Carcinogenesis Advance Access originally published online on January 6, 2009
Carcinogenesis 2009 30(2):366-375; doi:10.1093/carcin/bgn291
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Epstein-Barr virus nuclear antigen 2 disrupts mitotic checkpoint and causes chromosomal instability
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
1 National Health Research Institutes, Institute of Cancer Research, Miaoli 350, Taiwan
2 Department of Internal Medicine
3 Department of Pathology, National Taiwan University Hospital, Taipei 100, Taiwan
4 Present address: Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
* To whom correspondence should be addressed. Tel: +886 2 23123456 ext. 88285; Fax: +886 2 23915293; Email: wbwang{at}ntu.edu.tw
The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in transformation of B-lymphocytes mediated by Epstein-Barr virus (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 compromise mitotic spindle checkpoint (MSC) induced by the spindle inhibitor nocodazole and cause chromosomal instability (CIN) in HEp-2, U2-OS and BJAB cells. When EBNA2-expressing cells were treated with nocodazole, they exited mitosis prematurely and initiated another round of DNA synthesis. Nucleolocalization of EBNA2 was essential for EBNA2 to compromise MSC and to cause CIN. The metaphase chromosome spread data indicated that the EBNA2-expressing U2-OS cells showed a more heterogenous chromosome number distribution than the vector-transfected and parental cells. The median chromosome number for EBNA2-expressing, vector-transfected and parental U2-OS cells is 75, 65 and 64, respectively. EBNA2 was shown to be able to downregulate mitotic arrest deficient 2 (MAD2) 
2- to 3-fold and upregulate polo-like kinase 1 (PLK1) 2-fold. The dysregulation of MAD2 and PLK1 may lead to activation of anaphase promoting complex/ cyclosome and premature degradation of securin. Indeed, we found that when MSC was induced by nocodazole, securin was prematurely degraded in EBNA2-expressing cells. Finally, we show that EBNA2 could induce micronuclei and multinuclei formation in HEp-2 and U2-OS cells. Together, these studies reveal a new function of EBNA2 in cell-cycle regulation and may shed light on the role of EBNA2 in EBV-mediated tumorigenesis.
Abbreviations: APC/C, anaphase promoting complex/ cyclosome; BrdU, bromodeoxyuridine; CIN, chromosomal instability; EBV, Epstein-Barr virus; EBNA2, Epstein-Barr virus nuclear antigen 2; ER, estrogen receptor; FACS, fluorescence-activated cell sorting; MAD2, mitotic arrest deficient 2; MSC, mitotic spindle checkpoint; 4-OHT, 4-hydroxytamoxifen; PLK1, polo-like kinase 1
Received June 1, 2008; revised December 5, 2008; accepted December 20, 2008.