The focus is on """"""""high risk"""""""" types because they are commonly found with lesions associated with malignant progression. This project focuses on understanding the cellular factors that contribute to the HPVs role in progression to cancer and the induction of terminal differentiation of HPV containing cells so that the cancer cells can be eliminated. The """"""""high risk"""""""" HPV contains two genes, E6 and E7, which have transforming properties and are also the hallmark of cervical cancers. Thus, it becomes obvious that the preferred treatment for papillomavirus containing cervical carcinom. The sum of experimental, clinical, and epidemiological data associates specific human papillomavirus (HPV) types with human anogenital cancer, most notably cervical carcinoma, as includes abolishing the E6 and E7 gene products. Presence of the E6 protein increases immortalization frequency as reflected by the number of immortalized colonies in vitro and is known to interact and destabilize the p53 protein leading to the degradation of p53 through the ubiquitin pathway. Furthermore, interference with E6 because it is controlled by the same promoter as E7 may have an effect on both genes. Analysis of HPV-16-positive cervical carcinomas indicates that the E6 is a stable gene. Thus, the 3(prime) region which also associates with p53 presents an excellent target for disruption of the mRNA of both E6 and E7. Two hairpin ribozymes (patent pending) have been constructed and are directed against the 3(prime) end of the HPV-16 E6. These two ribozymes have been characterized biochemically and found capable of cutting the E6 mRNA in vitro. Although both ribozymes are under study, most of the work thus far has focused on one which is referred to as R434. R434 effects cell morphology compared to the vector transfected cells. RNAse protection assays demonstrate that the ribozyme is effectively expressed from the tRNA valine promoter but poorly expressed from a mouse promoter construct. Having demonstrated that the ribozyme does exist in the cell, additional experiments are underway to increase the efficiency of destroying the E6/E7 message. Because cellular response to DNA damage is influenced by the expression of the p53 gene, radiation response of human cervical carcinoma cells having or lacking HPV and with high or low amounts of p53 were examined for their radiation responsiveness to sense and antisense p53 retroviral constructs. The results show that alterations in endogenous levels of p53 by retroviral vectors with the wild type, full-length p53 cDNA in the sense configuration did not result in consistent differences in the set of responses to ionizing radiation. Thus, these data indicate that the level of wild type p53 is not a major participant in the mechanism response to ionizing radiation by these specific cervical carcinoma cell lines.
