Parker G A, Touitou R, Allday M J
Section of Virology and Cell Biology, Imperial College of Science, Technology and Medicine, London, UK.
Oncogene. 2000 Feb 3;19(5):700-9. doi: 10.1038/sj.onc.1203327.
Expression of EBNA3C is essential for the immortalization of B cells by EBV in vitro and, in co-operation with activated ras, EBNA3C has oncogenic activity in primary rodent fibroblasts. This suggested that this viral oncoprotein might disrupt the cyclin/CDK-pRb-E2F pathway, which regulates cell cycle progression at the restriction point (R-point) in G1 of the proliferation cycle. An assay was established in which transfected EBNA3C-positive cells could be sorted and simultaneously analysed for their distribution in the cell cycle. This revealed that in NIH3T3 fibroblasts compelled to arrest by serum-withdrawal, EBNA3C induces nuclear division that is often divorced from cytokinesis and so produces bi- and multinucleated cells. This was confirmed using the ecdysone-inducible system for expression of EBNA3C in human U2OS cells and by microinjection of expression vectors into NIH3T3 and U2OS. Further analysis revealed that in the inducible system, EBNA3C expression inhibits the accumulation of p27(K1P1) but not the dephosphorylation of pRb. Experiments using the microtubule destabilizing drug nocodazole, showed that EBNA3C could abrogate the mitotic spindle checkpoint.
EBNA3C的表达对于EB病毒在体外使B细胞永生化至关重要，并且与激活的ras协同作用时，EBNA3C在原代啮齿动物成纤维细胞中具有致癌活性。这表明这种病毒癌蛋白可能会破坏细胞周期蛋白/细胞周期蛋白依赖性激酶-pRb-E2F途径，该途径在增殖周期G1期的限制点（R点）调节细胞周期进程。建立了一种检测方法，通过该方法可以对转染的EBNA3C阳性细胞进行分选，并同时分析它们在细胞周期中的分布。结果显示，在因血清饥饿而被迫停滞的NIH3T3成纤维细胞中，EBNA3C诱导核分裂，而这种核分裂通常与胞质分裂分离，从而产生双核和多核细胞。使用蜕皮激素诱导系统在人U2OS细胞中表达EBNA3C以及将表达载体显微注射到NIH3T3和U2OS细胞中，证实了这一结果。进一步分析表明，在诱导系统中，EBNA3C的表达抑制p27（K1P1）的积累，但不抑制pRb的去磷酸化。使用微管破坏药物诺考达唑进行的实验表明，EBNA3C可以消除有丝分裂纺锤体检查点。
