| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 96 === The human Rad9 (Rad9) is the homologue of the fission yeast Schizosaccharomyces pombe Rad9 protein, a member of the checkpoint rad genes products. The Rad9 is a 391-amino acid protein that plays multiple roles in fundamental biological processes, including the regulation of the DNA damage response, cell cycle checkpoint control, DNA repair, apoptosis, transcriptional regulation and embryogenesis. Previous studies indicated that the biological functions of hRad9 in controlling cell cycle checkpoints, apoptosis, and genetic stability are much like p53, suggesting that Rad 9 and p53 might function coordinately in these important cellular bioprocesses. In our previous studies, we found that Rad9 could interact with MDM2 by using in vivo and in vitro assays. In this study, in vitro pull down assay was used to determine which domain of MDM2 has a Rad9-binding capacity. The results indicated that MDM2 interacts with Rad9 through its RING-finger motif, which is known to administer E3 ligase activity of MDM2, suggesting that Rad9 may affect the E3 ligase activity of MDM2 when Rad9 interacts with MDM2. To investigate this, Rad9 expression was knocked down by si-RNA, and the protein levels of p53 and E-cadherin, both of which are substrates for the MDM2 E3 ubiquitin ligase, were detected by Western blotting. The results revealed that Rad9-siRNA cells exhibited a significant decrease in the protein levels of p53 and E-cadherin. In addition, we also found that the Rad9-siRNA cells exhibited a significant increase in MDM2 protein level, indicating the Rad9 could influence MDM2 stability by interacting with MDM2. Since Rad9 is also known to act as a DNA damage sensor for DNA repair, we determined the protein levels of MDM2 and p53 in Rad9-siRNA cells under UV irradiation damage condition. Our present data reveals that protein levels of p53 increased, whereas protein levels of MDM2 decreased after UV irradiation in control cells. In contrast, after UV irradiation, inhibiting Rad9 by siRNA in HEK-293T cells resulted in lower levels of p53 expression and higher levels of MDM2 expression compared to those of control cells. Furthermore, after camptothecin treatment, inhibiting Rad9 by siRNA in HEK-293T cells resulted in markedly delayed in the accumulation of p53. To further investigate whether depletion of Rad9 could also impact on the functions of p53 in Rad9-siRNA cells, we examined the protein levels of p21 and PUMA, both of which are p53 downstream target proteins, and detected the caspase-3 activity, which is involved in the p53-dependent apoptosis pathway. We found that in Rad9-siRNA A549 cells, both p21 and PUMA had higher level expressions, but caspase-3 enzyme had lower activity. In addition, we also found that the Rad9-siRNA A549 cells possessed a higher cell migration activity, and the Rad9-siRNA cells possessed a higher cell invasion activity than that of the parental cell, MDA-MB-435S cell. To further study of the biological functions for Rad9 in regulating MDM2 and p53, the effects of Rad9 on transcriptional activity of p53 in Rad9-siRNA cells or in Rad9-overexpressing cells under normal condition are undertaken. In addition, the roles of Rad9 in apoptotic pathway and in tumorigenesis in response to DNA damage or anti-cancer drugs treatment will also be investigated.
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