Studies Of P53/MDM2/P21 And HBV Correlation In Hepatoma Cells

P53 is one of the most important tumor suppressor genes. When the cells exposed to various stresses, P53 can be activated immediately and then transactivate other genes for checkpoint control , preventing DNA replication and mitosis and protect cells from malignant transformation. P53 functions through "p53 net" composed by a series of P53-relataed genes. MDM2 and p21 are important members in p53 network and play important role for p53 regulation and function. Mutation or inactivation of P53 plays a central role in the development of many types of cancer. Loss of p53 function is also commonly found in human primary liver cancer, moreover, in view of the closely relative of HBV infection and hepatoma, relationship between HBV and p53 is always a research spot . A P53 response element like sequence in upstream of HBV enhancerl was found in our previous work and need to be investigated further.In this study, we observed firstly expression levels of p53 and its related genes-MDM2 and P21 in different hepatoma cell lines with different P53 or HBV states, investigated the relationship of the three genes, studied the correlation of P53 with DNA excision repair ability by unschedualed DNA synthesis (UDS) detection. In addition, correlation of HBV or P53 response element like sequence upstream of HBV enhancerl with P53 was studied. The current research is as follows: 1. Expression of P53/MDM2/P21 and their correlation analysis in hepatoma cellsHepG2, HepG2.2.15, PLC/PRF/5, HCC-9204 and Hep3B cells with different P53 or HBV states were studied. Expression of P53/P21/MDM2 was detected by western blot method. Reporter gene plasmids PG13-CAT and P21-LUC weretransfected into each cell line to observe the difference of p53 activity and its effect on p21 gene promoter. Results showed that PLC/PRF/5 cell has high level of P53 protein expression but low activity reflected by CAT activity following PG-13CAT transfection. This is probably because that mutation type of P53 in this cell line has prolonged half life but weak function. Unexpectedly, this cell line has a moderate P21 protein expression and high LUC activity following P21-LUC transfection. Results indicate that there exist a P53-independent p21 activation pathway in PLC/PRF/5 cell . There is no P53 and weak P21 protein expression in Hep3B cells , but P21-LUC reporter in this cell has a relatively higher activity than in HCC-9204 cell which expressing mutation type P53. Compared with HepG2, HepG2.2.15 has higher P53 or P21 expression and PG13-CAT or P21-LUC activity, which is thought to be related to HBV replication.In HBV-integrated cells such as PLC/PRF/5 , HepG2.2.15 and Hep3B, P21 protein or P21 reporter expression is high or moderate high, which indicate that some products of HBV may play upregulating role on P21.It seemed that there was a inverse association between MDM2 and P53 expression: MDM2 express stronger in cells having low P53 expression, whether P53 is wild or not, such as in PLC/PRF/5 and HepG2 (HepG2.2.15) cells. HCC-9204 and Hep3B cell have stronger MDM2 expression but low or no P53 expression. The results indicate that MDM2 could downregulate the wild or mutation type of P53.To examine the association of p53 , HBV and DNA repair capacity in hepatoma cell lines , DNA damage repair capacity was determined in HepG2, HepG2.2.15, Hep3B and PLC/PRF/5 by unscheduled DNA synthesis(UDS). Western blot was performed to observe P53 expression change after UVC exposure. Results showed that HepG2 has an increased P53 expression and was more competent to repair damaged DNA and had a higher survival rate . Results suggest that functional p53 may play an important role to keep cells in good condition to repair damaged DNA. HepG2 had relatively higher UDS than HepG2.2.15 which suggested that replication of HBV in HepG2.2.15 could downregulate DNA repair capacity . 2. Interaction effect of p53 with HBVTo observe whether there is an interaction of hepatitis virus B (HBV) and tumor suppressor P53, parental HBV-free HepG2 cells and HBV-transfec...