Establishment and characterization of immortalized nasopharyngeal epithelial cells with stable infection of Epstein-Barr virus

Abstract

Nasopharyngeal carcinoma (NPC), with a remarkably distinctive ethnic and geographic distribution, has the highest incidence among Southern Chinese. Unlike other types of head and neck cancers, a unique feature of NPC is its close association with Epstein-Barr virus (EBV). EBV infection represents an early event during the pathogenesis of NPC. Latent EBV infection is found in every cancer cell of virtually all cases of undifferentiated NPC in endemic regions. Genetic alterations in the premalignant epithelium may support the EBV latency. Although EBV-encoded proteins in its latent infection, including LMP1, LMP2A, etc., have been identified with crucial functions to initiate or promote oncogenesis, to date the exact role of EBV infection in the malignant transformation of nasopharyngeal epithelial (NPE) cells remains unclear. We have previously generated a panel of immortalized NPE cells by ectopic overexpression of hTert (catalytic human telomerase). However, reliable in vitro models of stable EBV infection are also necessary for further elucidating the regulatory network supporting the persistent latency of EBV in NPE cells, and also the potential roles of EBV in the pathogenesis of NPC. In the present study, we established NP361hTert-EBV cells with stable EBV infection via co-culture with Akata B cells harboring EBV, followed by the effective enrichment of EBV-infected cells, indicated by positive GFP, by FACS sorting. The loss of EBV episome in NPE cells was fast initially upon infection, as well as the first and second enrichment of EBV-positive cells. The relatively stable maintenance of EBV could be achieved after enrichment for three times. Results of western blotting analysis showed that the protein expression level of cyclin D1 was increased in NP361hTert-EBV cells, as compared to the non-infected NP361hTert cells. Meanwhile, we also observed increased expression of phosphorylated protein kinase Akt (Ser473) in NP361hTert-EBV cells, suggesting the Akt signaling was activated, and such activation might contribute to the persistence of EBV in NP361hTert cells. This stable EBV-infected NPE cells will serve as a powerful in vitro model for further study in elucidating the functional role of EBV in the carcinogenesis of NPC. This work was partly supported by General Research Fund (HKU 779713M, HKU780911M), and the AoE NPC grant (AoE/M-06/08) from Hong Kong Research Grant Council.