Study for inactivation mechanism of tumor-associated genes in sporadic colorectal cancers

散发性结直肠癌肿瘤相关基因失活机制研究

• 批准号: 13671348
• 负责人: HEMMI Hiromichi
• 金额: $ 2.3万
• 依托单位: TOHO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671348/
• 关键词: DNA mismatch repair genes; hMLH1 gene; hMSH2 gene; histone; acetylation; transcription factor; colorectal cancer; cell lines; ミスマッチ修復遺伝子; ヒストンH4; 転写活性; アセチル化状態

Accumulation of mutations in oncogenes and tumor suppressor genes is essential for generation of colorectal cancer. Defect of a DNA mismatch repair (MMR) gene is known to be responding to one of the accumulated mutations in these cancer-related genes. Seven human MMR genes including hMLH1 and hMSH2 have been identified. More than half of most HNPCC and certain fraction of sporadic colorectal cancers shows abnormality in the hMLH1 and hMSH2 genes. An epigeneic modification, namely hypermethylation, in the promoter region of the hMLH1 gene has been demonstrated as one of mechanisms of gene inactivation. Since we had inactivated sporadic cases without mutation in the gene nor hypermethylation, we looked for the another mechanism of inactivation. In this study, we focused two points: one is relationship between histone acetylation status and expression of hMLH1 and hMSH2; second is a regulatory mechanism of the hMLH1 gene expression.1) Acetylation status of the hMLH1 gene promoter in human colorectal cancer cell lines was examined by a ChIP method using anti histone H4 antibody. There was no correlation between protein expression and the acetylation status. However, TSA, an inhibitor of the deacetylation enhanced gene expression, indicating that acetylation enhances the expression.2) Essential sites in the promoter region of the hMLH1 gene for transcription has been identified. Then, we screened factors binding to the one of the essential sites by a yeast one-hybrid system. As on candidate, p29 protein, of which gene located in 1p31-32 where known to be frequent loss in colorectal cancer, has been found. The detailed investigation for the role and function of p29 are required in future.

癌基因和抑癌基因突变的积累对于结直肠癌的产生至关重要。已知 DNA 错配修复 (MMR) 基因的缺陷会对这些癌症相关基因中累积的突变之一做出反应。已鉴定出 7 个人类 MMR 基因，包括 hMLH1 和 hMSH2。超过一半的大多数 HNPCC 和某些散发性结直肠癌显示 hMLH1 和 hMSH2 基因异常。 hMLH1 基因启动子区域的表观遗传修饰（即高甲基化）已被证明是基因失活的机制之一。由于我们灭活了散发病例，但没有基因突变或高甲基化，因此我们寻找另一种灭活机制。本研究主要关注两点：一是组蛋白乙酰化状态与hMLH1和hMSH2表达的关系；二是组蛋白乙酰化状态与hMLH1和hMSH2表达的关系。其次是hMLH1基因表达的调控机制。1)使用抗组蛋白H4抗体通过ChIP方法检查人结直肠癌细胞系中hMLH1基因启动子的乙酰化状态。蛋白质表达和乙酰化状态之间没有相关性。然而，脱乙酰化抑制剂TSA增强了基因表达，表明乙酰化增强了表达。2)已经鉴定了hMLH1基因启动子区转录的必需位点。然后，我们通过酵母单杂交系统筛选了与重要位点之一结合的因子。作为候选蛋白，已发现p29蛋白，其基因位于1p31-32，已知该蛋白在结直肠癌中经常丢失。未来需要对p29的作用和功能进行详细研究。

项目成果

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