Expression of E6/E7 Transforming Genes is not Altered During Stratification and Differentiation of Two HPV -18 Transformed Cervical Carcinoma Cell Lines in an Organotypic Culture System
Noud, Patrick H.
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Human papillomaviruses (HPV) are important pathogens that cause papillomas: skin tumors that are often benign, but are occasionally malignant and life threatening. The study of HPV has been slow to date because culture systems that support HPV replication have been lacking. HPV replication is differentiation-dependent, and requires a culture system that approximates epithelial differentiation in vivo. Recently, organotypic raft systems have been described that support HPV replication. The focus of this study was to organotypically culture epithelial cells and HPV-positive cell lines, and achieve levels of differentiation adequate to support replication of HPV. This study also assessed the effects of differentiation on HPV-gene expression in two HPV -positive cell lines. To characterize the organotypic raft culture system, differentiation-specific markers were identified and monitored by SDS-P AGE and immunoblotting. In addition, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) techniques were used to follow HPV gene expression in the two HPV-positive cell lines as a function of the differentiation state of the cells. The differentiation that I observed with the organotypic raft system suggests that it will be suitable for the study of HPV in vitro. RT-PCR and immunoblotting proved to be useful tools for characterizing cellular differentiation and expression of HPV genes in this culture system. Transfection of HPV genomes into keratinocytes prepared for growth in organotypic rafts will provide an ultimate measure of the system's ability to support HPV replication.
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