COLON CELL DIFFERENTIATION--DCC ROLE IN MUC2 EXPRESSION

结肠细胞分化--DCC 在 MUC2 表达中的作用

• 批准号: 2895410
• 负责人: LEONARD H AUGENLICHT
• 金额: $ 19.02万
• 依托单位: MONTEFIORE MEDICAL CENTER (BRONX, NY)
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2895410
• 关键词: cell differentiation; colon neoplasms; gene expression; gene rearrangement; genetic enhancer element; genetic promoter element; goblet cells; human tissue; laboratory mouse; mucins; neoplasm /cancer genetics; phenotype; point mutation; protein biosynthesis; tissue /cell culture; transfection /expression vector; tumor suppressor genes

The DCC gene is a potential tumor suppressor gene whose deletion or inactivation plays a role in colonic tumor progression. Recent evidence suggests that it plays a role in colonic goblet cell differentiation and mucin production, consistent with its less can also be effected by inducers which interact with metabolic processes and signal transduction pathways. Some of these inducers may be related to dietary components. We are in a unique position to dissect this link among DCC, mucin synthesis and cell differentiation since we have initiated detailed studies on the mechanisms of regulation of the MUC2 gene, the gene which encodes the principal colonic mucin peptide backbone. We have now cloned, mapped and partially sequenced approximately 90 kilobases of the locus on chromosome II encompassing the MUC2 gene and its promotor and enhancer, and have found this contig contains another mucin backbone gene, probably MUC5b, which is also expressed in colonic cells both in vivo and in vitro, that could be coordinately regulated with MUC2. We will investigate the interaction between DCC and MUC2 expression at the RNA and protein level, as well as the elaboration and secretion of mucus and growth parameters. Colonic carcinoma cell lines which have deleted at least one copy of DCC will be used as recipients of expression vectors which contain the entire wild-type DCC cDNA, or which encode specific extracellular and intracellular domains of the DCC protein, and the influence on constitutive and inducible MUC2 expression determined. The experiments will be extended to mucin synthesis and secretion, since we have demonstrated that expression of the gene does not necessarily lead to production and secretion of mature mucins. Further, different promotor constructs will be used to define the relationship between specific levels of DCC expression and cell and molecular phenotype. The promoter and enhancer for MUC2 will be dissected to delineate sequence elements and their boundaries necessary to establish responsiveness of MUC2 to DCC. Finally, we will investigate this relationship of mutations in the promotor of the MUC2 gene to the sub-class of colonic carcinomas having a mucinous phenotype. These experiments will provide detailed understanding of the interaction of a gene important in the differentiation and transformation of colonic epithelial cells with the principal hallmark of differentiation along one lineage. In addition, since mucinous colonic tumors in which MUC2 is deregulated exhibit a poorer prognosis than most common colorectal tumors, understanding this interaction may define prognostic factors which are not reflected in tumor histology or pathology.

DCC基因是一种潜在的肿瘤抑制基因，其缺失或 失活在结肠肿瘤的进展中起着一定的作用。最近的证据 提示它在结肠杯状细胞分化和分化中起作用。 粘蛋白的产生也可以受到诱导剂的影响，这一点与其较少的粘蛋白一致 它们与新陈代谢过程和信号转导途径相互作用。 其中一些诱因可能与饮食成分有关。 我们处于一个独特的位置来剖析DCC、粘蛋白合成之间的这种联系 和细胞分化，因为我们已经启动了关于 MUC2基因的调节机制，该基因编码 主要为结肠粘蛋白多肽骨架。我们现在已经克隆、绘制和绘制了 对染色体上约90千碱基的基因座进行了部分测序 II包含MUC2基因及其启动子和增强子，并发现 这个重叠群包含另一个粘蛋白骨架基因，可能是MUC5B，它是 在体内和体外的结肠细胞中也有表达，这可能是 与MUC2协调调节。 我们将研究DCC和MUC2表达之间的相互作用 RNA和蛋白质水平，以及粘液的加工和分泌 和生长参数。缺失At基因的结肠癌细胞系 至少一个DCC拷贝将被用作表达载体的接收者 它包含整个野生型DCC cdna，或编码特定的 DCC蛋白的胞外和胞内结构域，以及 确定了对组成性和诱导性MUC2表达的影响。这个 实验将扩展到粘蛋白的合成和分泌，因为我们 已经证明了基因的表达并不一定会导致 成熟粘蛋白的产生和分泌。此外，不同的推动者 构造将用于定义特定级别之间的关系 DCC表达与细胞和分子表型的关系。发起人和 MUC2的增强子将被解剖以描绘序列元件和 建立MUC2对DCC的响应性所需的界限。 最后，我们将研究启动子突变的这种关系 MUC2基因对粘液性结肠癌亚类的作用 表型。 这些实验将提供详细的了解相互作用 一个在结肠分化和转化中起重要作用的基因 以分化为主要标志的上皮细胞 血统。此外，由于粘液性结肠肿瘤中MUC2是 与大多数常见的结直肠肿瘤相比，解除管制显示出更差的预后， 了解这种相互作用可能会定义一些不同的预后因素 反映在肿瘤组织学或病理学上。