This proposal is designed to fill a gap in our knowledge of the negative regulation of proliferation in cells of epithelial origin, the cell type in which the majority of human tumors occur. The MK mouse keratinocyte cell line provides an excellent model for these studies, as the cells are relatively easily obtained, possess many characteristics of normal keratinocytes, and both the positive and negative growth factor regulation has been characterized. Since it has been speculated that a loss of normal inhibitory signals may play a role in the expression of a malignant phenotype in epithelial cells, the proposed studies will concentrate on the actions of the antiproliferative growth factor, transforming growth factor type beta (TGF beta). The available evidence suggests that TGF Beta may exert its inhibitory effect by modulating levels of key RNA species that control cell proliferation. The molecular basis of TGF beta-mediated inhibition of growth will therefore be approached using hybridization-subtraction or differential screening of cDNA libraries from TGF Beta treated and untreated cells, methods that have been previously used to identify a number of potentially interesting genes involved in cellular proliferation, transformation, and differentiation. The experiments will be designed to identify genes that are induced by TGF Beta that may inhibit cellular growth (TGF Beta-inducible genes, TIGs), as well as genes required for proliferation that are repressed by TGF Beta (TGF Beta-repressible genes, or TRGs). Cell transfection experiments will be performed to differentiate between genes that play a direct, causal role in regulating cell proliferation from those altered as a consequence of the growth state. These experiments will identify genes that are necessary, and perhaps sufficient, for growth factor regulation of MK cell proliferation. Characterization of TGF Beta-modulated genes will provide a better understanding of the molecular regulation of epithelial cell growth and may ultimately aid in devising rational approaches to preventing or managing the uncontrolled growth characteristic of malignant neoplasia.
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