Establishment of a microdetection method for DNA mismatch repair deficiency in colorectal cancers and its clinicopathological study

结直肠癌DNA错配修复缺陷显微检测方法的建立及临床病理研究

• 批准号: 10671222
• 负责人: TSUJITA Kazunori
• 金额: $ 2.37万
• 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: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671222/
• 关键词: colorectal cancer; anti-hMLH1 antibody; anti-hMSH2 antibody; microsatellite instability; gene abnormality; 簡易検索法; 免疫組織化学的染色法; 臨床病理学的要因; hMLH1蛋白; hMSH2蛋白; RT-PCR; 臨床病理学要因

DNA mismatch repair (MMR) genes are responsible to hereditary nonpolyposis colorectal cancer and some of sporadic colorectal cancers. MMR-deficient tumor cells exhibit a low level of resistance to some anti-cancer agents, such as cisplatin, adriamycin, and alkylating agents. To know the MMR status or microsatellite instability (MSI) status of tumor cells is critical for choosing a therapeutical protocol. In this study, we established methods for microdetection of hMSH2 and hMLH1 proteins with monoclonal antibodies and MSI status determination. Further, clinicopathologic features of 99 cases of sporadic colorectal cancer was investigated.(1)MSI statusWe selected 16 microsatellite markers and determined an efficient detection procedure for MSI status by studying in 90 cases of sporadic colorectal cancer.(2)Expression of mismatch repair proteins, hMSH2 and hMLH1Expression of hMSH2 and hMLH1 proteins in 99 samples of sporadic colorectal cancer selected randomly was analyzes by Western blot … More and immunohistochemical staining with monoclonal antibodies developed in our laboratory. Totally 10 tumor samples with no/little expressing hMSH2 (4 samples) and hMLH1(6 samples) proteins were found, indicating approximately 10% of sporadic cases and 67% of the MSI tumor have some defects in the expression of these MMR proteins.(3)Mutations in hMSH2 and hMLH1 genesTo know the molecular mechanism of the suppression of the gene expression, mutation in the exons were studied. Two kinds of mutations, which affect protein function, in a hMSH2-deficient sporadic case and one non-sense mutation in hMSH2-deficient a familial case were found. No mutation was found in hMLH1-deficient cases.(4)Clinicopathologic featuresClinicopathologic features of 17 cases of MSI tumor showed similar characteristics to those frequently found in HNPCC cases such as right-sided location of the primary tumor, a lower frequency of well-differentiated adenocarcinoma, and Duke's staging A or B.Further study is required to clarify the molecular mechanism of suppression of MMR gene expression, especially hMLH1 gene. In addition, since MMR-deficient or MSI tumors show a low level of drug-resistance, the specified protocol for treatment of MSI tumor should be developed. Less

DNA 错配修复 (MMR) 基因导致遗传性非息肉病性结直肠癌和一些散发性结直肠癌，MMR 缺陷的肿瘤细胞对某些抗癌药物（如顺铂、阿霉素和烷化剂）表现出低水平的耐药性。了解肿瘤细胞的 MMR 状态或微卫星不稳定性 (MSI) 状态对于选择治疗方案至关重要。在本研究中，我们建立了微检测方法。 hMSH2和hMLH1蛋白单克隆抗体及MSI状态测定进一步研究了99例散发性结直肠癌的临床病理特征。(1)MSI状态我们通过对90例散发性结直肠癌的研究，选择了16个微卫星标记，并确定了MSI状态的有效检测程序。 (2)错配修复蛋白hMSH2和hMLH1的表达Expression of hMSH2 and随机选取99例散发性结直肠癌样本中的hMLH1蛋白，通过Western blot和本实验室开发的单克隆抗体进行免疫组化染色，共10例无/少表达hMSH2（4个样本）和hMLH1（6个样本）蛋白的肿瘤样本。发现，大约10％的散发病例和67％的MSI肿瘤在这些MMR蛋白的表达上存在一些缺陷。（3）突变hMSH2 和 hMLH1 基因中为了了解基因表达抑制的分子机制，研究了 hMSH2 缺陷散发病例中影响蛋白质功能的两种突变和 hMSH2 中的一种无义突变。 hMLH1缺陷病例中未发现1例家族性缺陷。(4)临床病理特征17例MSI肿瘤的临床病理特征相似。与 HNPCC 病例中常见的特征相比，如原发肿瘤位于右侧、高分化腺癌发生率较低、Duke 分期 A 或 B 等，需要进一步研究阐明抑制 MMR 基因表达的分子机制尤其是hMLH1基因，此外，由于MMR缺陷或MSI肿瘤表现出低水平的耐药性，因此应制定针对MSI肿瘤的治疗方案。

项目成果

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Masahiro Iwasaki：“散发性结直肠癌中异常 DNA 修复基因表达的分析”Vita。 14. 55-61 (1998)