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EBV-miR-BHRF1-1 contributes to Epstein-Barr virus lytic cycle induction via inhibits p53
Abstracts / Cell Biology International 32 (2008) S1eS67 pre-treatment of TGFb1 could induce the drug-resistance of SGC-7901 to mitomycin. Meanwhile, we found no difference of apoptosis rate in TGFb1treated SGC-7901 with its counterpart, which excluded the influence of apoptosis induced by TGFb1. Next, we tested the expression of MDR1, GST-p, two major MDR-related molecules, and found that they were up-regulated after treatment of TGFb1, indicating that TGFb1 induced drug-resistance might be correlated with MDR1, GST-p. At last, we used Smad4 siRNA and special inhibitor of MAPK signal pathway to explore the role of these signal pathways in this biological effect to investigate the mechanisms of TGFb1-induced GST-p and MDR1. As a result, we found that expression of GST-p induced by TGFb1 could be inhibited by Smad4 siRNA and PD98059 but not SB203580 and SP600125, indicating that TGFb1 could induce expression of GST-p through Smad and ERK signal pathways. As to MDR1, we found that it could be induced through JNK and ERK signal pathway by TGFb1. Based on our results, we conclude that TGFb1 might induce the drug resistance in SGC-7901, which might correlate with GST-p and MDR1; TGFb1 could induce the expression of GST-p through Smad4 and ERK signal pathway and the expression of MDR1 through JNK and ERK signal pathway respectively.
THE UBIQUITINATION OF P53 REGULATED BY EPSTEINBARR VIRUS ENCODED LATENT MEMBRANE PROTEIN 1 Li Li Li 1, Li Li Guo 1, Zi Jian Li 1, Xue Chen 1, Duo Ssha Hu 1, Xiang Jian Luo 1, Xiao Qian Ma 1, Ying Shi 1, Sai Wah Tsao 2, Ya Cao 1 1 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, P.R.China 2 Department of Anatomy, Faculty of Medicine, University of Hong Kong, Hong Kong, P.R.China EBV encoded latent memebrane protein 1 (LMP1), an oncogenic protein, plays an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). p53 protein accumulates but mutation of p53 gene is not common in NPC. The molecular mechanisms leading to the stabilization of p53 have not been completely elucidated. LMP1 regulated p53 phosphorylation on multiple sites via MAPK signaling pathway was determined previously, which is resulting in the increasing of p53 transcriptional activity, transactivation and stability. Ubiquitination is an emerging mechanism implicated in a variety of nonproteolytic cellular functions, which closely associated with protein phosphorylation. Here, we demonstrated that LMP1 induced Ub molecule Ubk63 binded with p53 via MAPK signaling pathway, which was associated with the p53 stability and activation. Among them, the regulation of p53 Ser20 via JNK pathway was more important than others. The phosphorylation of p53 could inhibit the combination of p53 to Ub molecule Ubk48, involving in the p53 stability. These results suggest that Ub molecules Ubk48 and Ubk63 could bind p53 to play different functions, which provides a novel view for us to understand the mechanism of p53 accumulation in the carcinogenesis of NPC.
GENE THERAPY OF TUMOR METASTASIS BY NON-VIRAL VECTOR Zheng Li, Juan Juan Xiang, Zhi Guo He, Xiao Ling Li, Xia Yu Li, Gui Yuan Li* Cancer Research Institute, Central South University, Changsha, Hunan 410078, China Metastatic disease represents one of the most difficult challenges in cancer therapy. Gene therapy provides modern medicine with new perspectives that were unthinkable before. Many non-viral vectors have been developed for therapeutic gene transfer for basic and clinical research. We previously reported a self-assembled non-viral gene vector, poly-L-lysine modified iron oxide nanoparticles (IONP-PLL), which is formed by modifying polyL-lysine to the surface of iron oxide nanoparticles and could transfer exogenous DNA to cells in vitro and in vivo. In this study, experimental pulmonary metastasis model was formed by injecting B16 cells into C57BL/6 mouse via tail lateral vein and nm23-H1-GFP gene encoding nm23-H1 protein and green fluorescent protein was used as therapeutic gene. The inhibition efficiency of metastasis was evaluated by intravenous injection of IONP-PLL/
S31
nm23-H1-GFP complexes into mouse and the number of metastases in the lung was calculated. At the same time, the common therapeutic efficiency of gene therapy and chemotherapy was evaluated by treating mice with IONP-PLL/nm23-H1-GFP complexes and CTX. After intravenous injection, IONP-PLL transferred therapeutic gene nm23-H1-GFP to lung and significantly reduced the number of metastases. Furthermore, when used with CTX, the fewer number of metastases was observed. These results suggest that IONP-PLL, an efficient gene vector, appeared to have potential for metastatic disease in cancer therapy and might be a helpful additional way to current chemotherapy.
EBV-miR-BHRF1-1 CONTRIBUTES TO EPSTEIN-BARR VIRUS LYTIC CYCLE INDUCTION VIA INHIBITS P53 Zi Jian Li, Li Li Li, Li Li Guo, Su Fang Liu, Zhong Xin Lu, Xue Chi Lin, Ya Cao* Cancer Research Institute, Xiangya School of Medicine, Central South University Epstein-Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), which are arranged in two clusters: 29 miRNAs are located in the introns of the viral BART gene while 3 are located adjacent to BHRF1. Here we demonstrated that ebv-miR-BHRF1-1, one of these miRNAs, was upregulated after inducing EBV from latent into lytic cycle by treatment of TPA (12-O-tetradecanoylphorbol-13-acetate) in NPC cell lines. On the contrary, p53 protein level and mdm2 protein level, which is one of p53’s downstream genes, were down-regulated after induction of the lytic cycle of EBV. And more interestingly, the percent of M-phase cells increased while apoptosis rate decreased accompanying with the down-regulation of p53 protein. Furthermore, bio-informatics discovery revealed that ebv-miR-BHRF1-1 could interact with the 3’-untranslated region of the p53 mRNA at two sites. These findings indicate that ebv-miR-BHRF1-1 contributed to EBV lytic cycle induction via inhibition of p53 in NPC cell.
LMP1 REGULATES Op18/STATHMIN SIGNALLING PATHWAY BY CDC2 MEDIATION IN NASOPHARYNGEAL CARCINOMA CELLS Xue Chi Lin, Su Fang Liu, Li Li Guo, Zi Iian Li, Xiao Qian Ma, Li Li Li, Xi Yun Deng, Ya Cao* Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China Op18/stathmin plays a crucial role in maintaining cell biological characters by regulating microtubule dynamics, especially entering into mitosis. The phosphorylated Op18/stathmin promotes microtubule polymerization to form the mitotic spindle which is essential for chromosome segregation and cell division. Cdc2 is a critical kinase to start M phase event in a cell cycle progression and a type of plus regulation factor of cell cycle. Latent membrane protein 1 (LMP1) is an EBV encoded oncogenic protein that is able to induce tumorigenesis by various mechanisms. Our studies focused on the regulation of Oncoprotein 18/stathmin (Op18/stathmin) signaling by LMP1 in nasopharyngeal carcinoma (NPC) cells, and showed that LMP1 could regulate Op18/stathmin signaling by cdc2 mediation. LMP1 up-regulated cdc2 kinase activity and Op18/sthamin phosphorylation at G2/M phase, promoted the interaction of cdc2 with Op18/stathmin and microtubule polymerization; Inhibition LMP1 expression attenuated the interaction of cdc2 and Op18/stathmin and promoted microtubule depolymerization; During mitosis, inhibition cdc2 kinase resulted in spindle disaggregation. All these results revealed a new pathway that LMP1 regulates Op18/stathmin signaling by cdc2 mediation. The new signaling pathway on LMP1 not only perfects the LMP1 regulation network, but also provides new insights for elucidating the molecular mechanism of LMP1 leading to tumorigenesis.
THE UBIQUITINATION OF P53 REGULATED BY EPSTEINBARR VIRUS ENCODED LATENT MEMBRANE PROTEIN 1 Li Li Li 1, Li Li Guo 1, Zi Jian Li 1, Xue Chen 1, Duo Ssha Hu 1, Xiang Jian Luo 1, Xiao Qian Ma 1, Ying Shi 1, Sai Wah Tsao 2, Ya Cao 1 1 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, P.R.China 2 Department of Anatomy, Faculty of Medicine, University of Hong Kong, Hong Kong, P.R.China EBV encoded latent memebrane protein 1 (LMP1), an oncogenic protein, plays an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). p53 protein accumulates but mutation of p53 gene is not common in NPC. The molecular mechanisms leading to the stabilization of p53 have not been completely elucidated. LMP1 regulated p53 phosphorylation on multiple sites via MAPK signaling pathway was determined previously, which is resulting in the increasing of p53 transcriptional activity, transactivation and stability. Ubiquitination is an emerging mechanism implicated in a variety of nonproteolytic cellular functions, which closely associated with protein phosphorylation. Here, we demonstrated that LMP1 induced Ub molecule Ubk63 binded with p53 via MAPK signaling pathway, which was associated with the p53 stability and activation. Among them, the regulation of p53 Ser20 via JNK pathway was more important than others. The phosphorylation of p53 could inhibit the combination of p53 to Ub molecule Ubk48, involving in the p53 stability. These results suggest that Ub molecules Ubk48 and Ubk63 could bind p53 to play different functions, which provides a novel view for us to understand the mechanism of p53 accumulation in the carcinogenesis of NPC.
GENE THERAPY OF TUMOR METASTASIS BY NON-VIRAL VECTOR Zheng Li, Juan Juan Xiang, Zhi Guo He, Xiao Ling Li, Xia Yu Li, Gui Yuan Li* Cancer Research Institute, Central South University, Changsha, Hunan 410078, China Metastatic disease represents one of the most difficult challenges in cancer therapy. Gene therapy provides modern medicine with new perspectives that were unthinkable before. Many non-viral vectors have been developed for therapeutic gene transfer for basic and clinical research. We previously reported a self-assembled non-viral gene vector, poly-L-lysine modified iron oxide nanoparticles (IONP-PLL), which is formed by modifying polyL-lysine to the surface of iron oxide nanoparticles and could transfer exogenous DNA to cells in vitro and in vivo. In this study, experimental pulmonary metastasis model was formed by injecting B16 cells into C57BL/6 mouse via tail lateral vein and nm23-H1-GFP gene encoding nm23-H1 protein and green fluorescent protein was used as therapeutic gene. The inhibition efficiency of metastasis was evaluated by intravenous injection of IONP-PLL/
S31
nm23-H1-GFP complexes into mouse and the number of metastases in the lung was calculated. At the same time, the common therapeutic efficiency of gene therapy and chemotherapy was evaluated by treating mice with IONP-PLL/nm23-H1-GFP complexes and CTX. After intravenous injection, IONP-PLL transferred therapeutic gene nm23-H1-GFP to lung and significantly reduced the number of metastases. Furthermore, when used with CTX, the fewer number of metastases was observed. These results suggest that IONP-PLL, an efficient gene vector, appeared to have potential for metastatic disease in cancer therapy and might be a helpful additional way to current chemotherapy.
EBV-miR-BHRF1-1 CONTRIBUTES TO EPSTEIN-BARR VIRUS LYTIC CYCLE INDUCTION VIA INHIBITS P53 Zi Jian Li, Li Li Li, Li Li Guo, Su Fang Liu, Zhong Xin Lu, Xue Chi Lin, Ya Cao* Cancer Research Institute, Xiangya School of Medicine, Central South University Epstein-Barr virus (EBV) was the first human virus found to encode microRNAs (miRNAs), which are arranged in two clusters: 29 miRNAs are located in the introns of the viral BART gene while 3 are located adjacent to BHRF1. Here we demonstrated that ebv-miR-BHRF1-1, one of these miRNAs, was upregulated after inducing EBV from latent into lytic cycle by treatment of TPA (12-O-tetradecanoylphorbol-13-acetate) in NPC cell lines. On the contrary, p53 protein level and mdm2 protein level, which is one of p53’s downstream genes, were down-regulated after induction of the lytic cycle of EBV. And more interestingly, the percent of M-phase cells increased while apoptosis rate decreased accompanying with the down-regulation of p53 protein. Furthermore, bio-informatics discovery revealed that ebv-miR-BHRF1-1 could interact with the 3’-untranslated region of the p53 mRNA at two sites. These findings indicate that ebv-miR-BHRF1-1 contributed to EBV lytic cycle induction via inhibition of p53 in NPC cell.
LMP1 REGULATES Op18/STATHMIN SIGNALLING PATHWAY BY CDC2 MEDIATION IN NASOPHARYNGEAL CARCINOMA CELLS Xue Chi Lin, Su Fang Liu, Li Li Guo, Zi Iian Li, Xiao Qian Ma, Li Li Li, Xi Yun Deng, Ya Cao* Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, PR China Op18/stathmin plays a crucial role in maintaining cell biological characters by regulating microtubule dynamics, especially entering into mitosis. The phosphorylated Op18/stathmin promotes microtubule polymerization to form the mitotic spindle which is essential for chromosome segregation and cell division. Cdc2 is a critical kinase to start M phase event in a cell cycle progression and a type of plus regulation factor of cell cycle. Latent membrane protein 1 (LMP1) is an EBV encoded oncogenic protein that is able to induce tumorigenesis by various mechanisms. Our studies focused on the regulation of Oncoprotein 18/stathmin (Op18/stathmin) signaling by LMP1 in nasopharyngeal carcinoma (NPC) cells, and showed that LMP1 could regulate Op18/stathmin signaling by cdc2 mediation. LMP1 up-regulated cdc2 kinase activity and Op18/sthamin phosphorylation at G2/M phase, promoted the interaction of cdc2 with Op18/stathmin and microtubule polymerization; Inhibition LMP1 expression attenuated the interaction of cdc2 and Op18/stathmin and promoted microtubule depolymerization; During mitosis, inhibition cdc2 kinase resulted in spindle disaggregation. All these results revealed a new pathway that LMP1 regulates Op18/stathmin signaling by cdc2 mediation. The new signaling pathway on LMP1 not only perfects the LMP1 regulation network, but also provides new insights for elucidating the molecular mechanism of LMP1 leading to tumorigenesis.