DiscussionIn this study, we reported that a p53 response element-like binding sequence, 5′-TGCC(G)T?TGCCT-3′ at the upstream of hepatitis B virus (HBV) enhancer I from 1047 to 1059 nucleotide. We found that HBV DNA could bind to P53 protein and form a DNA–protein complexes by EMSA and EMSSA. Our results also showed this complexes may increase the replication of HBV, CAT activity, tumor cell apoptosis, and cytoplasmic P53 accumulation in the hepatoma cells, which resulting in inactivation of p53 transactivation.In virally transformed cells, P53 combines with viral oncoproteins such as the simian virus 40 (SV40) large tumor antigen [29] and [30], the adenovirus E1B 55k D protein [31] and [32], the human papilloma virus E6 protein [33] and [34], and the Epstein–Barr virus-encoded nuclear antigen EBNA-5 [35]; these protein–protein interactions can inactivate p53 functions and thus are an important aspect of viral oncogenicity. Previously, we found that HBx protein was capable of binding to P53 protein to form protein–protein complexes, which appeared to interact with p53 in order to stimulate tumor cell growth [3] and [11]. Our finding has been further confirmed by several laboratories [36] and [37]. The fact that wtp53 transactivation of gene
Nutlin3a depends on the recognition of a specific DNA binding sequence, together with its ability to stimulate HBV replication, strongly suggests that similar sequences may exist in HBV DNA. In this experiment, p53-like response elements, TGCCT repeat sequences were observed in HBV DNA genome. This putative p53 binding sequence was found at the analogous position in all of the HBV subtypes and other hepadnavirus, indicating that p53 may exert its effect on the HBV promoter [20], [21] and [22]. HBx protein can enhance the activity of both cellular and viral promoters, transactivate HBc and HBx genes, and increase the replication of HBV [2] and [38], suggesting the characteristics of HBV DNA were similar to that of other DNA tumor viruses such as SV40, HPV or EBV [29], [30], [31], [32], [33], [34] and [35]. In the present study, we determined that HBV DNA can specifically bind to wild or mutant P53 protein, and form DNA–protein complex by EMSA and EMSSA methods.Furthermore, we found that the CAT activity and p21 promoter luciferase values were significantly increased in hepG2
cells when cotransfected with pCMV-p53 and pCMV-HBVa. Transactivation of p53 and HBV DNA was confirmed by gene-cotransfection and quantitative PCR analysis. Both p53 and HBV DNA could enhance the replication of HBV DNA, stimulate accumulation of P53, and promote apoptosis in HepG2 cells. Immunocytochemical staining indicated that P53 was prevented from entering the nucleus. Our previous results showed that the transfection of p53 could increase p21-promoter activity and the apoptosis index in SMMU-7721 cells, suggesting that p53 could promote p21 transcription and contribute to the apoptosis process of the cells [39]. It is generally accepted that the accumulation of active p53 in response to stress occurs mainly through post-translational mechanism, which stabilizes the p53 and increases the ability of nucleolus location [40]. These results demonstrated that the accumulation of p53 in cytoplasm can induce the transcriptional activities and the regulatory function of p53 when HBV DNA–P53 complex is formed, which in turn stimulate viral replication by transactivation. However, the biological consequence and the mechanisms with respect to the role this complex in HBV-associated HCC need further investigation.In conclusion, our observations confirm and extend the previous findings, and suggest that the interaction of HBV and p53 at the levels of protein–protein and DNA–protein. These complexes resulted in inactivation of p53 transactivation. Our results may provide a better understanding of the pathogenesis of HBV-associated hepatocellular carcinoma.Appendix A. Supplementary dataSupplementary Fig. 1. Supplementary Fig. 1Figure optionsDownload full-size imageDownload as PowerPoint slideSupplementary Fig. 2. Supplementary Fig. 2Figure optionsDownload full-size imageDownload as PowerPoint slideSupplementary data. Supplemental material 1Help with DOC filesOptionsDownload file (40 K)Supplementary data. Supplemental material 2Help with DOC filesOptionsDownload file (19 K)Supplementary data. Supplemental material 3Help with DOC filesOptionsDownload file ( K)
