Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study

PLoS One. 2013 Oct 22;8(10):e78395. doi: 10.1371/journal.pone.0078395. eCollection 2013.

Abstract

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma
  • Cell Line, Transformed
  • Epithelial Cells / metabolism
  • Epithelial Cells / pathology
  • Epithelial Cells / virology*
  • Epstein-Barr Virus Infections / metabolism*
  • Epstein-Barr Virus Infections / pathology
  • Female
  • Herpesvirus 4, Human / physiology*
  • Humans
  • MAP Kinase Signaling System
  • Male
  • Nasal Cavity / metabolism
  • Nasal Cavity / pathology
  • Nasal Cavity / virology*
  • Nasal Mucosa / metabolism
  • Nasal Mucosa / pathology
  • Nasal Mucosa / virology*
  • Nasopharyngeal Carcinoma
  • Nasopharyngeal Neoplasms / metabolism
  • Nasopharyngeal Neoplasms / pathology
  • Nasopharyngeal Neoplasms / virology
  • Pharynx / metabolism
  • Pharynx / pathology
  • Pharynx / virology*
  • Polycomb Repressive Complex 1 / metabolism
  • Virus Latency

Substances

  • BMI1 protein, human
  • Polycomb Repressive Complex 1

Grants and funding

This work was supported by the Hong Kong General Research Fund (HKU 777809M), the AoE NPC grant (AoE/M-06/08), Health and Medical Research Fund of Hong Kong grant (12110782) and CRCG funding support from the University of Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.