| Summary: | 博士 === 國立臺灣大學 === 微生物學研究所 === 89 === Epstein-Barr virus (EBV) is highly associated with nasopharygeal carcinoma (NPC) and B-cells proliferative diseases. Tyrosine kinases constitute the signaling cascades and play a pivotal role in cell fate determination. This suggests that tyrosine kinases are likely to be the targets regulated by EBV. To search the EBV-regulated tyrosine kinase, we used kinase display assays in three types of systems. First, we used lymphoblastoid cell lines (LCL) that were established by EBV-immortalized primary B cells. Compared with freshly isolated primary B cells, three differential expressed genes, tkt, frk, and ron were isolated and confirmed. Second, EBV-infected Burkitt’s lymphoma cell lines showed no difference in the amount or types of gene expression, comparing to the parental cell line. Third, EBV single gene expression cell lines were explored to identify their difference in the amount of kinase transcripts. Significant increment of tkt expression was found in EBV Zta-expressing cell lines. We also tested many different kinases in EBV EBNA 1-, LMP1-, LMP2-expressing cell lines. Some candidates were suggested but further confirmation is required.
About 5% of EBV-immortalized LCLs spontaneously go into lytic cycle, and this process needs Zta protein expression. The tkt gene was upregulated both in Zta-expressing cells and LCLs, therefore we proposed that Zta could transactivate TKT in both single gene and whole virus expression cell lines. To further address this issue, the expression of TKT was confirmed by gene specific RT-PCR in both Zta constitutive and inducible expressing cell lines. The increase of tkt in Zta cell line was not only detected in transcriptional level, but also in translational level, as demonstrated by northern and western blot analyses. In addition, the deletion analysis of Zta revealed that the transactivation and dimerization domains were both essential for tkt upregulation. Further findings of TKT expression level in the lytic cycle of AKATA cells provided more evidence in Zta activating TKT within the whole virus system.
Interestingly, upregulation of phosphorylated TKT and TKT downstream effector matrix metalloproteinase-1 (MMP-1) in Zta-expressing cells suggested that TKT could initiate a signaling cascade in this study. Moreover, a correlation existed between the expression levels of Zta and MMP-1 transcripts in NPC biopsy specimens. This clearly provides evidence for Zta regulating cellular gene expression in vivo. Previous studies also showed MMPs are the key factors in extracellular matrix turnovers and tumor progression. Therefore, we hypothesize that upregulation of TKT and MMP may contribute to the oncogenesis of NPC.
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