Abstract
P53 gene is well known as a tumor suppressor gene which plays an important role in conserving stability by preventing genome mutation and inducing apoptosis. Fishes also have p53 gene as mammal. Reports showed that induction of apoptosis by γ irradiation in zebrafish is suppressed in p53 mutant embryos, and cancer occurred in p53-/- zebrafish (Berghmans S, Proc Natl Acad Sci U S A. 2005 Jan 11). And similar reports about medaka showed that, in p53-/- cells apoptosis is suppressed observed by time lapse, and cancer occurred in medaka (Genome Biology 2006, 7:R116). In our study, to make the role of medaka p53 gene clear, we compared the phenotype of medaka wild type cells and p53-/- cells, and transfected p53 over-expressed plasmid into p53-/- cells for rescue experiments. At first, we analyzed apoptosis occurred in p53-/- cells. As a result, the proportion of apoptotic cells in p53-/- cells is much smaller than in wild type cells. We also added Pifithrin-α. Hydrobromide, a p53 gene inhibitor of mice, to wild type cells, and found that the proportion of apoptotic cells decreased significantly, the p53 gene inhibitor of mice is also effective in medaka. To investigate the relationship between p53 gene and DNA repair, we used comet assay to compare the DSBs repair induced by γ ray irradiation between wild type cells and p53-/- cells. As a result, we found that the tail moment which values the ability of DNA repair reduced significantly after few hours passed irradiation in wild type cells, while there is no significant change in p53-/- cells. So we get a conclusion that the ability of DNA repair in p53-/- cells is weaker than wild type cells. In order to investigate the relationship between p53 and the first stage of DNA repair, we used γ H2AX focus assay to compare the focus formation in wild type cells and p53-/- cells after irradiation. At result, there is no significant difference between normal cells and p53-/- cells. At present, we are using p53 gene transfected p53-/- cells to investigate the resumption of the functions lost in p53-/- cells.
