| Budget Amount *help |
¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1999: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
Gene expression and genome amplification of human papillomavirus (HPV) are tightly regulated by the status of host cell differentiation. In basal layer cells, weak expression of viral early genes and low copy number of viral genome are detectable. In the course of keratinocyte differentiation, the levels of viral gene expression and DNA amplification are upregulated, and the viral particle production can be detected only in fully differentiated cells. It is necessary, therefore, to analyze the mechanisms of HPV gene function and replication in the system that reflects the keratinocyte differentiation program.
The organotypic culture of keratinocytes is developed to reconstitute the skin structure, in which HPV DNA can express viral genes and replicate in differentiation specific manner. We adapted an organotypic culture system developed by Dr.Kuroki et al. for HPV analysis. HPV18 genomic DNA was introduced into human primary keratinocytes by lipofection, and then the cells were used for
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organotypic culture. In the basal layer cells, HPV DNA was difficult to detect by in situ hybridization, although it was detectable by Southern blot analysis. The DNA amplification could be found in the differentiated cells, in which transglutaminase, one of the differentiation markers, was expressing. mRNA expression level was also enhanced through cell differentiation. The result indicated that the organotypic culture system was suitable for the analysis of differentiation specific HPV function.
The gene function of HPV was analyzed in the organotypic culture system. Two major viral oncoproteins, E6 and E7, were expressed in keratinocytes and the effects on cell proliferation and differentiation were analyzed. In normal condition, cell division was restricted only in the basal layer, but E6/E7 expressing cells could proliferated even in partially differentiated layer. However, differentiation program was maintained intact even with E6/E7 expression. We are now expanding the analysis for other viral genes and low-risk type E6/E7 genes.
We observed a linkage between gene expression and DNA replication in the organotypic culture. Both functions are regulated mainly by LCR of HPV genome, in which viral E2 protein is involved. We obtained evidence that the E2 protein functioned as a mediator between both activities. We are now investigating the involvement of E2 protein in differentiation specific HPV replication mechanism. Less
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