EB病毒核抗原第二型對細胞週期與染色體穩定性的影響

Abstract

Epstein-Barr 病毒 (EB病毒, EBV) 第二型核抗原 (EBNA2) 在EBV造成的B細胞轉形扮演關鍵的角色，同時在轉殖鼠中亦可以誘發腫瘤的形成。然而，EBNA2造成癌化的確切機制仍不清楚。這裡我們報告了EBNA2可以透過干擾CDK1-Cdc25C的途徑而減緩細胞週期在G2時期的進行。擾亂細胞週期的進行會造成染色體的不穩定性，進而造成癌化，是為大家所熟知的。我們也發現EBNA2可以在HEp-2、U2-OS、BJAB等細胞中干擾有絲分裂紡錘絲檢查點 (MSC)，並且造成多套體。因為多套體是造成非整倍體的原因，因此我們想知道EBNA2是否能造成非整倍體。的確，利用karyptyping的技術，我們發現EBNA2表現的細胞非整倍體的數目會大量增加。利用BrdU嵌合分析，我們發現EBNA2表現的細胞在nocodazole存在下不會停在metaphase，反而會離開有絲分裂而開始另一次的DNA複製。EBNA2會減少Mad2並增加Plk1的表現，而Mad2、Plk1不正常調控會造成APC/C的活化以及securin提早被降解。的確，我們發現在EBNA2表現的細胞中，當nocodazole誘發MSC時，securin會提早被降解。我們也發現了EBNA2在HEp-2與U2-OS細胞中會誘導微核以及多核的形成。最後我們也證明了EBNA2誘發的多套體是由於DNA的內複製以及細胞質分裂失敗所造成的。綜合以上，這些研究發現了EBNA2在調控細胞週期及染色體穩定上的新功能，並且提供了EBNA2在EBV造成的癌化過程扮演新的角色。

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays a key role in transformation of B-lymphocytes mediated by EBV and can induce tumor formation in transgenic mice. However, the precise mechanism underlying EBNA2-mediated tumorigenesis remains elusive. Here we report that EBNA2 can retard cell cycle at G2/M phase by disrupting CDK1-Cdc25C pathway. It is well known that dys- regulating cell cycle causes chromosomal instability and results in turmerigenesis. We found that EBNA2 can compromise mitotic spindle checkpoint (MSC) and cause polyploidy in HEp-2, U2-OS and BJAB cells. Because polyploidy is thought the route to aneuploidy, we also tested whether EBNA2 can induce aneuploidy. Indeed, by using karyotyping, we found aneuploidy is increased in EBNA2-expressing cells. Furthermore, EBNA2-expressing cells initiated another round of DNA synthesis during nocodazole-induced metaphase arrest by using BrdU incorporation assay. EBNA2 was shown to be able to down-regulate Mad2 and up-regulate Plk1. The dysregulation of Mad2 and Plk1 may lead to activation of APC/C and premature degradation of Securin. Indeed, we found that when MSC was induced by nocodazole, Securin was prematurely degraded in EBNA2-expressing cells. We also showed that EBNA2 could induce micronuclei and multinuclei formation in HEp-2 and U2-OS cells. Finally, we demonstrated that EBNA2-induced polyploidy is due to both DNA endo-replication and cytokinesis failure. Together, these studies reveal a new function of EBNA2 in cell cycle regulation and chromosomal instability, and may shed light on the role of EBNA2 in EBV-mediated tumorigenesis.