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Analysis of HPV regulatory gene function with organotypic culture of epithelial cells.

通过上皮细胞器官型培养分析 HPV 调节基因功能。

基本信息

批准号：
13214048
负责人：
SAKAI Hiroyuki
金额：
$ 21.95万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

There are many cancers associated with the infection of human papillomavirus (HPV), among which cervical cancer is considered the most important one. To prevent the HPV-induced cancer development, it is essential to understand the replication cycle of HPV on the molecular basis. However, the analysis of HPV life-cycle has been hampered by a fact that the HPV replication strongly depends on the differentiation program of epithelial cells. We developed an HPV replication system with organotypic culture of keratinocyte (raft culture) and a novel HPV replicon, which enable us to analyze the HPV replication and the HPV-induced hyperplasia in vitro.With this system, we could obtain following results: (1) the efficient replication system of HPV could be adapted to the screening of anti-virus drugs, (2) activation of Ha-ras pathway conferred a malignant cell phenotype on the HPV-infected cells, (3) high-risk type E7 was responsible for the hyperplasia induction by HPV, in which the inactivation of both pRb and pl30 and the activation of JNK pathway might be involved, (4) continuous expression of E7 in the HPV-positive cervical cancer cell was essential for the cell viability and for the prevention of senescence induction, (5) E4 gene product could induce G2/M arrest in cell division cycle without preventing cellular DNA synthesis, suggesting the involvement of E4 in the viral DNA amplification in the differentiated layer of epidermis.

许多癌症都与人乳头瘤病毒（HPV）感染有关，其中宫颈癌被认为是最重要的一种。为了预防 HPV 诱发的癌症发展，有必要从分子基础上了解 HPV 的复制周期。然而，HPV 复制强烈依赖于上皮细胞的分化程序，这一事实阻碍了 HPV 生命周期的分析。我们开发了一种具有角质形成细胞器官型培养（筏培养）的HPV复制系统和一种新型HPV复制子，使我们能够在体外分析HPV复制和HPV诱导的增殖。通过该系统，我们可以获得以下结果：（1）HPV的高效复制系统可以适应抗病毒药物的筛选，（2）激活 Ha-ras途径赋予HPV感染细胞恶性细胞表型，(3)高危型E7负责HPV诱导增生，其中pRb和pl30的失活以及JNK途径的激活可能参与其中，(4)E7在HPV阳性宫颈癌细胞中的持续表达对于细胞活力和预防衰老至关重要 (5)E4基因产物可以诱导细胞分裂周期G2/M期阻滞而不阻止细胞DNA合成，表明E4参与了表皮分化层中病毒DNA的扩增。