Regulation Of Survivin By P53 Mediated By Epstein-Barr Virus Encoded Latent Membrane Protein 1

Tumor is considered the result of turbulence of cell regulation pathways. Compared with normal cells, some signaling pathways are activated abnormally, while others are blocked in tumor. In a word, loss control of proliferation, inhibition of apoptosis, invasion and metastasis are the results of the abnormal signaling pathways in tumor.Epstern-Barr virus (EBV) is the first identified DNA virus that is associated with human malignancies, such as Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma (NPC), lung cancer, gastric carcinoma and breast cancer etc. Latent membrane protein 1 (LMP1) is the only one with oncogenic properties among EBV encoded proteins. Our previous studies have been focused on the signaling transduction pathways mediated by LMP1. Based on the LMP1-inducible expression system, we elucidated that LMP1 was involved in multiple biological functions, such as cell proliferation, apoptosis, invasion and metastasis, through initiating NF-κB, AP-1 and JAK/STAT signaling pathways, which affect their downstream genes, including survivin and p53.Survivin is a member of the inhibitor of apoptosis (IAP) family identified in 1997. Differently from other IAP proteins, survivin is expressed widely in fetal tissues and most tumor tissues, but becomes restricted during development, and appears to be negligibly expressed in the majority of adult tissues. The Baculovirus IAP Repeat (BIR) domain in survivin has been identified to be associated with its anti-apoptotic function. The expression of survivin is also cell cycle-regulated, and is highly expressed in G2/M. Survivin has dual functions of cell cycle regulation and inhibition of apoptosis. Survivin, as a mitotic substrate of cdc2/cyclinB1, can be phosphorylation on Thr34, which contributes to regulation of cell division via an interaction with caspase-9. Also, survivin can promote G1/S phase progress by translocating into nuclei and combining with CDK4. This suggests that survivin can regulate either G2/M or G1/S phase of cell cycle. The role of survivin in inhibition of apoptosis has a similar degree of complexity, connecting to multiple parallel pathways that regulate gene expression, protein-protein interactions and mitochondrial functions.Our previous study has demostrated that LMP1 could mediate the activity of survivin through NF-κB and AP-1. LMP1 could mediate the translocation of survivin into nuclei, which promotes Rb phophosrylation and S phase progress and leads to cell proliferation. Also, LMP1 could induce the expression of survivin and CDK4 simultaneously, promote their translocation into nuclei and mediate their combination, which contributes to the G1/S phase progress. LMP1 could inhibit apoptosis by survivin. The expression of antisense RNA for survivin is sufficient to inhibit cell liferation and induce apoptosis. All these study in our lab provides us with important basis for the mechanisms of the regulations of survivin mediated by LMP1.Survivin overexpression in many tumor cells is associated with the loss or mutation of wild type p53. Mutation of p53 may stimulate the expression of survivin through one or more signaling pathways. Wild type p53 can repress survivin transcriptionally. Survivin promoter has a p53 binding element. It is possible that p53 directly binds survivin promoter alone or in combination with other protein(s), such as E2F, or sin3-HDAC to repress survivin. p53 also represses the activity of cdc2/cyclinB1 by binding to the catalytic subunit of cdc2/cyclinB1, resulting in downregulation of survivinThr34. However, survivin may also influence p53 activity through regulation of caspase/Mdm2 and AuroraB etc.Over half of human tumors are associated with p53 mutations, indicating its pivotal role as a tumor suppressor protein. P53, a nuclear phosphorylation protein, is associated with regulation of cell cycle, DNA repair, cell differentiation, apoptosis and so on. But in NPC, unlike most human tumors, mutation rate of p53 is less than 10%. Analysis of the expression of the p53 protein by immunohistochemistry on NPC biopsies indicates that p53 accumulation is significantly correlated to LMP1. Many data suggest that p53 has the functional activation in the carcinogenesis of NPC. We confirmed that there were lots of free, wide-type p53 in immortalized NP69 cell model, and interestingly, p53 is activated. LMP1 could promote p53 nuclear accumulation, up-regulate transcriptional activity and expression of p53. MAP kinases signaling pathway plays a direct role in LMP1-induced phosphorylation of p53 at multiple sites, which contributes to the promotion of p53 transcriptional activity, transactivition activity. In other NPC models, we also confirmed that LMP1 upregulated expression of p53 and its transactivition activity, downregulated the catalytic activity of cdc2/cyclinB1, which led to cells blocked in G2/M phase, without apoptosis.On the basis of our work and related researches, we assumed that LMP1 may regulate the expression of survivin by p53. Firstly, we used NPC cell lines containing mutation of p53 in exon 8 (CNE1, CNE1-LMP1) and immortalized NP69 cell model containing wild type p53 (NP69, NP69-pLNSX, NP69-LMP1) as our research models. We found that LMP1 could upregulate survivin, survivinThr34 phosphorylation, p53 and p53Ser20 phosphorylation by Western blot in both cell models. However, after being transfected with p53siRNA, survivin protein levels were decreased. These data confirmed our assumptions that LMP1 could regulated survivin by p53, which might have no relation with mutation of p53 in exon 8.Furthermore, we focused on the mechanisms that LMP1 regulated survivin by p53. To examine the transactivation activity of survivin promoter by p53 and p53-survivinDNA binding activity mediated by LMP1, we used reporter gene technology and EMSA. We found that LMP1 could upregulate survivin promoter activity obviously, however, after p53 expression was blocked by siRNA, survivin promoter activity decreased; we also found LMP1 could promote p53-survivinDNA binding activity. These data suggested that LMP1 could upregulate survivin by p53 transcriptionally.Also, we used immunofluorescence , cytoplasm and nucleus Western blot, Co-IP to test the celluar location of survivin and p53, the interaction of survivin and p53 proteins. We found that LMP1 could promote survivin and p53 translocation into nulei; survivin and p53 proteins could interact, but with no differences in LMP1 negtive and positive cells. These suggested that LMP1 upregulated survivin by p53 mainly transcriptionally.Finally, we compared the apoptosis rate of cells untreated with transfected p53 siRNA by flow cytometric. We found that apoptosis rate increased after p53 expression blocked by siRNA, which was the result of decreased survivin.In this study, we identified that LMP1 could upregulate survivin by transcriptional factor p53. LMP1 could upregulate survivin promoter activity by p53; LMP1 could promote p53-survivinDNA binding activity, which contributed to increased survivin expression mediated by LMP1. These provided us with a novel view of comprehensive understanding in p53 function activated by LMP1 and the oncogenic function of LMP1 in the carincogenesis of NPC, which will lead to the identification of novel targets for drug discovery and provide a basis for gene therapy.