P