Epstein–Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells

Li, H M; Zhuang, Ze-Hao; Wang, Quan-Fang; Pang, Jesse C. S.; Wang, X H; Wong, Hai Ming; Feng, Huichen; Jin, Dong-Yan; Ling, Ming-Tat; Wong, Yong Chuan; Eliopoulos, Aristides G.; Lw, Young; Huang, Dolly P.; Tsao, Sai‐Wah

doi:10.1038/sj.onc.1207580

Cited by 46 publications

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“…Furthermore, downregulation of RASSF1A by transient transfection of LMP1 was shown to be dose-dependent in HeLa cells, confirming that LMP1 is directly involved in the downregulation of RASSF1A expression ( Figure 1c). The concentrations of LMP1 plasmids used for transfection were 0.3-1.2 mg/10 6 cells, which is within the normal range of LMP1 concentrations used in cell signaling studies (Eliopoulos et al, 1999b;Li et al, 2004). Suppression of RASSF1A expression in LMP1 expressing cells could also be demonstrated at the transcription level by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) (Figure 1d).…”

Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 59%

“…To explore the potential involvement of these signaling pathways in mediating LMP1-induced downregulation of RASSF1A, the effects of specific inhibitors on RASSF1A expression were examined. The concentrations and treatment times of these inhibitors for these signaling pathways have been optimized in previous studies (Li et al, 2004;Wu et al, 2006). The effectiveness of each inhibitor in suppressing the kinase activities of MAPK, JNK and PI3K was confirmed by examining the phosphorylation status of their respective downstream targets (Figure 2A, a-c).…”

Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 94%

“…The wild-type (wt) and mutant LMP1 plasmids used in this study were kindly provided by Professor Lawrence Young, University of Birmingham, UK; which include the pSG5 vector, pSG5B95.8 LMP1, the pSG5-B95.8LMP1 AxAxA (mutated CTAR1), pSG5-B95.8LMP1 378STOP (deleted in CTAR2) and pSG5-B95.8LMP1 AxAxA/378STOP (mutated CTAR1 and deleted CTAR2). This set of mutants have been used previously in defining the intracellular signaling of LMP1 (Eliopoulos and Young, 2001;Li et al, 2004). The experiments were performed in both HeLa and HaCaT cells.…”

Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 99%

“…Various deletion fragments of the 3 kb RASSF1A promoter region (À3101/À1, À1190/À1, À431/ À1, À431/ þ 15, À300/À1, À202/À1) were subcloned into the pGL3-basic vector, upstream of the luciferase coding sequence and examined for the promoter usage in transfected cells. The suppressing effect of LMP1 on RASSF1A promoter activity was assayed as previously described (Li et al, 2004). For a typical transfection, 0.25 mg of RASSF1A reporter, 0.01 mg of control reporter (pRL-SV40) and 0.1 mg of LMP1 expression plasmid were co-transfected with Fugene 6 transfection reagents (Roche Molecular Biochemicals Inc., USA).…”

Section: Transient Reporter Assaysmentioning

confidence: 99%

“…Cells were harvested and protein concentrations were determined as previously described (Li et al, 2004). Protein (30 mg) was resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a nitrocellulose membrane (Immobilon-P; Millipore, Bedford, MA, USA).…”

Section: Western Blotting Analysismentioning

confidence: 99%

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Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

et al. 2006

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Epstein-Barr virus (EBV) infection is closely associatedwith nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumorsuppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-jB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASS-F1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

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Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 59%

Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 94%

Section: Lmp1 Downregulates Rassf1a Expressionmentioning

confidence: 99%

Section: Transient Reporter Assaysmentioning

confidence: 99%

Section: Western Blotting Analysismentioning

confidence: 99%

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Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

et al. 2006

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Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase

Man

Jin

et al. 2006

Intl Journal of Cancer

111

136

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Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase‐immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16INK4A locus and downregulation of RASSF1A expression. The deletion of the p16INK4A locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array‐based CGH revealed a gain of a 17q21‐q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16INK4A locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF‐κB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development. © 2006 Wiley‐Liss, Inc.

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Epstein‐Barr virus infection in immortalized nasopharyngeal epithelial cells: Regulation of infection and phenotypic characterization

Tsang

Zhang

Seto

et al. 2010

Intl Journal of Cancer

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Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-b effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBVencoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBVinfected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-a. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development. Infection of Epstein-Barr virus (EBV) is ubiquitous and more than 95% of the adult population world-wide is infected with this virus.1 EBV infection is life-long and largely asymptomatic. EBV is implicated as an etiological agent in several human malignancies of either lymphoid or epithelial origin. These include Burkitt's lymphoma, Hodgkin's lymphoma, nasal T/NK lymphomas, undifferentiated nasopharyngeal carcinomas (NPC), gastric adenocarcinomas, smooth muscle tumors and various B-cells lymphomas in AIDS patients or transplant recipients with compromised immune functions. 2-4EBV infection can be detected in most, if not all, undifferentiated NPC, regardless of geographical and ethnical origin of NPC patients.2 Expression of latent EBV genes including EBER, EBNA1, LMP1 and LMP2A as well as lytic EBV genes, such as BZLF1, could be detected in cancer cells inside the nasopharyngeal carcinoma specimens. Clonal infection of EBV has been detected in precancerous lesions of NPC, implicating its involvement at the early stage of NPC pathogenesis.5 EBV infection has long been postulated to play an important role in transformation of premalignant ...

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Epstein–Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells

Cited by 46 publications

References 36 publications

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase

Epstein‐Barr virus infection in immortalized nasopharyngeal epithelial cells: Regulation of infection and phenotypic characterization

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