Transcriptional Regulation in hMLH1-Silenced Colon Cells

hMLH1 沉默的结肠细胞中的转录调控

基本信息

批准号：
7232712
负责人：
W DAVID SEDWICK
金额：
$ 28.67万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-06-01 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7232712
关键词：
Acute Antisense RNA Azacitidine Biological Assay Cell Line Cell Nucleus Cells Characteristics Chromosomes Chromosomes, Human, Pair 3 Colon Colon Carcinoma Colonic Neoplasms Condition DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases Defect Deoxycytidine Development Enzymes Exhibits Gene Cluster Gene Expression Gene Expression Regulation Gene Silencing Genes Genus Cola Goals Histone Deacetylase Histone Deacetylation Histones Human In Situ Individual Interphase Laboratories Link Localized MLH1 gene Maintenance Malignant Neoplasms Maps Methylation Microsatellite Instability Mismatch Repair Nuclear Pattern Pharmaceutical Preparations Play Process Promoter Regions Regulation Repression Role Site Structure Subgroup Testing Time Transcriptional Regulation base cohort colon cancer cell line comparative gene repression neoplastic cell promoter research study response tumor

项目摘要

DESCRIPTION (provided by applicant): The studies in this proposal will dissect conditions leading to escape from aberrant methylation in a cohort of genes localized on chromosome 3 in a cell line representative of a major subgroup of colon cancers following treatment of cells with 2-deoxy-5- azacytidine [AzadC]. Detailed microarray analyses in our laboratory of a human colon tumor cell line in which the mismatch repair gene, hMLH1 is aberrantly silenced for expression have defined a set of genes on chromosome 3 that are co-induced for expression with the hMLH1 gene. Proposed experiments will utilize rolling circle probes to localize these genes in interphase nuclei. Proposed studies will determine if there is a structural basis for co-expression of these genes that are widely separated from each other on chromosome 3 and will probe how their regulation differs from other AzadC responsive genes. Further studies will dissect the fine structure of the promoter regions of each of these genes for their CpG methylation patterns in gone silenced cells and after transient AzadC-induced release from this repression, and exploit a group of constitutively expressing subclones isolated after AzadC exposure. These studies will test the hypothesis that these genes are representative of a co-regulated gene cohort resulting from the aberrant methylation process. They will further contrast conditions leading to abrogation of regulatory mechanisms in this subset of genes with those required for AzadC-induced modulation of other gene sites. The mechanisms leading to gene silencing and escape from these gene repression processes will be further examined by analyzing the intersection of genes altered for expression in cells treated with AzadC versus antisense RNA to the three major human DNA methyltransferase enzymes, DNMT1, 3a and 3b. Mechanistic differentiation of methylation-related gene expression regulation will also be further dissected employing antisense RNAs to specific histone deacetylases. The cell lines that are a focus of the proposed studies are representative of 15-20% of human colon cancers that exhibit microsatellite instability [MSI], which is almost universally synonymous with defects in DNA MMR. 2/3 of these MSI cancers are classified as cancers of sporadic origin because their occurrence does not correlate with a known familial defect.

描述（由申请人提供）：本提案中的研究将剖析导致在用 2-脱氧处理细胞后代表结肠癌主要亚组的细胞系中位于 3 号染色体上的一组基因逃避异常甲基化的条件。 -5-氮胞苷[AzadC]。我们实验室对人类结肠肿瘤细胞系进行了详细的微阵列分析，其中错配修复基因 hMLH1 的表达异常沉默，确定了 3 号染色体上的一组基因，这些基因与 hMLH1 基因共同诱导表达。拟议的实验将利用滚环探针将这些基因定位在间期细胞核中。拟议的研究将确定这些在 3 号染色体上彼此广泛分离的基因的共表达是否存在结构基础，并将探讨它们的调控与其他 AzadC 响应基因有何不同。进一步的研究将剖析这些基因启动子区域的精细结构，了解其在沉默细胞中以及在 AzadC 诱导的短暂释放后的 CpG 甲基化模式，并利用在 AzadC 暴露后分离的一组组成型表达亚克隆。这些研究将检验以下假设：这些基因代表异常甲基化过程产生的共同调控基因组。他们将进一步对比导致该基因子集中调节机制失效的条件与 AzadC 诱导的其他基因位点调节所需的条件。通过分析用 AzadC 处理的细胞中表达改变的基因与三种主要人类 DNA 甲基转移酶 DNMT1、3a 和 3b 的反义 RNA 的交叉点，将进一步检查导致基因沉默和逃避这些基因抑制过程的机制。甲基化相关基因表达调控的机制分化也将使用特定组蛋白脱乙酰酶的反义 RNA 进一步剖析。拟议研究的重点细胞系代表 15-20% 的人类结肠癌，这些细胞表现出微卫星不稳定性 [MSI]，这几乎普遍与 DNA MMR 缺陷同义。这些 MSI 癌症中有 2/3 被归类为散发性癌症，因为它们的发生与已知的家族缺陷无关。