Functional studies of Epstein-Barr virus latent membrane protein 1 in nasopharyngeal epithelial cells

by Li, Hoi-ming

(Uncorrected OCR) Abstract of thesis entitled

Functional studies of Epstein-Barr Virus Latent Membrane

Protein 1 in nasopharyngeal epithelial cells

Submitted by

Li, HoiMing

for the degree of Doctor of Philosophy at The University of Hong Kong

on 31 March 2004

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong

and Southern provinces of China. The incidence rate of NPC could reach to

25-50 per 100,000 per year. NPC is closely associated with Epstein-Barr virus

(EBV) infection. Over 95% of the world's population carries latently infected

EBV in their B cells. EBV genome could be detected in most of the NPC cells.

Role ofEBVin the pathogenesis ofNPC is largely unknown. Understanding the

mechanisms of tumorigenesis by EBV is essential for better prevention and

treatment of NPC. Previous studies have found that several of the EBV latent

genes modulate cellular phenotype and growth characteristics, in which the

oncogene Latent Membrane Protein 1 (LMP1) is believed to play important role

in malignant transformation.

The main focus of my research is on the mechanisms of LMPI In

transformation of nasopharyngeal epithelial cells. In this study, a novel function ofLMPl to induce Idl expression in nasopharyngeal epithelial cells (NP69) and HEK293 is reported. Idl is a negative transcriptional regulator of p16INK4a. Expression of Idl facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical and genetic inhibitors of cell signaling, induction of Idl by LMPI was shown to be dependent on its NF-KB activation domain at the carboxy-terminal region, CTARI and CTAR2. Induction of Idl by LMPI may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.

Previous studies have shown that LMPI activates Cdc42, which mediate some phenotypic changes such as actin reorganization. Such changes are known to relate to cell cycle progression, cytokinesis and adhesion, which are involved in carcinogenesis. LMPI was shown to be able to activate Cdc42 in 3T3 fibroblasts. Here Cdc42 was found to be activated by LMPI in HEK293 and nasopharyngeal carcinomatoid SUNEI. This supports a role of LMPI in carcinogenesis through constitutive activation of Cdc42 in nasopharyngeal epithelial cells.

Previous studies have also shown that loss or alteration of E-cadherin

occurs frequently in many different epithelial cancers. It has also been found to

be downregulated in NPC. E-cadherin is a plasma membrane protein that functions in cell-cell adhesion. In my study, E-cadherin was found to be downregulated by LMPI in nasopharyngeal carcinomatoid CNE2 and SUNEI. In NPC cells, inhibition of its expression was not related to the CTAR2 domain

ofLMPl.

Currently, most of the functional studies on the transformation ability of EBV were demonstrated in either non-nasopharyngeal epithelial or transformed cell lines, which have aberrant cellular signaling systems that complicate the experiments. Hence, in this study, a near normal human nasopharyngeal epithelial cell line (NP460 hTert) has been established with the assistance of

constitutive telomerase expression. The immortalized telomerase expressing cells have up-regulation in Idl, increased phosphorylation of IKBa, undetectable p 16INK4a expression and amplification at 17 q21-q25 on 11 p 15 chromosome. The phenotypic alterations of the immortalized nasopharyngeal epithelial cells were similar to the functional properties of EBV-encoded LMPI suggested that LMPI may partly contribute to the immortalization of nasopharyngeal epithelial cells.