60Coγ-ray irradiation induces the Phosphorylation of Histone H2AX foci formation in human bladder cancer EJ cells

Full text: DNA double-strand breaks caused by cellular expose to different chemicals and ionizing irradiation are widely believed to be responsible for cell killing and carcinogenesis. It can initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and DNA repair. Phosphorylation of Histone H2AX foci formation is one of the earliest events and is very important for DNA damage and repair activities. In the present study, we investigate whether γ-ray irradiation could induce the formation of γH2AX foci in human bladder cancer cell line EJ cells and whether this change has dependent relationship between foci numbers and irradiated doses and time after expose. Western blot and FACsan were used to detect the changes of γH2AX protein expression in EJ cells, which showed that γH2AX protein expression were significantly increased after exposure to different doses of γ-ray irradiation compared with sham-radiation control. The dose- effect relationship was not remarkable by using these two methods. Neutral comet assay was also employed to measure the DNA double strand breaks, which only sensitivity to high doses (lower than 0.5 Gy was not sensitive). γH2AX foci formation were detected by using zeiss immunofluorescent microscopy, monoclonal anti-phospho-histone H2AX antibody and FITC labeled secondary antibody were used, the result showed that γH2AX foci can be induced byγ-ray irradiation in a dose-dependent and time-dependent manners. The foci number and size in nuclei of EJ cells was significantly increased after exposed to different doses of γ-ray irradiation and the foci remained detectable at 24 h after expose to irradiation, the dose range which foci can be clearly detected was from 0.1 Gy to 4 Gy in our study. We conclude that γH2AX foci enumerating is the most sensitive method and indicator for DNA damage and repair in mammalian cells, it has a wonderful application prospect in future irradiation damage provenience and it may be a new bio-marker for radiological emergency. (author)