1.Interaction of Tumor Necrosis Factor Receptor-associated Factors with the Latent Membrane Protein 1 Is Essential for Activation of NF-κB
Chengxing WANG ; Xiaoyan LI ; Huanhua GU ; Xiyun DENG ; Ya CAO
Progress in Biochemistry and Biophysics 2001;28(2):240-245
The Epstein-Barr virus latent membrane protein 1 (LMP1) oncopro tein causes multiple cellular changes, including activation of the NF-κB trans cription factor. To elucidate its possible mechanism, the interaction between LM P1 and the tumor necrosis factor receptor associated factor (TRAF) molecules was detected by the immunoprecipitation-Western blotting assay. Results showed tha t LMP1 was co-precipitated with TRAF1,2,3 in the LMP1-HNE2 cell line. In the m eantime, κB reporter gene analysis revealed that over expression of TRAF1 or TR AF2 augmented LMP1-mediated NF-κB activation from LMP1, suprisingly, overexpr ession of either TRAF3 or an dominant negative TRAF3 inhibited the NF-κB activ ation, indicating that TRAF1 or TRAF2 is a positive modulator of LMP1-mediated NF-κB activation, whereas,TRAF3 is a negative modulator. Rather both CTAR1 (carboxy-terminal activating region 1) and CTAR2 domains of LMP1 can independently activate NF-κB by interacting with TRAF proteins. These data indicate that LMP1 interacts TRAF1,2,3 which are important for LMP1-mediated N F-κB activation, and further suggest that signaling from TRAFs may be involved in the progression to malignancy in cells of epithelial origin such as nasophar yngeal carcinoma (NPC).
2.Inhibitory effect of JIP on AP-1 activity induced by LMP1 in nasopharyngeal carcinoma cells and its mechanism
Zhi HU ; Feijun LUO ; Xiyun DENG ; Liqun YIN ; Yan ZHAO ; Faqing TANG ; Min TANG ; Huanhua GU ; Wei YI ; Ya CAO
Chinese Journal of Pathophysiology 2000;0(10):-
AIM: To investigate the mechanism of the AP-1 signal transduction pathway inhibited by JIP in nasopharyngeal carcinoma cells. METHODS: AP-1 activity was triggered by Dox-induced LMP1 expression in Tet-on-LMP1-HNE 2 cells (L7). The retention of phospho-JNK in the cytoplasm caused by JIP was examined with immunofluroscence assay. RESULTS: 24 h after transfection of L7 cells with the JIP expression plasmid, the translocation of activated JNK was inhibited, which resulted in the retention of phospho-JNK in the cytoplasm and down-regulation of the AP-1 activity. CONCLUSION: JIP down-regulates the activity of AP-1 through the inhibition of the translocation of JNK.
3.Point mutation of 2.8 kb EcoRI fragment of the NPC transforming genein NPC cell lines
Xiyun DENG ; Ya CAO ; Li CAO ; Lee M LEO ; Kaitai YAO
Chinese Medical Journal 1998;111(3):0-0
Objective To determine whether the previously isolated transforming sequence, designated the Tx gene, which was derived from a Chinese human nasopharyngeal carcinoma (NPC) cell line CNE-2, is mutated in NPC cell lines and biopsy tissues.Methods DNA samples prepared from various cell lines and 11 cases of NPC biopsies were subjected to analysis with the modified polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) silver staining technique to screen for possible point mutation(s).Results Mobility shifted bands were revealed in two NPC cell lines, CNE-2 and HONE-1, but not in non-NPC cell lines, including stomach cancer cell line HGC-1, colon cancer cell line SW-480 and cervical cancer cell line HeLa. No sequence changes or polymorphisms were found in 11 cases of primary NPC biopsies.Conclusions In most cases of NPC, activation of this transformation-associated sequence does not involve a mechanism of gene mutation. The role played by this gene in NPC is to be further determined.
4.LMP1 activates NF-κB via degradation of IκBα in nasopharyngeal carcinoma cells
Liqun YIN ; Wei LIAO ; Xiyun DENG ; Min TANG ; Huanhua GU ; Xiaoyan LI ; Wei YI ; Ya CAO
Chinese Medical Journal 2001;114(7):718-722
Abstract:Objective To elucidate the mechanisms by which Epstein-Barr virus-encoded latent membrane protein 1 activates NF-κB in nasopharyngeal carcinoma cells.Methods A tetracycline-regulated LMP1-expressing nasopharyngeal carcinoma cell line, Tet-on-LMP1-HNE2, was used as the cell model. The kinetics of the expression of proteins, including LMP1, IκBα and IκBβ, was analyzed by Western blotting. The subcellular localization of NF-κB (p65) was detected by indirect immunofluorescence assay. The NF-κB transactivity was studied by transient transfection and reporter gene assay. Results IκBα was phosphorylated and degraded after the inducible expression of LMP1, although the total protein levels remained stable. The steady-state level of total IκBβ protein may have resulted from the initiation of an autoregulation loop after the activation of NF-κB. No change in the IκBβ level was detected. NF-κB (p65) was translocated from the cytoplasm to the nucleus following degradation of IκBα. After the introduction of the dominant-negative mutant of IκBα (Del 71) into Tet-on-LMP1-HNE2 cells, both nuclear translocation and transactivation of NF-κB induced by LMP1 was significantly inhibited. Conclusions The results indicated that in nasopharyngeal carcinoma cells, LMP1 activated NF-κB via phosphorylation and degradation of IκBα, but not IκBβ. The dominant-negative mutant of IκBα (Del 71) could completely inhibit both the nuclear translocation and transactivation of NF-κB induced by LMP1.
5.Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence.
Jing YANG ; Faqing TANG ; Huanhua GU ; Xiyun DENG ; Xinxian WENG ; Min TANG ; Ya CAO
Chinese Medical Journal 2002;115(6):803-809
OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.
METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.
RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.
CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.
Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology
6.Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus LMP1 via NF-kappa B or AP-1 signaling pathway in nasopharyngeal carcinoma cells.
Chengxing WANG ; Xiyun DENG ; Xiaoyan LI ; Huanhua GU ; Wei YI ; Xinxian WENG ; Linqing XIA ; Ya CAO
Chinese Journal of Oncology 2002;24(1):9-13
OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.
METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.
RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.
CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.
Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology
7.Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-kappaB signaling pathway in nasopharyngeal carcinoma.
Chengxing WANG ; Midan AI ; Wei REN ; Hui XIAO ; Xiaoyan LI ; Faqing TANG ; Huanhua GU ; Wei YI ; Xinxian WENG ; Xiyun DENG ; Ya CAO
Chinese Medical Journal 2003;116(7):1022-1028
OBJECTIVESTo identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).
METHODSA stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.
RESULTSLMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.
CONCLUSIONLMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.
Apoptosis ; physiology ; Humans ; NF-kappa B ; physiology ; Nasopharyngeal Neoplasms ; physiopathology ; Protein Biosynthesis ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 1 ; Tumor Cells, Cultured ; Viral Matrix Proteins ; physiology
8.EB virus encoded latent membrane protein 1 modulates the phosphorylation of epidermal growth factor receptor in nasopharyngeal carcinoma cell line.
Yongguang TAO ; Xiyun DENG ; Zhi HU ; Min TANG ; Huanhua GU ; Wei YI ; Chengxing WANG ; Feijun LUO ; Ya CAO
Chinese Journal of Oncology 2002;24(3):226-229
OBJECTIVETo elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma cell line.
METHODSThe levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment.
RESULTSIn pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense.
CONCLUSIONEB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.
Blotting, Western ; Epidermal Growth Factor ; metabolism ; Herpesvirus 4, Human ; metabolism ; Humans ; Nasopharyngeal Neoplasms ; pathology ; virology ; Phosphorylation ; Receptor, Epidermal Growth Factor ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; metabolism
9.Study of oral microbial adhesion and biofilm formation on the surface of nano-fluorohydroxyapatite/polyetheretherketone composite
Lixin WANG ; Huan ZHANG ; Yi DENG ; Zuyuan LUO ; Xiyun LIU ; Shicheng WEI
Chinese Journal of Stomatology 2015;50(6):378-382
Objective To develop novel polyetheretherketone(PEEK) based nanocomposites which possess the favorable antibacterial property,and to investigate the oral microbial adhesion and biofilm formation on the surfaces of PEEK,nano-fluorohydroxyapatite(n-FHA)-PEEK and nano-hydroxyaptite (n-HA)-PEEK.Methods The bacterial adhesion and biofilm formation on the surfaces of n-FHA-PEEK,n-HA-PEEK were investigated via microbial viability assay kit and laser scanning confocal microscope (LSCM),respectively,with pure PEEK as control group.Results No significantly statistical difference were found in the bacterial adhesion amounts on the surfaces of n-FHA-PEEK,n-HA-PEEK and PEEK at 1 h and 4 h.However,the number of bacteria on the n-FHA-PEEK surface decreased dramatically at 2 h(0.496±0.008) compared with n-HA-PEEK groups(0.543± 0.015,P<0.01).Although the biofilms formation on surfaces observed by LSCM had similar morphology and thickness at 3,7,14 d,that on the n-FHA-PEEK surface showed the highest dead-to-live bacteria ratio among the three materials at 14 d.Conclusions The combination of n-HA,especially for the n-FHA could inhibit the bacteria adhesion and accelerate the bacterial death,eventually may have an influence on the structure of biofilms and reduce the risk of periimplantitis.Therefore,n-FHA-PEEK nanocomposites presented a good prospect for clinical applications as dental implant materials.