Proteome Analysis Of Transformation Potential Of The Epstein-Barr Virus-encoded Latent Membrane Protein1 In Nasopharyngeal Epithelial Cells NP69

Background & Objective Epstein-Barr virus (EBV) is frequently implicated in the etiology of nasopharyngeal carcinoma (NPC). EBV genomes and its expression products have been identified in various types of NPC tissues. EBV latent membrane protein1 (LMP1) is known for the only oncogenic protein coded by EBV genome in transformation and tumorigenesis, suggesting a very important role for LMP1 on the development of NPC. There are three carboxy terminal activating regions (CTAR1, CTAR2 and CTAR3) associatied with the role of LMP1 by mediating complicated signaling pathways. Previous studies have shown that CTAR2 (the TRADD binding domain) is important for LMP1-mediated transformation and tumorigenesis of certain cells. But it is scanty of the systemic and integrity proof to support the molecular mechanism. In order to globally understand and confirm the signal pathways mediated by LMP1, to explain the molecular net involved in LMP1 CTAR2-mediated transformation and tumorigenesis of nasopharyngeal epithelial cells, this study is to plan to separate and identify the differential protein associated with LMP1 transforming nasopharyngeal epithelial cells by proteome approach, and to provide further evidence for the role of EBV on the development of NPC.Method pLNSX (as controlled vector), pLNSX-LMP1^WT and pLNSX-LMP1^TRADD were transfected into the ecotropic retrovirus packaging cell line PA317 with Lipofectamine, respectively. The transfected PA317 cells were selected with G418 sulfate, two weeks later, the resistant cells were collected and expanded as the virus-producing cell lines (RV-LNSX, RV- LMP1^WT and RV-LMP1^TRADD) to use in subsequent experiment. Then, nasopharyngeal epithelium cells NP69 were infected by the concentrated retrovirus respectively. After selected with G418 sulfate, G418 sulfate-resistant clones were selected and pooled to establish the stable transfected cell lines NP69-LNSX, NP69-LMP1^WT, and NP69-LMP1^TRADD. Cellular morphology, cellular growth curve, colony formation, soft agar formation, cell cycle and the ability of inhibition apoptosis were observed and detected. Two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins of NP69-LNSX, NP69-LMP1^WT and NP69-LMP1^TRADD cells, respectively. Well-resolved and reproducible 2-D patterns of the above three-group cells were acquired. Differentially expressed proteins were identified by MALDI-TOF-MS and database searching. The differential expression levels of the partial proteins were determined by real-time quantitative RT-PCR and/or Western blot.Results (1) The morphous of NP69-LMP1^WT cells graduallydeveloped from a typical epithelial polygon, cobblestone to an elongated and fibroblastoid shape with a marked reduction in cell-cell contact. But NP69-LMP1^TRADD exhibited approximately polygonal morphous and grew in a more compact pattern with tighter cell-cell contact. The growth velocity, soft agar focus formation ability, the proportion of S stage, and antiapotosis capability of NP69-LMP1^WT cells obviously increased than NP69-LMP1^TRADD cells and NP69-LNSX cells; (2) 21 differentially expressed proteins between NP69-LNSX and NP69-LMP1^WT and 18 differentially expressed proteins between NP69-LMP1^WT and NP69-LMP1^TRADD were identified. Most of them were characterized as cellular structure proteins, the members of enzymes and transcription and translation associated proteins. (3) In the transformed cells, 6 proteins were confirmed with real-time quantitative RT-PCR on the level of transcription, 2 proteins were confirmed with Western blot on the level of protein expression. Both results were coincident with the results of the proteomics.Conclusions (1) LMP1 has the ability to assign NP69 cells morphological transformation, enhance the ability to grow, proliferate, form soft agar focus and inhibit apoptosis, et al. (2) LMP1 promotes NP69 cells transforming with the up-regulation of calreticulin, vimentin and lamin A/C and the down-regulation of CK5, CK19 and annexin n, et al. (3) The TRADD domain of LMP1 was pivotal participating in the above effects with the regulation of the expression of CK19, annexin II, calreticulin, lamin A/C, and so on.