Regulation of the Stages of Carcinogenesis
Current general research interests involve in vitro and in vivo study of the transition of normal human genital epithelial cells to the maligant state. For this purpose a human carcinogenesis model utilizing recombinant human papillomavirus DNA and a serum free medium developed by the laboratory are being utilized. This model facilitates the study of cell growth, gene expression, and cytogenetic alterations in normal and human papillomavirus (HPV)-immortalized cervical epithelial cells and provides for analysis of the mechanisms of modulation of cellular and viral gene transcription by cytokines and hormones. Retroviral vectors have been constructed for investigating the role of specific HPV oncoproteins and tumor suppressor genes in epithelial cell growth and differentiation. The emphasis is on HPV-16 and HPV-18 because of their association with the majority of cervical carcinomas. The HPVs are transcriptionally complex and encode a series of transcriptional regulatory factors. Because HPVs contribute to the carcinogenic process by uncoupling the process of cell growth and differentiation, it becomes important to identify the stages at which virus containing cells lose the ability to undergo squamous differentiation. The objective is to obtain information critical to planning interventive measures to cervical cancer by identifying factors that modulate in vitro transformation processes which lead to malignancy of human cells.
The primary projects involve the development of terminal cellular differentiation and the study of potential carcinogens. Because of the importance of HPV E6 and E7 gene products, current studies are examining how differentiation is capable of downregulating products of these two genes. The current approach involves the use of catalytic RNAs, known as ribozymes, to destroy the products of these genes and thus induce terminal diffeerentiation. The region being analyzed is also associated with p53. In collaboration with Dr. A. Hampel of Northern Illinois University two hairpin motifs have been constructed to interrupt E6/7 mRNA. A patent application is pending for the hairpin ribozyme.
It is well established that HPV containing cells usually escape recognition by cell mediated immunity . The HPV E5 gene is important because it may prevent immunologic recognition of HPV positive cells by downregulating E6/7 antigen epitope presentation. To examine the role of E5 in this context a panel of E5 mutants have been constructed in regions conserved in different HPVs. These E5 mutants are being transfected into epithelial cells previously immortalized by HPV-16 E6/7. Transfection with wild type E5 causes inhibition of MHC-1 cell surface expression. FACS analysis of MHC-1 antibody binding capacity indiciates that a mutant E5 causes less inhibtion of MHC-1 expression than does wild type E5. E5 mutants thus provide the opportunity to identify the gene segment responsible for downregulating E6/7 antigen presentation.
Terminal diffentiation of keratinocytes results in formation of cornified envelops and production of involucrin. The regulation of human involucrin gene transcription is controlled by a 2456 nucleotide 5' noncoding region which is active in multiplying keratinocytes and is enhanced by calcium. Calcium responsiveness is associated with the distal portion of the non-coding region and several cellular factors. The absence of a model for studying HPV late gene expression necessitates an alternate model be used. Because cellular genes and HPV late genes are activated during terminal differntiation the current studies with involucrin represent a model for understanding regulation of HPV late gene expression by specific transcription factors.
Our cervical carcinogenesis model has also proved useful for the study of co-carcinogens. HPV-16 immortalized genital cells are responsive to the genotoxic action of known chemical carcinogenes (polycyclic hydrocarbons, alkylating agents or cigarette smoke condensate), but differ from rodent cells such as hamster cells in that they are not converted to malignancy. Ras oncogene and human herpes virus-2 convert HPV immortalized cells to malignancy whereas human herpes virus-6 infection only increases HPV expression. Human immunodeficieincy virus is unable to infect genital cells. Investigations are underway to identify the gene segments contributing to the co-carcinogenesis.