Accordingly, the overall goal of the current proposal involves elucidation of mechanisms underlying signaling of specific TGFb responses in intestinal epithelial cells (IEC's) and human colon carcinoma cells (HCCC's). A clearer understanding of the signaling components regulated by TGFb will ultimately facilitate efforts to identify deficiencies in such cellular components associated with colon cancer progress. In this proposal, the hypothesis that the major classes of TGFb responses in IEC's and HCCC's are dependent upon the activities of the signaling components Ras and Erk will be tested. More specifically, inducible expression of dominant-negative mutant versions of Ras and Erk2 will be employed as tools to assess the role of these signaling components in mediating specific TGFb responses in IEC's and HCCC's, i.e., growth inhibition, repression of the activity of the cell cycle component cyclin-dependent kinase 2 (Cdk2), up-regulation of the Cdk inhibitor p21Cip1, production of the extracellular matrix protein fibronectin and its receptors (integrins), induction of the colon cell differentiation marker carcinoembryonic antigen, and modulation of gene expression. Additional studies involving transfection of autocrine TGFb-positive and negative HCCC's with dominant-negative Erk2 will be used to test the hypothesis that the TGF -mediated decreases in anchorage-independent growth and tumorigenicity of the autocrine TGFb-positive HCCC's are dependent upon the function of Erk2. Finally, the investigators will examine whether alterations exit in TGF regulation of mitogen- activated protein kinases (MAPK's) in TGFb-resistant HCCC's with intact TGFb receptors.
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