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

与人乳头瘤病毒（HPV）感染有关的癌症有很多，其中宫颈癌被认为是最重要的一种。为了预防HPV诱导的癌症发展，从分子基础上了解HPV的复制周期是至关重要的。然而，HPV的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。我们开发了一种角质细胞器官型培养（筏培养）和新型HPV复制子的HPV复制系统，使我们能够在体外分析HPV复制和HPV诱导的增生。使用该系统，我们可以得到以下结果：(1) HPV的高效复制系统可以适应抗病毒药物的筛选；(2)Ha-ras通路的激活使HPV感染的细胞具有恶性细胞表型；(3)高危型E7负责HPV诱导增生，其中可能涉及pRb和pl30的失活以及JNK通路的激活。(4)在hpv阳性宫颈癌细胞中连续表达E7对细胞存活和防止衰老具有重要作用；(5)E4基因产物可诱导细胞分裂周期中G2/M阻滞，但不妨碍细胞DNA合成，提示E4参与了表皮分化层病毒DNA扩增。

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.