Regulation of intestinal epithelial cell proliferation impacts on multiple problems relevant to surgical oncologists. The stress of major surgical procedures, nutritional deprivation and systemic inflammation that occur in patients with cancer all lead to atrophic changes in gut epithelium. Disruption of normal growth regulatory mechanisms occurs in carcinogenesis. Colon cancer is the second leading cause of cancer death in the U.S.A. The major long-term goal of this project is to better understand the intracellular mechanisms that govern normal and malignant gut epithelial cell proliferation. Transforming growth factor-Beta (TGF- Beta) has been proposed as a negative regulator of normal intestinal epithelial cell growth and numerous studies suggest that TGF-Beta blocks cell cycle progression in middle G1. The central novel hypothesis for this proposal is 1) that TGF-Beta inhibition of intestinal cell proliferation results from inhibition of cyclin D1. Secondary hypotheses based upon preliminary data are: 2) that aberrant regulation of D cyclins has an important role in intestinal carcinogenesis and 3) TGF- Beta inhibits transcription of cyclin D1. Each of the two components of this IRPG contains five specific aims designed to test predictions that emanate from the hypotheses. The proposal in hand will address hypotheses 1 and 2.
The specific aims for this proposal are: 1) to determine whether knockout of cyclin D1 with antisense or antibody microinjection blocks intestinal cell growth. Cyclin D knockout in cultured intestinal epithelial cells will be accomplished by two different methods; administration of antisense oligodeoxynucleotides and by microinjection of D cyclin antibodies or antisense plasmid to determine whether inhibition of the cyclin D1 (as well as D2, D3 and cdk4) is sufficient to inhibit cell proliferation. 2) To determine whether expression of D cyclins or cdk4 under the control of a heterologous promoter results in resistance to the growth-inhibitory effects of TGF-Beta. Both constitutive and inducible overexpression of D cyclins or cdk4 in intestinal epithelial cells will be achieved by stable transfections. TGF-Beta responsiveness and tumorigenicity will be determined in the transfectants. 3) To characterize the regulation of D cyclins and their associated kinases by TGF-Beta in intestinal epithelial cells. The effects of TGF-Beta on association of the D-type cyclins with their kinases and the CcnD/cdk4 kinase activities will be determined. Post-translational tyrosine phosphorylation changes of D cyclins in response to TGF=-Beta will be determined. 4) To determine whether inhibiting cyclin D1 expression with TGF-Beta or with antisense causes similar abrogation of late G1 events. pRb phosphorylation nd cyclin E-dependent histone H1 kinase activities will be analyzed. 5) To determine whether aberrant regulation of cyclin D expression occurs in intestinal carcinogenesis. Cyclin D1 constitutive overexpression in IEC-6 cells will be assessed for the ability to cause TGF-Beta resistance and transformation. Correlation between malignant transformation and disruption of cyclin D regulation by TGF-Beta will be determined in vivo and in vitro. Dr. E. A. Thompson's companion IRPG proposal will test the third hypothesis. Our long-term objective is to better understand how the intestinal cell cycle is regulated and whether aberrant regulation accounts for early tumor progression.
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