Study of the Mechanism Involved in the LMP-1-mediated Repression of E-cadherin Promoter in Epithelial Cells

• Main Authors: Ka Po Tse, 謝家寶
• Other Authors: Hwan-You Chang
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/10906691661270713869

碩士 === 國立清華大學 === 生命科學系 === 89 === E-cadherin is an important tumor suppressor, loss of E-cadherin lead to increased invasiveness of cancer. Nasopharyngeal carcinoma (NPC) is a cancer notorious for its high metastatic ability and reduction of E-cadherin happens when metastasis occurred. The underlying mechanism is still not clear. NPC is closely associated with Epstein-Barr virus (EBV) and in tumor cells, a viral oncoprotein LMP-1 is frequently expressed. Previous reports suggest that LMP-1 reduces E-cadherin expression in RHEK cells. LMP-1 can also induce Ets-1, which promotes cell invasiveness in MDCK cells. In this study, the LMP-1 expressing-MDCK and NPC076 cells exhibit a spindle-shaped morphology, as compared to a cobble-like phenotype of the parental cells. Immunofluorescence study and Western blot analysis showed that E-cadherin, b-catenin and g-catenin of the cadherin-catenin complex are down-regulated. On the other hand, the mesenchymal markers such as vimentin and fibronectin are induced. These changes correlate with increased cell migration ability as examined by wound-induced migration assay. Down-regulation of E-cadherin is mainly at the transcription level, potentially through binding sites regulated by transcription factors Snail, Ets-1 and Sp1. To further investigate if LMP-1 down-regulate E-cadherin through these sites, LMP-1 was co-transfected with a series of luciferase reporter constructs containing E-cadherin promoter and its transcription factor binding site-specific mutants-1 into MDCK or MCF-7 cells. These results demonstrate that E-cadherin is down-regulated by LMP-1 at the transcription level, and the repression is independent of Snail, Ets-1 and Sp1. In addition, hypermethylation of E-cadherin- gene promoter can result in down-regulation of E-cadherin. Treatment of LMP-1-expressing cells with 5’-Aza-2’-deoxycytidine restores E-cadherin expression. Therefore, the result indicate that promoter hypermethylation is involved in the LMP-1 mediated repression of E-cadherin. Conclusively speaking, E-cadherin, b-catenin and g-catenin were down-regulated by LMP-1 whereas mesenchymal markers such as vimentin and fibronectin were up-regulated. In addition, LMP-1 may down-regulate E-cadherin at the transcription level by promoter hypermethylation. It is the first report that indicates expression of LMP-1 in epithelial cells could lead to promoter hypermethylation. The mechanism that may be involved in LMP-1-mediated hypermethylation of E-cadherin promoter and its role in NPC metastasis will be discussed. ABSTRACT 3 INTRODUCTION 5 MATERIALS AND METHODS 19 RESULTS: 30 DISCUSSION 39 REFERENCE: 44