This K08 award will provide the financial support necessary for Tien C. Ko, M.D. to obtain scientific training in the laboratory of E. Aubrey Thompson, Ph.D. in the Department of Human Biological Chemistry and Genetics. The long-term goal of this proposal is to enable Dr. Ko to become an independent investigator, addressing problems relevant to surgical oncologists. During five years of this proposal, Dr. Ko will acquire knowledge and technical skills in plasmid synthesis, cell transfections and promotor cloning. The specific purpose of this proposal is to better understand the molecular mechanisms that govern normal and malignant gut epithelial cell proliferation. Colon cancer is the third leading cause of cancer death in both women and men in the U.S.A. Disruption of normal growth regulatory mechanisms occurs in carcinogenesis. The new information derived from this proposal will help develop novel strategies to combat colon cancer. We will specifically focus on the effect of transforming growth factor beta (TGF-beta) on intestinal cell proliferation. TGF-beta has been proposed as a negative regulator of normal intestinal cell proliferation and a suppressor of malignant transformation. Numerous studies suggest that TGF-beta blocks cell cycle progression in middle G1. Based on our preliminary data, we propose three novel hypothesis: I) that the antiproliferative effects of TGF-beta on intestinal epithelial cells results from inhibition of cyclin D1 (CcnD1) expression, 2) that overexpression of CcnD1 has an important role in intestinal carcinogenesis and 3) that TGF-beta inhibits transcription of the CcnD 1 gene. To test these hypothesis, we have three specific aims. The first specific aim is to determine whether antisense knockout of CcnD1 inhibits intestinal epithelial cell proliferation. Cyclin D1 knockout in cultured intestinal epithelial cells will be accomplished by two different antisense methods; administration of antisense oligonucleotide and transfection with an inducible antisense plasmid. The second specific aim is to determine whether overexpression of CcnD1 occurs during intestinal carcinogenesis. first, human colon carcinoma cell lines and Ras-transfected normal intestinal cell line will be examined for overexpression of CcnD1. Next, cyclin D1 will be overexpressed in IEC-6 cells by stable transfection with plasmid containing sense CcnD1 under control of the cytomegalovirus (CMV) promoter. Malignant transformation and responsiveness to TGF-beta will be examined in transfected cells.
The third aim i s to determine whether TGF-beta inhibits CcnD I promoter activity. CcnD 1 promoter will be cloned with a probe generated by 5'-RACE PCR technique. The promoter will be linked to a reporter gene and transfected in IEC-6 cells in transient assays. The effects of TGF-beta on reporter gene expression will be examined. During the next five years, l hope to acquire knowledge and technical skills in molecular biology which will enable me to study the regulation of intestinal cell cycle. My long-term goal is to become an independent investigator in a university environment. l hope to address problems relevant to surgical oncologists both as a clinician and a basic scientist.
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