Transcriptional Regulation in hMLH1-Silenced Colon Cells

描述（由申请人提供）：本提案中的研究将分析在用2-脱氧-5-氮杂胞苷[AzadC]治疗细胞后，导致位于结肠癌主要亚群的细胞系3号染色体上的一组基因逃避异常甲基化的条件。在我们的实验室中，对错配修复基因hMLH1异常沉默表达的人类结肠肿瘤细胞系进行了详细的微阵列分析，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来定位这些基因在间期细胞核中的位置。拟议的研究将确定3号染色体上这些彼此广泛分离的基因是否存在共表达的结构基础，并将探索它们的调控与其他AzadC应答基因的不同之处。进一步的研究将解剖这些基因的启动子区域的精细结构，以了解它们在沉默细胞和短暂的AzadC诱导的从这种抑制中释放后的CpG甲基化模式，并利用一组在AzadC暴露后分离的组成表达亚克隆。这些研究将验证这样的假设，即这些基因是由异常甲基化过程引起的共同调节基因群的代表。他们将进一步对比导致该基因亚群的调节机制被废除的条件与azadc诱导的其他基因位点的调节所需的条件。通过分析AzadC与反义RNA对三种主要的人类DNA甲基转移酶DNMT1、3a和3b处理后细胞中表达改变的基因的交叉，将进一步研究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利用反义rna对特定组蛋白去乙酰化酶进行进一步剖析。作为拟议研究重点的细胞系代表了15-20%表现出微卫星不稳定性[MSI]的人类结肠癌，这几乎普遍等同于DNA MMR缺陷。三分之二的MSI癌症被归类为散发性起源的癌症，因为它们的发生与已知的家族缺陷无关。

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.