TGF-BETA REGULATION OF INTESTINAL EPITHELIAL CELLS

在美国，结直肠癌是导致癌症死亡的第二大原因。鉴定结直肠肿瘤细胞中的特定分子“损伤”提高了设计更具特异性的治疗方法的前景。两个特定的分子损伤之间的界面建议在此进行研究。总的目标是确定RAS基因，一个在大约50%的结直肠癌中被突变激活的基因，通过什么机制降低一个强大的肿瘤抑制系统的活性，转化生长因子β(TGFβ)配体/受体轴。最终的结果是，由于对转化生长因子β的抵抗，导致了不受监管的增长。将使用肠上皮细胞系RIE-1和一组转RIE-1克隆和人结肠癌细胞的速率。提出了六个具体的目标：1)逐步分析RAS过度表达对从细胞膜到细胞核的Smad通路中转化生长因子β信号的影响；2)对人结肠癌细胞中的转化生长因子β信号进行相关观察，并与RAS活性相关；3)和4)。将研究两条RAS刺激的生长因子途径，即表皮生长因子途径和TGFbeta配体的产生对TGFbeta耐药性的影响；5)通过将功能性TGFbetaRII基因导入RIE-RAS细胞来检测RAS转化的可逆性和肿瘤表型的逆转；6)本文描述的初步研究支持RAS介导的TGFbetaRII通过Raf不依赖的途径降低TGFbetaRII的耐药性，使我们进一步研究传统的Raf/MAPKK/MAPK信号通路、磷脂酰肌醇3激酶途径和Rho通路。这里使用的技术通常是标准技术，例如细胞转染法、生长分析、Northern分析和Western分析。该方案的独特之处在于它收集和使用了大量表达RAS效应器系统关键相关组件的转基因细胞系。该项目的长期目标是确定适用于结直肠癌治疗干预的新策略的信号通路，并进一步了解RAS和TGFβ信号之间的相互作用。

Colorectal neoplasia is the second leading cause of cancer death in the United States. Identification of specific molecular "lesions" in colorectal tumors cell has elevated the prospects for design of more specific treatments. The interface between two specific molecular lesions are proposed for study herein. The general goal is to identify mechanisms by which Ras, a mutationally activated gene in about 50 percent of colorectal cancers, decreases the activity of a potent tumor suppressor systems, the transforming growth factor beta (TGFbeta) ligand/receptor axis. The end result is unregulated growth due to TGFbeta resistance. The rate intestinal epithelial cell line, RIE-1 and a battery of transfected RIE-1 clones and human colon carcinoma cells will be used. Six Specific Aims are proposed: 1) a step-wise analysis of the impact of Ras over expression of TGF beta signaling in the Smad pathway from the cells membrane to the nucleus will be performed; 2) related observations on TGFbeta signaling will be made in human colon carcinoma cells and correlated with Ras activity; 3) and 4). The effects of two Ras- stimulated growth factor pathways, the epidermal growth factor pathway and TGFbeta ligand production on TGFbeta resistance will be studied; 5) the reversibility of Ras transformation and reversion of the neoplastic phenotype will be tested by transfection of a functional TGFbetaRII gene into RIE-Ras cells and 6) preliminary studies described herein support Ras-mediated TGFbeta resistance reduction of TGFbetaRII occurs by a Raf- independent pathway leading us to further study of the conventional Raf/MAPKK/MAPK signaling pathway, the phosphatidylinositol 3 kinase pathway and the Rho pathway. The techniques employed herein are generally standard techniques such as cellular transfection, growth assays, Northern analysis, and Western analysis. The proposal is unique in its collection and use of a large variety of transfected cell lines expressing key, relevant components of the Ras effector system. The long term goal of this project is to identify signaling pathways eligible for novel strategies for therapeutic intervention in colorectal cancer and to further understanding of the interaction between Ras and TGFbeta signaling.