TGF-BETA REGULATION OF INTESTINAL EPITHELIAL CELLS

结直肠肿瘤是美国癌症死亡的第二大原因。 结直肠肿瘤细胞中特异性分子“病变”的鉴定提高了设计更特异性治疗的前景。 两个特定的分子病变之间的界面提出了研究。总体目标是确定Ras（约50%的结直肠癌中的突变激活基因）降低有效肿瘤抑制系统（转化生长因子β（TGF β）配体/受体轴）活性的机制。 最终结果是由于TGF β抗性而导致的不受调节的生长。 将使用大鼠肠上皮细胞系RIE-1和一组转染的RIE-1克隆和人结肠癌细胞。 提出了六个具体目标：1）将进行Ras过度表达对从细胞膜到细胞核的Smad途径中TGF β信号传导的影响的逐步分析; 2）将在人结肠癌细胞中进行TGF β信号传导的相关观察并与Ras活性相关; 3）和4）。 将研究两种Ras刺激的生长因子途径，表皮生长因子途径和TGF β配体产生对TGF β抗性的影响; 5）Ras转化的可逆性和肿瘤表型的逆转将通过将功能性TGF β RII基因转染到RIE-Ras细胞中来测试，和6）本文所述的初步研究支持Ras介导的TGF β RII的TGF β抗性降低通过Raf-1介导发生。这一独立的信号通路的发现使我们进一步研究了传统的Raf/MAPKK/MAPK信号通路、磷脂酰肌醇3激酶通路和Rho通路。 本文所用的技术通常是标准技术，例如细胞转染、生长测定、北方分析和Western分析。 该提案在收集和使用大量表达Ras效应子系统的关键相关组分的转染细胞系方面是独特的。 该项目的长期目标是确定适合用于结肠直肠癌治疗干预的新策略的信号通路，并进一步了解Ras和TGF β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.