MOLECULAR REGULATION OF THE GUT EPITHELIAL CELL CYCLE

肠道上皮细胞周期的分子调控

• 批准号: 2545361
• 负责人: TIEN C KO
• 金额: $ 9.03万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1999-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2545361
• 关键词: animal genetic material tag; antisense nucleic acid; athymic mouse; carcinogenesis; carcinogenesis inhibitor; cell growth regulation; colon neoplasms; cyclins; fusion gene; gastrointestinal epithelium; gastrointestinal pharmacology; gene induction /repression; genetic promoter element; human genetic material tag; laboratory rat; molecular cloning; neoplasm /cancer genetics; oligonucleotides; reporter genes; tissue /cell culture; transfection; transforming growth factors

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 is 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.

该K08奖将为Tien C提供必要的财政支持。 医学博士KO，获得E. Aubrey实验室的科学培训 汤普森博士在人类生物学系和 遗传学。该建议的长期目标是使Ko博士能够 成为独立研究者，解决与 外科肿瘤学家。在此提案的五年中，Ko博士将 获得质粒合成的知识和技术技能，细胞 转染和启动子克隆。 这个的具体目的 提案是更好地了解控制的分子机制 正常和恶性肠道上皮细胞增殖。结肠癌是 男女癌症死亡的第三大主要原因 美国正常生长调节机制的破坏发生在 致癌作用。该提案得出的新信息将有助于 制定与结肠癌作斗争的新型策略。我们将具体 专注于转化生长因子β（TGF-β）对 肠细胞增殖。 TGF-beta已被提出为负 正常肠细胞增殖和抑制剂的调节剂 恶性转化。大量研究表明TGF-beta块 中间G1中的细胞周期进展。根据我们的初步数据，我们 提出三个新的假设：i） 肠上皮细胞上的TGF-β是由细胞周期蛋白抑制引起的 D1（CCND1）表达式，2）CCND1的过表达具有重要 在肠道癌中的作用和3）TGF-β抑制 CCND 1基因的转录。为了检验这些假设，我们有三个 具体目标。第一个具体目的是确定是否反义 CCND1的敲除抑制肠上皮细胞增殖。 培养的肠上皮细胞中的细胞周期蛋白D1敲除将是 通过两种不同的反义方法完成；管理 反义寡核苷酸和诱导型反义转染 质粒。第二个具体目的是确定是否过表达 CCND1发生在肠道癌发生期间。首先，人类结肠 癌细胞系和RAS转染的正常肠细胞系将 对CCND1的过表达进行检查。接下来，Cyclin D1将是 通过用质粒稳定转染在IEC-6细胞中过表达 在控制巨细胞病毒（CMV）的控制下包含有义务的CCND1 发起人。恶性转变和对TGF-beta的反应能力将 在转染的细胞中进行检查。第三目的是确定是否 TGF-β抑制CCND I启动子活性。 CCND 1启动子将 用5'-RACE PCR技术产生的探针克隆。发起人会 与报告基因链接并在瞬态中的IEC-6细胞中转染 测定。 TGF-β对报告基因表达的影响将是 检查。在接下来的五年中，我希望获得知识和 分子生物学技术技能，这将使我能够研究 肠细胞周期的调节。我的长期目标是成为一个 大学环境中的独立调查员。我希望解决 与外科肿瘤学家有关的问题既是临床医生又是基本的问题 科学家。