The long-range goal of this project is to understand epitheial cell transformation, currently a poorly comprehended area but a highly significant one since the majority of human neoplasias arise from cells of epithelial origin. The basic approach combines the inherent flexibility and potential of the murine mammary gland system with the advantagess offered by the well-characterized transforming agent, SV40. We have established that SV40 can reproducibly transform mouse mammary epithelial cells. The roles of SV40 gene functions in determination of transformed mammary epithelial cell phenotypes will be delineated. Dedifferentiated cells on plastic and differentiated cells on floating collagen membranes will be transformed by SV40 mutants affected in T(t)-antigens. Transformants recovered will be inspected for epithelial cell markers and phenotypic properties, including growth in low concentrations of serum or calcium, anchorage-independent growth, response to exogenous growth factors, distribution of cytoskeleton components, expression of SV40 and MMTV antigens, and transplantability in vivo. Correlations between in vitro markers and tumorigennicity will be sought. Interactions between SV40 early proteins and epithelial cell proteins under various growth conditions will be analyzed, with a major emphasis being focused on the host-coded 53K non-viral T-antigen. The effect of SV40 gene expression on differentiation of mammary epithelial cells will be determined. The response of mutant SV40 transformants will be compared to the ability of normal mammary cells cultured on floating gels in the presence of appropriate hormones to undergo differentiation culminating in casein synthesis. Potential cocarcinogenic effects of MMTV expression on transformation of mammary epithelial cells by SV40 will be studied using preneoplastic HAN cells which express MMTV antigens as target cells. The expression and interactions among SV40, MMTV, and 53K proteins will be monitored in mammary cell transformants and correlated with cell phenotypes. Finally, attempts will be made to develop a biological assay for MMTV using cultured mammary epithelial cells. The outgrowth patterns and growth requirements of virus-exposed cells embedded in collagen gels will be scrutinized for alternations in behavior as one potential assay system. this project will provide insights into the cell biology of breast cancer and epthelial cell transformation in general.
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