Genetic Analysis of Beta-Catenin Function in Human CA

人类 CA β-连环蛋白功能的遗传分析

• 批准号: 7154043
• 负责人: TODD A WALDMAN
• 金额: $ 26.07万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-12 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7154043
• 关键词: APC gene; Address; Affect; Agar; Alleles; Animal Model; Antibodies; BMP4; Binding; Binding Sites; Biochemical; Biological; Biological Models; Biology; Brush Border; Caco-2 Cells; Cancer cell line; Cells; Colon Carcinoma; Confocal Microscopy; Cytoplasm; Doctor of Philosophy; Drosophila melanogaster; Electrical Resistance; Electron Microscopy; Electrophysiology (science); Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Event; Gelshift Analysis; Gene Expression; Gene Targeting; Genes; Genetic; Growth; HCT116 Cells; Half-Life; Homologous Gene; House mice; Human; Immunodeficient Mouse; Immunohistochemistry; In Vitro; Kinetics; Lead; Light; Maintenance; Malignant - descriptor; Malignant Neoplasms; Measurement; Measures; Mediating; Microscopy; Mutation; Northern Blotting; Nuclear; Numbers; Oncogenes; Oncogenic; Pathogenesis; Pattern; Phenotype; Phosphorylation Site; Pilot Projects; Point Mutation; Protein Overexpression; Proteins; Range; Rate; Reporter; Sampling; Scanning Transmission Electron Microscopy Procedures; Signal Transduction; Staining method; Stains; Structure; Subcutaneous Injections; System; TCF Transcription Factor; TCF7L2 gene; Testing; Thinking; Tight Junctions; Tissues; Transcription Coactivator; Transcriptional Activation; Tumor Cell Line; Tumorigenicity; Western Blotting; Xenograft procedure; activating transcription factor; anti-cancer therapeutic; beta catenin; bone morphogenetic protein 4; cancer cell; carcinogenesis; cell type; colon cancer cell line; gene function; genetic analysis; immunocytochemistry; in vivo; inhibitor/antagonist; light microscopy; malignant state; monolayer; mutant; promoter; protein function; research study; restoration; small molecule; three dimensional structure; transcription factor; tumor; tumor xenograft; vector

Oncogenic activation of g-catenin is common to the pathogenesis of a wide range of human cancers. As is the case for many cancer-related genes, some of the most valuable studies in 13-catenin biology have been undertaken in genetically tractable model organisms such as Drosophila melanogaster and Mus musculus. By exploiting recent technological advances in gene targeting it is now possible to undertake similar studies of gene function in human cancer cells themselves. To do this, we have created high-efficiency human promoterless 13-catenin gene-targeting vectors and employed them to create isogenic sets of human cancer cells that differ only in the presence or absence of their endogenous mutant and/or wild-type fS-catenin genes. Initial functional studies have shed light on several aspects of the function of 13-catenin in human cancer. In particular, we have shown that activated 13-catenin is necessary for TCF-mediated transcriptional activation in human cancer cells. We have also demonstrated that deletion of activated g-catenin causes cancer cells to form dome-like structures, recapitulating a phenotype seen in differentiating Caco-2 cell monolayers. Pilot experiments have suggested that oncogenic 13-catenin is not required for the human colon cancer cells to form xenograft tumors in immunodeficient mice. Also surprisingly, we have detected no discernible differences in the steady state levels and intracellular localization of wild-type and mutant, activated 13-catenin, as detected by Western Blot and immunocytochemistry analysis. Finally, we have identified and characterized BMP-4, a TGF-f3 homolog, as a putative effector of activated 13-catenin signaling in human cancer using Affymetrix Genechip analysis. We propose to further utilize these isogenic human cancer cell systems to study the functions of activated 13-catenin in the pathogenesis of human cancer. In particular, we plan to: 1) Determine the cell biological and pathobiologieal phenotypes of activated 13-catenin in human cancer cells and tissues, 2) Determine whether oncogenic activation of/3-catenin is required for maintenance of tumorigenicity in human cancer cells containing multiple genetic hits, 3) Determine the biochemical mechanism(s) of g-catenin activation in human cancer, and 4) Identify and characterize genes regulated by the activated B-catenin transcription factor.

G-catenin的致癌激活对于广泛的人类癌症的发病机理是共同的。和 许多与癌症相关的基因的病例，13-Catenin生物学中一些最有价值的研究已经 在遗传上的模型生物（例如果蝇和肌肉）中进行。经过 利用最新的基因靶向技术进步，现在有可能对 人类癌细胞本身的基因功能。为此，我们创造了高效的人 无启动子13-catenin基因靶向载体，并使用它们来创建异源性人类癌症集 仅在存在或不存在内源性突变体和/或野生型FS-catenin基因的细胞。 最初的功能研究揭示了13-catenin在人类癌症中功能的几个方面。在 特别是，我们已经表明，激活的13-catenin对于TCF介导的转录激活是必需的 人类癌细胞。我们还证明，活化的G-catenin的缺失导致癌细胞 形成圆顶状结构，概括了在区分CACO-2细胞单层中看到的表型。飞行员 实验表明，人类结肠癌细胞不需要致癌13-catenin 免疫缺陷小鼠中的异种移植肿瘤。同样令人惊讶的是，我们没有发现 如检测到的 通过Western印迹和免疫细胞化学分析。最后，我们已经确定并表征了BMP-4，A TGF-F3同源物作为使用Affymetrix激活的13-catenin信号传导的推定效应子 转基因分析。我们建议进一步利用这些等生的人类癌细胞系统来研究 活化的13-catenin在人类癌发病机理中的功能。特别是，我们计划：1）确定 人类癌细胞和组织中活化13-catenin的细胞生物学和病原体表型，2） 确定是否需要/3-catenin的致癌激活才能维持人的肿瘤性 含有多个遗传命中的癌细胞，3）确定G-catenin的生化机制 人类癌症的激活，以及4）识别和表征由活化的B-catenin调节的基因 转录因子。