| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所 === 96 === Epstein-Barr virus (EBV) is closely associated with many human cancers, including Hodgkin’s disease, immunocompromise-associated B- and T-cell lymphoma, and nasopharyngeal carcinoma (NPC). EBNA2 is a viral oncoprotein and is required for EBV-induced B-cell immortalization. However, its carcinogenic role in epithelial cells is unclear. Our previous study showed that EBNA2 could activate p53 and p21WAF1, and retard epithelial cell growth. Recently we found that H2AX phosphorylation, a DNA damage marker, was increased and G2/M cell cycle progression was suppressed in EBNA2-expressing cells. Based on these results, we propose that EBNA2 may interfere with DNA repair and induce DNA damage, which result in G2/M cell cycle arrest. To examine this possibility, we established an EBNA2-ER inducible cell line (E2ER18), then investigated the signs of DNA damage by H2AX phosphorylation and examined genomic instability using micronucleus test when nucler translocation of EBNA2-ER was induced by 4-hydroxytamoxifen (4-OHT). The results showed that H2AX phosphorylation and micronucleus formation were increased when nucler translocation of EBNA2-ER was induced. In addition, ATM and p53 were hyperphosphorylated upon 4-OHT treatment. These data indicated that EBNA2 could induce DNA damage response and genomic instability. To further investigate the correlation between EBNA2-induced DNA damage response and micronucleus, we found that EBNA2 could repress BRCA1 expression. Using a functional DNA repair assay, we demonstrated that homologous recombination repair (HRR) was repressed by EBNA2. Besides, the repair kinetics of camptothecin-induced DNA damage was delayed in EBNA2-expressing cells. This might account for EBNA2’s capacity in inducing DNA damage and genomic instability. Next, we examined the EBNA2 domain responding to HRR suppression. The data showed that an uncharacterized region between amino acid 200 and 285 was important for EBNA2-mediated repression of HRR. Finally, we found that centrosome number was increased in EBNA2-expressing cells, suggesting that genomic instability was common in these cells.
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