Transcriptional Regulation in hMLH1-Silenced Colon Cells

描述(申请人提供)：这项建议中的研究将剖析在用2-脱氧-5-氮胞苷[AzadC]治疗细胞后，在代表结肠癌主要亚组的细胞系中，定位于染色体3的一组基因中导致逃脱异常甲基化的条件。我们实验室对错配修复基因hMLH1异常沉默表达的人结肠肿瘤细胞系进行了详细的微阵列分析，确定了3号染色体上与hMLH1基因共同诱导表达的一组基因。拟议的实验将利用滚动的圆形探针来定位间期核中的这些基因。拟议的研究将确定这些基因在3号染色体上彼此广泛分离的共同表达是否有结构基础，并将探索它们的调控与其他AzadC反应基因的不同之处。进一步的研究将剖析这些基因启动子区域的精细结构，以寻找它们在沉默的细胞中和在AzadC诱导的短暂释放这种抑制后的CpG甲基化模式，并利用在AzadC暴露后分离的一组结构性表达的亚克隆。这些研究将检验这样一种假设，即这些基因代表了由异常甲基化过程产生的共同调节的基因队列。他们将进一步将导致这一基因子集调控机制丧失的条件与AzadC诱导的其他基因位点调节所需的条件进行对比。通过分析在AzadC处理的细胞中改变表达的基因与人类三种主要DNA甲基转移酶DNMT1、3a和3b的反义RNA的交集，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。利用针对特异组蛋白脱乙酰酶的反义RNA也将进一步剖析甲基化相关基因表达调控的机制分化。作为拟议研究的重点的细胞系代表了15%-20%表现出微卫星不稳定[MSI]的人类结肠癌，这几乎是DNA MMR缺陷的同义词。这些MSI癌中有三分之二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。

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.