CYCLIN GENES AND PROLIFERATION OF GUT EPITHELIAL CELLS

细胞周期蛋白基因与肠道上皮细胞的增殖

• 批准号: 2712708
• 负责人: E. AUBREY THOMPSON
• 金额: $ 19.82万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-24 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2712708
• 关键词: animal genetic material tag; biological signal transduction; carcinogenesis; cell cycle; cell differentiation; cyclins; gastrointestinal epithelium; gene induction /repression; genetic promoter element; genetic transcription; neoplastic transformation; nuclear runoff assay; tissue /cell culture; transforming growth factors

The morbidity of colon cancer reflects the lack of an effective chemotherapeutic strategy for treatment of the disseminated form of the disease. Our long term objective is to devise strategies that permit intervention at the level of the primary regulatory defect that leads to malignant transformation. Our strategy is based upon four observations concerning growth factor regulation of epithelial cell proliferation. l) TGF-beta is a major effector of gut epithelial cell differentiation. 2) Undifferentiated colon adenocarcinoma is unresponsive to TGF-beta, as are intestinal epithelial cells that have been transformed with activated Ras genes. 3) Growth factors, including TGF-beta, regulate the expression of D cyclins, which are important in maintaining the proliferative state. 4) D cylcin genes are oncogenic in many different types of adenocarcinoma. Our immediate objective is to elucidate the role of TGF-beta in regulating gut epithelial cell proliferation and the role of D cyclins in that process. Our initial studies will focus upon IEC-6 intestinal epithelial cells, which, like normal gut epithelial cells, cease to proliferate in the presence of TGF-beta. Our data indicate that the gene encoding cyclin D1 (CcnD1) is the primary focus of TGF-beta inhibition of IEC-6 cells. TGF-beta inhibits transcription of CcnD1 resulting in G0 arrest. Antisense oligonucleotides directed against cyclin D1 mRNA can recapitulate the effects of TGF-beta. Inducible overexpession of cyclin D1 renders the cells resistant to TGF-beta. Two related hypotheses are derived from our initial observations. Hypothesis l: We propose that TGF-beta inhibition of cyclin D1 expression accounts for the normal cessation of proliferation that accompanies differentiation of the gut epithelium. This hypothesis will be tested by experiments described in the companion IRPG proposal, submitted by R.D. Beauchamp, M.D. Hypothesis 2: We propose that TGF-beta inhibition of epithelial cell proliferation is due to inhibition of CcnD1 transcription. The proposal in hand addresses the second hypothesis. Five specific aims are proposed to test predictions that emanate from the working hypothesis. Specific Aims l deals with analysis of nuclear run-on transcription and mRNA turnover in IEC-6 cells in the presence and absences of TGF-beta. Specific Aims 2A address the cloning and analysis of CcnD7 promoter structure. Specific Aim 5 addresses the hypothesis that cyclin D1 is both the target of TGF-beta and a component of the TGF-b signal transduction pathway. Our objective is to understand CcnD7 regulation in detail sufficient to the framing of hypotheses concerning means to block cyclin D1 expression in tumor cells.

结肠癌的发病率反映了缺乏有效的 化学治疗策略用于治疗散布形式的 疾病。我们的长期目标是制定允许的策略 在主要调节缺陷水平上的干预措施导致 恶性转化。我们的策略基于四个观察 关于上皮细胞增殖的生长因子调节。 l） TGF-β是肠道上皮细胞分化的主要效果。 2） 未分化的结肠腺癌对TGF-β没有反应， 用活化的Ras转化的肠上皮细胞 基因。 3）包括TGF-β在内的生长因子调节的表达 D细胞周期蛋白，这对于维持增殖状态很重要。 4） D cylcin基因在许多不同类型的腺癌中具有致癌性。 我们的直接目标是阐明TGF-beta在调节中的作用 肠道上皮细胞的增殖和D细胞周期蛋白的作用 过程。 我们的最初研究将重点放在IEC-6肠上皮上 像正常的肠道上皮细胞一样，细胞不再在 TGF-beta的存在。我们的数据表明编码细胞周期蛋白的基因 D1（CCND1）是IEC-6细胞TGF-β抑制的主要重点。 TGF-β抑制CCND1的转录，导致G0停滞。反义 针对细胞周期蛋白D1 mRNA的寡核苷酸可以概括 TGF-beta的影响。 可诱导细胞周期蛋白D1的过度裸露使 细胞抗TGF-β。两个相关的假设来自我们 初始观察。假设L：我们提出TGF-β抑制 Cyclin d1表达占据了正常停止增殖 伴随肠道上皮的分化。这个假设 将通过同伴IRPG提案中描述的实验进行测试， 由R.D. Beauchamp，医学博士假设2：我们提出TGF-Beta 抑制上皮细胞增殖是由于抑制了CCND1 转录。手中的提议解决了第二个假设。五 提出了具体目的来测试从 工作假设。具体目的l涉及核跑步的分析 在存在的IEC-6细胞中的转录和mRNA周转，并且 缺失TGF-beta。具体目的2a解决了克隆和分析 CCND7启动子结构。具体目的5解决了以下假设 Cyclin D1既是TGF-beta的靶标，也是TGF-B的成分 信号转导途径。 我们的目标是了解CCND7 详细的规定足以构建有关假设 用于阻断肿瘤细胞中细胞周期蛋白D1表达的方法。