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The Epstein-Barr virus latent membrane protein 1 participates in mediating the Janus kinase 3 signaling passageway in the nasopharyngeal carcinoma cell
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Abstracts / Cell Biology International 32 (2008) S1eS67
phosphorylated proteins between NP69-PLNSX and NP69-LMP1WT cell lines and 11 differentially tyrosine-phosphorylated proteins between NP69LMP1WT and NP69-LMP1TRADD cell lines. Some of which had previously been implicated in LMP1 signal pathway. The other proteins, including Heat shock protein 70, Cytoskeletal 7, Phosphotidylethanolamin binding protein 1, Tubulin, et al were novel signaling molecules and targets with no previously known function in LMP1 signal transduction. These data will be helpful to elucidate the molecular mechanism of LMP1 in EBV-associated nasopharyngeal carcinogenesis and guide the discovery of new drug targets and the rational utilization of pathway-specific chemotherapies. This study was supported by grants from National Science Foundation of China (30470668).
THE EPSTEIN-BARR VIRUS LATENT MEMBRANE PROTEIN 1 PARTICIPATES IN MEDIATING THE JANUS KINASE 3 SIGNALING PASSAGEWAY IN THE NASOPHARYNGEAL CARCINOMA CELL Zhi Wei Zhang, Qiong Zhang, Jie Qiong Liu, Yan Hui Yu, Zhi Min He Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, china The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a critical oncogenic protein in EBV-related tumorigenesis and usurps cellular signaling pathways resulting in the induction of NFkB and AP1 via two C-terminal activating regions (CTAR1 and CTAR2), To make sure whether there exists a third activating region (CTAR3) in its C-terminal and whether there is a relation between CTAR3 and JAK3/STAT1 pathway, a plasmid encoding a mutant LMP1 defective in binding sequence (aa 232e351) for Janus kinase3 (JAK3) protein and a reporter plasmid containing JAK3 promoter and luciferase gene fused sequence were constructed. This LMP1D232-351 gene was almost completely defective in JAK3 activation in comparison with wild type LMP1 (wt LMP1) and was almost consistent with wtLMP1 in NFkB and AP1 activation. When wtLMP1 and LMP1D232-351 were introduced into nasopharyngeal cancer cell line CNE-2 through retrovirus, respectively, the CNE2-wt LMP1 cells showed an increased expression of non-phoshated and phosphated JAK3 than CNE-2 control cells; while CNE-2- LMP1D232-351 cells showed a lower expression of two kinds of JAK3 than CNE-2-wtLMP1 cells. When CNE-2-wt-LMP1 cells were treated with WHIP-131, an inhibitor of JAK3, no change in non-phosphated JAK3 expression and decreasing expression in phosphated JAK3 was observed. Further assays showed wt-LMP1 had a STAT1-binding and anti-apoptosis activity, while LMP1D232-351 was missing the activity in CNE-2 cells. These results substantiate that LMP1 could activate the JAK3/STAT1 signaling pathway and inhibit cellular apoptosis of CNE-2 cells depending on CTAR3 and the action may be one of the mechanisms of EBV-LMP1 participating in nasophayngeal carcinogenesis. This study was supported by grants from National Science Foundation of China (30470668).
THE EFFECT OF SURVIVIN ON MULTIDRUG RESISTANCE BY EPIDERMAL GROWTH FACTOR RECEPTOR 2 VIA NF-kB ACTIVATING IN BREAST CANCER CELLS Ren Kui Ding, Wei Ding, Cheng Kun Wang, Hui Lu, Zhi Min He Cancer Research Institute, Xiangya school of medicine, Central South University, Changsha 410078, Hunan, P.R.China Survivin is a structurally unique inhibitor of apoptosis (IAP) and might potentially play a key role in resistance to anti-cancer drugs. However, there is no information on the role of survivin in multidrug resistance (MDR) in the presence of Her-2 in cancer cells. HER2 is a member of the EGFR family, which is associated with poor chemotherapeutic response. In this study we set up HER2-overexpressing breast cancer cell lines (MCF7/HER2) and investigated the effects and mechanism of survivin on MDR by Her-2. The results showed that MCF7/HER2 cells had an increased expression of survivin mRNA and protein and the resistance index (IR) of this cells to Taxel, MMC, VP16, 5Fu and Mit increased 21, 5.45, 3.12, 2.32, 2.87 folds than control cells, respectively. When HER2 was silenced the survivin expression decreased and the IR of aforementioned drugs decreased 14.3, 4.8, 5.64, 2.62 and 3.43 folds respectively. When survivin was silenced IR of MCF7/HER2 cells to the drugs
decreased 6.48; 3.12; 2.18; 1.96 and 2.54 folds, respectively. HER2 could activates survivin by 2e6 fold with the increasing concentration of HER2 in cotransfection studies with survivin luciferase reporter, while tyrosine kinase inhibitor AG825 could inhibit the activity by 1.5 - 8 fold. HER2 also could activate NF-kB in cotransfection studies with NF-kB luciferase reporter. When IkBa were cotransfected with HER2, NF-kB activation was significantly inhibited. When treated with dexamethasome, a potent inhibitor of NF-kB, the expression of suvivin mRNA and protein in MEF7/HER2 cells were decreased. These results suggest that survivin took part in HER2- and NF-kB emediated MDR of breast cancer cells and might be a potent therapeutic target in reversing MDR of tumor cells. This study was supported by grants from Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700) and the Medicinal and Pharmic Science Foundation of Hunan Province, China (Z02-1).
ESTABLISHMENT OF A PYM-RESISTANT HUMAN TONGUE CARCINOMA CELL LINE TCA8113/PYM AND CLONING OF RESISTANCE-RELATED GENES Min Zhou, Xiao Rong Liu, Bo Peng, Shan Sha Fan, Yan Hui Yu, Yong Mei Ouyang, Zhi Min He Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, 410078, Hunan, P. R. China Pingyangmycin(PYM) is an anti-cancer antibiotic frequently used in chemotherapy of tongue carcinoma before or after surgical. Innate and acquired resistance of tongue carcinoma to PYM interfere seriously the effects of PYM in chemotherapy. To probe into the resistant mechanism, we first set up a PYMresistant human tongue carcinoma cell line Tca8113/PYM which showed an 55-fold higher resistance to PYM than parental cell (Tca8113) by incubation of the cells with increasing concentration of PYM. Tca8113/PYM cells did not present either significant differences in growth rate or cell cycle distribution, but were cross-resistant to a subset of clinically relevant anti-cancer agents, cDDP, VCR, THP, TAXEL and MMC, but not to ADM, VP-16 and 5-Fu in comparison with parental cells. Secondly, differential expressions of genes between Tca8113/PYM and parental cells were examined by cDNA microarray. To our surprise, no changes in MDR1, MRP or LRP mRNA expression were found, but a number of genes associated with a variety of cellular functions and EST showed differential expression. We cloned and identified two novel human sequences from an EST by bioinformatics binding RT-PCR methods and nominated as TCRP1 variant1 and variant2 (Genbank No. are EF197985 and EF362480). Further bioinformatics analysis indicates both splice variants encoding a predicted 129-amino-acid and a putative 235amino-acid might be membrane proteins. Enforce expression of two fulllength cDNA in parental Tca 8113 cells confers resistance of two cell lines to PYM and cDDP. The newly established Tca8113/PYM cell line and the accumulation of its gene expression data will provide potentially useful tools in gaining insights into the mode of action of PYM and elucidating the mechanisms of acquired resistance, as well as in investigating and developing methods to prevent and overcome resistance to this drug. This study was supported by grants from Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700).
DOdFMG, A GENISTEIN DERIVATIVE, AS EFFECTIVE ANTI LUNG ADENOCARCINOMA CELL AGENTS IN VITRO AND IN VIVO Bo Peng 1,2, Cao Jian Guo 2, Cheng Kun Wang 1,2, Sha Sha Fan 1, Zhi Min He 1 1 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, 410078, China 2 Cancer Research Institute, Nanhua University, Hengyang, 421001, China It has aroused a general interest in the utilization of natural products, or their synthetic analogs to find new anti-cancer agents. Genistein, which is abundant in soybeans, has attracted more and more interest because of its ability to inhibit a number of kinds of tumor cell growth in vitro. However, genistein has not been considered for development as a chemotherapeutic drug because of its poor bio-availability and high rate of metabolism in vivo. To improve the
Abstracts / Cell Biology International 32 (2008) S1eS67
phosphorylated proteins between NP69-PLNSX and NP69-LMP1WT cell lines and 11 differentially tyrosine-phosphorylated proteins between NP69LMP1WT and NP69-LMP1TRADD cell lines. Some of which had previously been implicated in LMP1 signal pathway. The other proteins, including Heat shock protein 70, Cytoskeletal 7, Phosphotidylethanolamin binding protein 1, Tubulin, et al were novel signaling molecules and targets with no previously known function in LMP1 signal transduction. These data will be helpful to elucidate the molecular mechanism of LMP1 in EBV-associated nasopharyngeal carcinogenesis and guide the discovery of new drug targets and the rational utilization of pathway-specific chemotherapies. This study was supported by grants from National Science Foundation of China (30470668).
THE EPSTEIN-BARR VIRUS LATENT MEMBRANE PROTEIN 1 PARTICIPATES IN MEDIATING THE JANUS KINASE 3 SIGNALING PASSAGEWAY IN THE NASOPHARYNGEAL CARCINOMA CELL Zhi Wei Zhang, Qiong Zhang, Jie Qiong Liu, Yan Hui Yu, Zhi Min He Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, china The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a critical oncogenic protein in EBV-related tumorigenesis and usurps cellular signaling pathways resulting in the induction of NFkB and AP1 via two C-terminal activating regions (CTAR1 and CTAR2), To make sure whether there exists a third activating region (CTAR3) in its C-terminal and whether there is a relation between CTAR3 and JAK3/STAT1 pathway, a plasmid encoding a mutant LMP1 defective in binding sequence (aa 232e351) for Janus kinase3 (JAK3) protein and a reporter plasmid containing JAK3 promoter and luciferase gene fused sequence were constructed. This LMP1D232-351 gene was almost completely defective in JAK3 activation in comparison with wild type LMP1 (wt LMP1) and was almost consistent with wtLMP1 in NFkB and AP1 activation. When wtLMP1 and LMP1D232-351 were introduced into nasopharyngeal cancer cell line CNE-2 through retrovirus, respectively, the CNE2-wt LMP1 cells showed an increased expression of non-phoshated and phosphated JAK3 than CNE-2 control cells; while CNE-2- LMP1D232-351 cells showed a lower expression of two kinds of JAK3 than CNE-2-wtLMP1 cells. When CNE-2-wt-LMP1 cells were treated with WHIP-131, an inhibitor of JAK3, no change in non-phosphated JAK3 expression and decreasing expression in phosphated JAK3 was observed. Further assays showed wt-LMP1 had a STAT1-binding and anti-apoptosis activity, while LMP1D232-351 was missing the activity in CNE-2 cells. These results substantiate that LMP1 could activate the JAK3/STAT1 signaling pathway and inhibit cellular apoptosis of CNE-2 cells depending on CTAR3 and the action may be one of the mechanisms of EBV-LMP1 participating in nasophayngeal carcinogenesis. This study was supported by grants from National Science Foundation of China (30470668).
THE EFFECT OF SURVIVIN ON MULTIDRUG RESISTANCE BY EPIDERMAL GROWTH FACTOR RECEPTOR 2 VIA NF-kB ACTIVATING IN BREAST CANCER CELLS Ren Kui Ding, Wei Ding, Cheng Kun Wang, Hui Lu, Zhi Min He Cancer Research Institute, Xiangya school of medicine, Central South University, Changsha 410078, Hunan, P.R.China Survivin is a structurally unique inhibitor of apoptosis (IAP) and might potentially play a key role in resistance to anti-cancer drugs. However, there is no information on the role of survivin in multidrug resistance (MDR) in the presence of Her-2 in cancer cells. HER2 is a member of the EGFR family, which is associated with poor chemotherapeutic response. In this study we set up HER2-overexpressing breast cancer cell lines (MCF7/HER2) and investigated the effects and mechanism of survivin on MDR by Her-2. The results showed that MCF7/HER2 cells had an increased expression of survivin mRNA and protein and the resistance index (IR) of this cells to Taxel, MMC, VP16, 5Fu and Mit increased 21, 5.45, 3.12, 2.32, 2.87 folds than control cells, respectively. When HER2 was silenced the survivin expression decreased and the IR of aforementioned drugs decreased 14.3, 4.8, 5.64, 2.62 and 3.43 folds respectively. When survivin was silenced IR of MCF7/HER2 cells to the drugs
decreased 6.48; 3.12; 2.18; 1.96 and 2.54 folds, respectively. HER2 could activates survivin by 2e6 fold with the increasing concentration of HER2 in cotransfection studies with survivin luciferase reporter, while tyrosine kinase inhibitor AG825 could inhibit the activity by 1.5 - 8 fold. HER2 also could activate NF-kB in cotransfection studies with NF-kB luciferase reporter. When IkBa were cotransfected with HER2, NF-kB activation was significantly inhibited. When treated with dexamethasome, a potent inhibitor of NF-kB, the expression of suvivin mRNA and protein in MEF7/HER2 cells were decreased. These results suggest that survivin took part in HER2- and NF-kB emediated MDR of breast cancer cells and might be a potent therapeutic target in reversing MDR of tumor cells. This study was supported by grants from Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700) and the Medicinal and Pharmic Science Foundation of Hunan Province, China (Z02-1).
ESTABLISHMENT OF A PYM-RESISTANT HUMAN TONGUE CARCINOMA CELL LINE TCA8113/PYM AND CLONING OF RESISTANCE-RELATED GENES Min Zhou, Xiao Rong Liu, Bo Peng, Shan Sha Fan, Yan Hui Yu, Yong Mei Ouyang, Zhi Min He Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, 410078, Hunan, P. R. China Pingyangmycin(PYM) is an anti-cancer antibiotic frequently used in chemotherapy of tongue carcinoma before or after surgical. Innate and acquired resistance of tongue carcinoma to PYM interfere seriously the effects of PYM in chemotherapy. To probe into the resistant mechanism, we first set up a PYMresistant human tongue carcinoma cell line Tca8113/PYM which showed an 55-fold higher resistance to PYM than parental cell (Tca8113) by incubation of the cells with increasing concentration of PYM. Tca8113/PYM cells did not present either significant differences in growth rate or cell cycle distribution, but were cross-resistant to a subset of clinically relevant anti-cancer agents, cDDP, VCR, THP, TAXEL and MMC, but not to ADM, VP-16 and 5-Fu in comparison with parental cells. Secondly, differential expressions of genes between Tca8113/PYM and parental cells were examined by cDNA microarray. To our surprise, no changes in MDR1, MRP or LRP mRNA expression were found, but a number of genes associated with a variety of cellular functions and EST showed differential expression. We cloned and identified two novel human sequences from an EST by bioinformatics binding RT-PCR methods and nominated as TCRP1 variant1 and variant2 (Genbank No. are EF197985 and EF362480). Further bioinformatics analysis indicates both splice variants encoding a predicted 129-amino-acid and a putative 235amino-acid might be membrane proteins. Enforce expression of two fulllength cDNA in parental Tca 8113 cells confers resistance of two cell lines to PYM and cDDP. The newly established Tca8113/PYM cell line and the accumulation of its gene expression data will provide potentially useful tools in gaining insights into the mode of action of PYM and elucidating the mechanisms of acquired resistance, as well as in investigating and developing methods to prevent and overcome resistance to this drug. This study was supported by grants from Basic Research Special Program of the Ministry of Science and Technology of China (2003CCC00700).
DOdFMG, A GENISTEIN DERIVATIVE, AS EFFECTIVE ANTI LUNG ADENOCARCINOMA CELL AGENTS IN VITRO AND IN VIVO Bo Peng 1,2, Cao Jian Guo 2, Cheng Kun Wang 1,2, Sha Sha Fan 1, Zhi Min He 1 1 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, 410078, China 2 Cancer Research Institute, Nanhua University, Hengyang, 421001, China It has aroused a general interest in the utilization of natural products, or their synthetic analogs to find new anti-cancer agents. Genistein, which is abundant in soybeans, has attracted more and more interest because of its ability to inhibit a number of kinds of tumor cell growth in vitro. However, genistein has not been considered for development as a chemotherapeutic drug because of its poor bio-availability and high rate of metabolism in vivo. To improve the