Expression of Epstein‐Barr virus‐encoded LMP1 and hTERT extends the life span and immortalizes primary cultures of nasopharyngeal epithelial cells

Cell immortalization is regarded as an early and pre‐requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein‐Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV‐encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c‐Myc, Bmi‐1, and Id‐1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT‐immortalized nasopharyngeal epithelial cells. The LMP1/hTERT‐immortalized NP446 cells were non‐tumorigenic in immunosuppressed nude mice and retained anchorage‐dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV‐encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma. J. Med. Virol. 82:1711–1723, 2010. © 2010 Wiley‐Liss, Inc.

[1]  S. Law,et al.  Genetic alterations in a telomerase-immortalized human esophageal epithelial cell line: implications for carcinogenesis. , 2010, Cancer letters.

[2]  G. Teoh,et al.  Association of Epstein-Barr virus with nasopharyngeal carcinoma and current status of development of cancer-derived cell lines. , 2008, Annals of the Academy of Medicine, Singapore.

[3]  K. Choy,et al.  Authentication of nasopharyngeal carcinoma tumor lines , 2008, International journal of cancer.

[4]  N. Raab-Traub,et al.  EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas , 2007, PLoS pathogens.

[5]  R. Feederle,et al.  Epstein‐Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection , 2007, International journal of cancer.

[6]  S. Tsao,et al.  Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells , 2007, Oncogene.

[7]  A. Taylor,et al.  Bmi-1 is induced by the Epstein-Barr virus oncogene LMP1 and regulates the expression of viral target genes in Hodgkin lymphoma cells. , 2007, Blood.

[8]  M. Fujita,et al.  Efficient immortalization of primary human cells by p16INK4a‐specific short hairpin RNA or Bmi‐1, combined with introduction of hTERT , 2007, Cancer science.

[9]  P. Meltzer,et al.  Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase , 2006, International journal of cancer.

[10]  M. Allday,et al.  Epstein-Barr Virus Selectively Deregulates DNA Damage Responses in Normal B Cells but Has No Detectable Effect on Regulation of the Tumor Suppressor p53 , 2006, Journal of Virology.

[11]  Yun-Fei Xia,et al.  Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells. , 2006, Cancer research.

[12]  S. Uriarte,et al.  Porphyromonas gingivalis enhances FasL expression via up-regulation of NFkappaB-mediated gene transcription and induces apoptotic cell death in human gingival epithelial cells. , 2006, Microbiology.

[13]  W. Hahn,et al.  Creating oral squamous cancer cells: A cellular model of oral–esophageal carcinogenesis , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[14]  D. Stevenson,et al.  Epstein-Barr virus latent membrane protein 1 (CAO) up-regulates VEGF and TGF alpha concomitant with hyperlasia, with subsequent up-regulation of p16 and MMP9. , 2005, Cancer research.

[15]  S. Tsao,et al.  Amplification and overexpression of Aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells , 2005, Molecular carcinogenesis.

[16]  G. Glinsky,et al.  Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. , 2005, The Journal of clinical investigation.

[17]  Li Dong Wang,et al.  Distinct profiles of critically short telomeres are a key determinant of different chromosome aberrations in immortalized human cells: whole-genome evidence from multiple cell lines , 2004, Oncogene.

[18]  D. McCance,et al.  Degradation of p53, Not Telomerase Activation, by E6 Is Required for Bypass of Crisis and Immortalization by Human Papillomavirus Type 16 E6/E7 , 2004, Journal of Virology.

[19]  S. Tsao,et al.  Epstein–Barr virus latent membrane protein 1 (LMP1) upregulates Id1 expression in nasopharyngeal epithelial cells , 2004, Oncogene.

[20]  S. Tsao,et al.  Phenotypic alterations induced by the Hong Kong‐prevalent Epstein‐Barr virus‐encoded LMP1 variant (2117‐LMP1) in nasopharyngeal epithelial cells , 2004, International journal of cancer.

[21]  C. Prives,et al.  Decreased p21 Levels Are Required for Efficient Restart of DNA Synthesis after S Phase Block* , 2004, Journal of Biological Chemistry.

[22]  M. Allday,et al.  Epstein–Barr virus can inhibit genotoxin-induced G1 arrest downstream of p53 by preventing the inactivation of CDK2 , 2003, Oncogene.

[23]  W. Hammerschmidt,et al.  The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  L. Young,et al.  Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Activates the Phosphatidylinositol 3-Kinase/Akt Pathway to Promote Cell Survival and Induce Actin Filament Remodeling* , 2003, The Journal of Biological Chemistry.

[25]  K. Münger,et al.  NF-kappaB-mediated induction of p21(Cip1/Waf1) by tumor necrosis factor alpha induces growth arrest and cytoprotection in normal human keratinocytes. , 2003, Molecular cancer research : MCR.

[26]  S. Tsao,et al.  A new method for improving metaphase chromosome spreading , 2003, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[27]  N. Raab-Traub.  Epstein-Barr virus in the pathogenesis of NPC. , 2002, Seminars in cancer biology.

[28]  S. Tsao,et al.  The significance of LMP1 expression in nasopharyngeal carcinoma. , 2002, Seminars in cancer biology.

[29]  P. Johnson,et al.  Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from southern Chinese , 2002, International journal of cancer.

[30]  William C Hahn,et al.  Establishment : adaptation to culture and immortalization , 2002 .

[31]  S. Tsao,et al.  Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes. , 2002, Biochimica et biophysica acta.

[32]  L. Young,et al.  LMP1 structure and signal transduction. , 2001, Seminars in cancer biology.

[33]  S. Tsao,et al.  Nonrandom chromosomal imbalances in human ovarian surface epithelial cells immortalized by HPV16-E6E7 viral oncogenes. , 2001, Cancer genetics and cytogenetics.

[34]  D. Polsky,et al.  The transcriptional repressor of p16/Ink4a, Id1, is up-regulated in early melanomas. , 2001, Cancer research.

[35]  T. Yoshizaki,et al.  Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  W. Tolleson,et al.  Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes. , 2001, Cancer research.

[37]  G. Tse,et al.  High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. , 2000, Cancer research.

[38]  W. Keith,et al.  Mapping of the gene for the human telomerase reverse transcriptase, hTERT, to chromosome 5p15.33 by fluorescence in situ hybridization. , 2000, Neoplasia.

[39]  Zhiming He,et al.  LMP1 of Epstein–Barr virus suppresses cellular senescence associated with the inhibition of p16INK4a expression , 2000, Oncogene.

[40]  S. Lowe,et al.  LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene , 2000, Journal of Virology.

[41]  R. Reddel,et al.  Telomere maintenance mechanisms and cellular immortalization. , 1999, Current opinion in genetics & development.

[42]  K Kornfeld,et al.  Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase. , 1999, Genes & development.

[43]  R. DePinho,et al.  The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus , 1999, Nature.

[44]  V. Godfrey,et al.  Expression of the Epstein-Barr virus latent membrane protein 1 induces B cell lymphoma in transgenic mice. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  E. Kieff,et al.  The Epstein–Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-κB , 1997 .

[46]  M. Ueffing,et al.  Latent membrane protein 1 of Epstein–Barr virus mimics a constitutively active receptor molecule , 1997, The EMBO journal.

[47]  Kathryn Flynn,et al.  Clonal proliferations of cells infected with Epstein-Barr virus in preinvasive lesions related to nasopharyngeal carcinoma. , 1995, The New England journal of medicine.

[48]  W. Miller,et al.  The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor , 1995, Journal of virology.

[49]  M. Newton,et al.  Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells. , 1994, Genes & development.

[50]  Gerard I. Evan,et al.  Induction of apoptosis in fibroblasts by c-myc protein , 1992, Cell.

[51]  W. Hammerschmidt,et al.  The transforming domain alone of the latent membrane protein of Epstein-Barr virus is toxic to cells when expressed at high levels , 1989, Journal of virology.

[52]  S. Tsao,et al.  Regulation of p63 expression in primary and immortalized nasopharyngeal epithelial cells. , 2008, International journal of oncology.

[53]  N. Raab-Traub,et al.  LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice. , 2006, Oncogene.

[54]  Ching‐Hwa Tsai,et al.  Regulation of matrix metalloproteinase-1 by Epstein-Barr virus proteins. , 2003, Cancer research.

[55]  P. Johnson,et al.  Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[56]  M. Israel,et al.  Immortalization of primary human keratinocytes by the helix-loop-helix protein, Id-1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[57]  A. Berns,et al.  Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF. , 1999, Genes & development.

[58]  C. Tzeng,et al.  Clonal karyotype abnormalities in EBV-associated hemophagocytic syndrome. , 1997, Haematologica.

[59]  E. Kieff,et al.  The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[60]  E. Kieff.  Epstein-Barr virus and its replication , 1996 .

[61]  B. Christensson,et al.  Expression of Ki67 antigen, epidermal growth factor receptor and Epstein-Barr virus-encoded latent membrane protein (LMP1) in nasopharyngeal carcinoma. , 1994, European journal of cancer. Part B, Oral oncology.