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

Abstract

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 ...

CRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells

“…1b). HEK293-BX1 cells, which constitutively carry the GFP-expressing BX1 strain of EBV and were established through co-culture with Akata-BX1 cells (Chen et al, 2005b;Tsang et al, 2010), were chosen as the target cells for CRISPR/Cas9 editing because HEK293 cells are highly transfectable, and have been extensively used for production and analysis of recombinant EBVs created on BACs Geiser et al, 2011). The EBV genome copy number in HEK293-BX1 cells as analysed by fluorescence in situ hybridization (FISH) was 79±52.…”

“…NP460 is an immortalized nasopharyngeal epithelial cell line . Infection of NP460 with EBV through co-culture with infected B cells might shed light on early events of cancer CRISPR/Cas9 editing of the EBV genome development (Tsang et al, 2010(Tsang et al, , 2012. It is noteworthy that C666-1 and NP460-EBV are difficult-to-transfect cells and that the copy numbers of the EBV genome (30-50 for C666-1 and 10-30 for NP460-EBV) in these cells are relatively high (Lun et al, 2012;Yip et al, 2013).…”

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells

“…Additionally, monoclonal EBV episomes are detected in NPC, suggesting that NPC tumors are the clonal outgrowth of an initially infected cell likely predisposed to oncogenic transformation from additional genetic and environmental cofactors, such as the loss of p16 and exposure to dietary nitrosamines (2,3). In contrast to the immortalizing properties of EBV to primary B cells, the contribution of EBV infection to epithelial cell oncogenesis is less understood, as infection alone is insufficient to immortalize or induce oncogenic potential in preneoplastic cell lines from the nasopharynx (5,6).…”

“…In the present study, the oncogenic potential of LMP1 and LMP2A were investigated by expression in the EBVnegative NPC cell line HK1 and examined for potentially combined effects in response to cell death activation, cell growth, and motility. HK1 cells are one of the few authenticated NPC cell lines that are sensitive to serum deprivation and chemotherapeutics but can resist these cell death treatments by infection with EBV (6,23,31). Additionally, HK1 cells are free of HeLa contamination, which confounds the interpretation of many NPC-derived cell lines (32,33).…”

Profiling of Epstein‐Barr virus‐encoded microRNAs in nasopharyngeal carcinoma reveals potential biomarkers and oncomirs

“…19 Both C666 and NP460hTERTþEBV harbor latent EBV infection. 20,21 EBV-negative cell line CNE2 was derived from poorly differentiated NPC, 22 whereas NP69 is an immortalized nasopharyngeal epithelium cell line. 23 C666 and CNE2 were cultured in RPMI-1640 medium (Sigma, St Louis, Mo) supplemented with 10% fetal bovine serum.…”