HUMAN PAPILLOMAVIRUS GENE EXPRESSION IN CERVICAL CANCER

This project will test the hypothesis that mapping the viral RNA and characterizing the early viral proteins of human papillomavirus (HPV) will improve our understanding of how these viruses alter the normal keratinocyte and how this may relate to carcinogenesis of the cervix. HPV gene expression will be studied in fresh tumor and wart tissues, in tumor cell lines, and in vector systems designed to express viral genes after transfection into cultured cells. It is proposed that: 1. HPV messenger RNA will be mapped and quantitated in benign warts and invasive tumors. Comparisons will be made between different tumors and between benign and malignant lesions so as to obtain insight into which genes may be important in carcinogenesis and to try to explain why some viral types such as HPV 16 and 18 are commonly associated with malignancy while other types such as HPV6 and 11 are almost exclusively associated with benign lesions. 2. The early proteins of HPV will be identified and characterized with special emphasis on the E6 and E7 proteins, the major viral proteins in cervical cancer. Antibodies will be made against the E6, E7, E2, and E4 proteins of various types of HPV. The major messenger RNA in benign genital lesions appears to code for E4 and therefore E4 may be present in large enough amounts to be detectable by immunoperoxidase methods, and thus may be useful for clinical diagnosis. 3. When HPV DNA is introduced into cultured cells it is not replicated in a stable fashion, nor are the viral genes expressed in detectable amounts. In order to overcome this block in expression two approaches will be taken. First, vectors will be made whereby the early viral genes will be placed under the control of exogenous strong or inducible promoters. Expression of viral proteins and RNA will be looked for in transient transfection conditions and after permanent transformation of cells. HPV DNA has been shown to immortalize primary keratinocytes, a phenotypic change that may relate to carcinogenesis. These vectors will be used to develop a system for analysing what genes are important in the immortalizion process. The second approach will attempt to enhance viral gene expression by cotransfecting the HPV genome along with a vector expressing the viral E2 gene behind a strong exogenous promoter. E2 is a transcriptional trans-activator and supplying large amounts in trans may overcome the block in vitro viral gene expression and may even allow viral DNA replication.