Induction of G2 Retardation and Polyploidy by Epstein-Barr Virus EBNA2 Protein in Human Epithelial Cells

Epstein-Barr Virus (EBV) 是一種廣泛存在於人類的疱疹病毒(herpes virus)，與許多人類惡性腫瘤有關，如伯奇氏淋巴瘤 (Burkett’s lymphoma) 及鼻咽癌 (nasopharyngeal carcinoma) 等。前人的結果顯示，EBV有八個潛伏蛋白質 (latent protein) 與B細胞的轉形癌化有關，其中之一即是EBNA2，EBNA2是一個轉錄活化子，可以在轉殖鼠中，誘發腫瘤形成。雖然先前的研究顯示EBNA2轉錄活化的功能與其引起細胞轉形癌化的功能有關，但是EBNA2如何引起腫瘤的相關機制目前還不清楚。 過去我們實驗室曾報導，EBNA2可能會藉著活化p53刺激p21的表現，進而讓上皮細胞生長停滯。為了更進一步了解EBNA2對細胞增殖的影響，我們利用流式細胞儀來分析EBNA2會使細胞停滯在哪一個時期，結果發現EBNA2會使細胞週期停滯在G2/M phase。為了更精確的知道EBNA2是影響細胞週期的G2 phase或M phase，藉著分析M phase的標記蛋白質phospho-Histone H3後，我們確定EBNA2會讓細胞停滯在G2 phase。這個結論也由分析Cdk1活化的時間點得到證明。 之前的文獻中指出，當細胞生長停滯在G2 phase時，常常會伴隨著多套體及多核的產生。由於EBNA2會讓細胞週期停滯在G2 phase，因此我們偵測EBNA2是否會讓細胞形成多套體。我們利用流式細胞儀分析發現，EBNA2的確能引起多套體的產生。為了解EBNA2引起多套體的機制，我們想知道EBNA2是否會干擾mitotic spindle checkpoint。藉著分析Histone-H3的磷酸化狀態以及mitotic index，我們發現EBNA2的確會干擾spindle checkpoint。EBNA2有可能藉由干擾mitotic spindle checkpoint讓染色體無法平均的分配到子細胞中。這些帶有不正常染色體的細胞進行再複製後，即會造成多套體。 綜合以上結果我們發現，EBNA2具有一個未被發現的新功能，就是它會引起細胞基因體的不穩定。我們相信EBNA2的這個功能，對於EBNA2引起腫瘤的形成是非常重要的。Epstein-Barr Virus (EBV) is a herpesvirus which exists commonly in the human population and has been shown to be associated with many human malignancies, notably Burkett’s lymphoma and nasopharyngeal carcinoma. Eight latent proteins of EBV have been shown to be critical for EBV transformation of B-cells. Among them, EBV nuclear antigen 2 (EBNA2), a transcriptional activator, has been shown to be able to induce tumor in transgenic mice. Although the transactivation function of EBNA2 has been proposed to contribute to its transformation function, the mechanism underlying EBNA2 tumor-inducing function remains unclear. Previously we reported that EBNA2 can retard epithelial cell growth possibly through stimulating p53 activity and inducing p21 expression. To further investigate the effects of EBNA2 on cell proliferation, we used flow cytometric analysis to determine which phase of cell cycle was affected by EBNA2. We found that EBNA2 could retard cell cycle progression at G2/M phase. To more specifically pinpoint whether G2 or M phase was affected by EBNA2, we measured the phosphorylation status of histone H3 after cells were released from G1/S block. Our data indicated that EBNA2 specifically retarded cell cycle progression at G2 phase. This conclusion was also confirmed by the data that measured the phosphorylation status of Cdk1 after cells were released from G1/S block. Previous studies indicated that agents that induce cell cycle G2 arrest often cause cells to become polyploid and contain multinuclei. Since EBNA2 could induce G2 arrest, we also test whether EBNA2 could induce polyploidy. Indeed, we found that EBNA2 could induce polyploidy by flow cytometric analysis. To investigate the mechanism underlying EBNA2 induction of polyploidy, we asked whether EBNA2 could disrupt mitotic spindle checkpoint. By analyzing the phosphorylation status of histone H3 and mitotic index, we found that EBNA2 indeed could disrupt the mitotic spindle checkpoint. Through disrupting spindle checkpoint, EBNA2 may cause chromosomes to be unevenly divided into daughter cells. Polyploidy cells may then arise after endoreduplication of these unevenly divided chromosomes. Multinucleation is frequently accompanied with disruption of spindle checkpoint and polyploidy. We also found that many EBNA2-expressing cells contained multinuclei. Together, the above data reveal a novel function of EBNA2, that is, its ability to induce genome instability. We believe that this function of EBNA2 may be more important than other known functions of EBNA2 in inducing tumor formation.中文摘要……………………………………………………………… 1 英文摘要……………………………………………………………… 3 序言…………………………………………………………………… 5 研究目標………………………………………………………………17 材料與方法……………………………………………………………18 結果……………………………………………………………………30 討論……………………………………………………………………41 附圖……………………………………………………………………48 參考資料………………………………………………………………7