CYCLIN GENES AND PROLIFERATION OF GUT EPITHELIAL CELLS

结肠癌的发病率反映出缺乏有效的治疗方法。 播散型卵巢癌的化疗策略 疾病。我们的长期目标是制定战略，允许 在导致主要监管缺陷的层面上进行干预 恶变。我们的策略基于四点观察 关于生长因子对上皮细胞增殖的调控。l) 转化生长因子-β是肠上皮细胞分化的主要效应因子。2) 未分化结肠腺癌对转化生长因子-β无反应 活化的RAS基因转化的肠上皮细胞 基因。3)生长因子，包括转化生长因子-β，调节血管内皮生长因子的表达 D细胞周期蛋白，这对维持增殖状态很重要。4) D-细胞周期蛋白基因在许多不同类型的腺癌中都有致癌作用。 我们的近期目标是阐明转化生长因子-β在调节 肠上皮细胞增殖及D细胞周期蛋白在其中的作用 进程。我们的初步研究将集中在IEC-6肠道上皮细胞 细胞，就像正常的肠道上皮细胞一样，停止在 转化生长因子-β的存在。我们的数据表明，编码细胞周期蛋白的基因 D_1(CCND_1)是转化生长因子β抑制IEC-6细胞的主要靶点。 转化生长因子-β抑制CCND1的转录，导致G0期停滞。反义 针对细胞周期蛋白D1mRNA的寡核苷酸可以概括 转化生长因子-β的作用。细胞周期蛋白D1的可诱导过度表达使 对转化生长因子-β耐药的细胞。两个相关的假设是从我们的 初步观察。假设L：我们认为转化生长因子-β抑制血管生成 细胞周期蛋白D1的表达与细胞增殖的正常停止有关 伴随着肠道上皮的分化。这一假设 将通过配套的IRPG提案中描述的实验进行测试， 假设2：我们提出转化生长因子-β 抑制上皮细胞增殖是由于抑制了CCND1 抄写。手头的提案解决了第二个假设。五 提出了具体的目标来测试从 工作假说。《特定目标》L谈核跑动分析 联合应用对IEC-6细胞转录和信使核糖核酸的影响 缺乏转化生长因子-β。特定的AIMS 2A涉及克隆和分析 CcnD7启动子结构。具体目标5解决了这样的假设 细胞周期蛋白D1既是转化生长因子-β的靶标，又是转化生长因子-β的一个组成部分 信号转导途径。我们的目标是了解CcnD7 详细的监管，足以形成关于以下方面的假设 阻断肿瘤细胞中细胞周期蛋白D1表达的手段。

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.