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

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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.