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。本文提出了两种特定分子损伤之间的界面进行研究。总的目标是确定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 (tgfβ)配体/受体轴的活性的机制。最终的结果是由于tgf - β抗性导致的不受调节的生长。将使用速率肠上皮细胞系、RIE-1和一系列转染的RIE-1克隆和人结肠癌细胞。提出了六个具体目标：1)逐步分析Ras对TGF β信号在Smad从细胞膜到细胞核通路中的影响；2) tgfβ信号将在人结肠癌细胞中进行相关观察，并与Ras活性相关；3)和4)。研究Ras刺激的两种生长因子途径表皮生长因子途径和tgfβ配体产生对tgfβ抗性的影响；5) Ras转化和肿瘤表型逆转的可逆性将通过将功能性TGFbetaRII基因转染到RIE-Ras细胞中进行测试。6)本文所述的初步研究支持Ras介导的TGFbeta抗性降低是通过不依赖Raf的途径发生的，因此我们将进一步研究传统的Raf/MAPKK/MAPK信号通路、磷脂酰肌醇3激酶途径和Rho途径。本文采用的技术通常是标准技术，如细胞转染、生长测定、北方分析和西方分析。该提案的独特之处在于它收集和使用了大量表达Ras效应系统关键相关成分的转染细胞系。该项目的长期目标是确定适合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信号传导之间的相互作用。

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.