A significant amount of information has been collected utilizing various cell model systems infected with the oncogenic DNA tumor virus, Simian virus 40 (SV40). The cells, both established lines and cell strains, acquire the neoplastic phenotype after a finite period, and are able to produce tumors when inoculated into an appropriate animal. Primary cell strains, rodent of human, are inefficiently transformed requiring quantitative as well as qualitative methods to follow the emerging transformed population. With the availability of SV40 transformed and primary tumor lines and monoclonal antibodies to various epitopes of the SV40 large T antigen and host protein p53, it is feasible to characterize the relationship of the neoplastic phenotype to these oncogenes. In previous studies, flow cytometry has been used to analyse the events related to a permissive and nonpermissive infection with SV40 virus. Flow cytometry allows multiple parameter analysis on single cells or populations to obtain, 1) percentage of antigen positive cells, 2) average content of antigen per cell population, and, 3) the distribution of antigen per cell or population related to other parameters, such as DNA content.
The specific aims propose to further characterize by quantitative flow cytometry and other technologies the relationship of viral and host proteins to the permissive and transforming infection with SV40. 1) To quantitate per cell and population, expression of various SV40 T antigen epitopes in permissive CV-1 cells, and nonpermissive, human diploid fibroblasts (IMR-90), mouse embryo cells, and Chinese hamster embryo cells. 2) To correlate cellular DNA content (cell cycle) and quantity of p53 and T antigen to transformation with wild type and tsA SV40 virus. 3) to characterize and correlate the quantity of cell surface T antigen with the establishment of transformation. These studies would provide definitive evidence regarding the relationship to the quantity and presence of this antigen and its interaction with the cellular protein, p53. The flow cytometry studies will be correlated with other techniques to characterize the emerging transformed population. Other techniques employed will be immunoprecipitation, DNA content changes, and phenotypic expression of transformation. The data will help elucidate the functional involvement of T antigen and p53 to the emergence of the transformed population. This analysis should provide insight into the cellular and viral events necessary for cell transformation with SV40 virus.
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