| Nasopharyngeal carcinoma (NPC) is a human malignant tumor derived from nasopharyngeal epithelial cells with high incidence in several provinces of Southern China. The development of NPC is a multifactor and multistage process, mainly related with Epstein-Barr virus (EBV), chemical carcinogens and genetic factors. The evidence of both serological and histological study show that EBV is closely associated with NPC. EBV is observed in NPC tumor cells and dysplasia lesions but is absent in normal nasopharyngeal epithelial cells, which suggests that EBV infects epithelial cells at the early stage of NPC development. However, the infection mechanism and the function of EBV in tumor development remain unclear. Three-stage hypothesis of carcinogenesis pointed out that tumor promoter might play a key role in tumor development. It has been demonstrated that okadaic acid (OA), as a potent tumor promoter, is a highly selective inhibitor of protein phosphatases type 1(PP1) and type 2A(PP2A), subsequently that causes dramatic increases in phosphorylation of numerous proteins. For that reason, OA is an extremely useful tool for studying the mechanism of tumor development.Proteomics associated analysis technology belongs to a new, high throughput post-genome research method, which is widely used in human diseases, especially in the tumor research. The classical procedure of proteomics research is composed of separating the proteins by two-dimensional electrophoresis (2-DE) and acquiring the peptide mass fingerprints by mass spectrum analyses and getting the information ofproteins through Internet search. To investigate the relationship between NPC and EBV by proteomics technique will do benefit to a new exploration of NPC carcinogenesis.The present investigation was designed as a 2 X 2 test which consisted of one negative control group and three experimental groups including the treatment with EBV, OA, EBV+OA respectively. The main purpose is to study the protein expression profile and changes among those groups, and to provide some new clues of relationship between EBV and NPC in protein level.We did the following work in this paper.Firstly, we studied various conditions for performing 2-DE by using HNE-1 cells, after optimizing the methods of protein extraction and the conditions in the first dimensional electrophoresis, and then acquired repeatable 2-DE maps.Secondly, HNE-1 cells were treated with EBV, OA and EBV+OA, The effect of OA on the growth of HNE-1 cells was observed by MTT test. The EBV infection was verified by PCR, Southern blot, in situ hybridization and electronic microscope. The results of PCR, Southern blot indicated that BamHI W fragment of EBV was present in EBV and EBV+OA treated cells. The result of in situ hybridization showed that the infection ratio was 9.294% in EBV treated cells, 9.816% in EBV + OA treated cells. The statistical analysis ( x 2 test) showed that there was no significant difference between two groups. The EBV could be observed in the EBV treated HNE-1 cells under electronic microscope.Finally, we analyzed the protein expression pattern alternation of those groups. In contrast to the control group, seven proteins disappeared and seven new proteins were expressed in EBV treated group. Three proteins vanished and eleven proteins newly appeared in OA treated group. Five proteins disappeared and nine proteins appeared in EBV + OA group. It was noticeable that five of nine newly expressed proteins were expresseduniquely in EBV + OA group. We acquired the proteins information byInternet search according to the Mr/Pi of proteins. The results showed that EBV might make a great impact on the pathway of DNA synthesis regulation, gene transcription and so on by its gene expression products, and then altered protein expression pattern, consequently promoted cell growth and suppressed cell apoptosis, while OA could inhibit phosphatase to cause the over-phosphorylation of related proteins which affected pathways of the signal transduction in the cells. In addition, EBV... |
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