Comprehensive Analysis of Genetic Alterations in Human Cancers by the Simultaneous Multiple SSCP Analysis on the Entire Coding Region of Each Gene.

作为本研究的前提，我们在本项目期间建立了一个包含约800对来自各种人类肿瘤患者的标本的肿瘤库。利用这些材料，我们开发了两种检测肿瘤特异性遗传变异的新方法，一种是Alu间长链PCR扩增的综合基因组扫描方法，另一种是基因整个区域的多重SSCP分析。这些新技术的应用，我们在遗传不稳定（MI+）结直肠癌的一个亚组中发现了编码E2F4转录调节因子的基因中的一种新的肿瘤特异性突变。该突变是该基因编码外显子内CAG重复数的减少，导致E2F4蛋白的13个连续丝氨酸残基中的一个或两个氨基酸减少。在日本和美国患者中，MI+结直肠癌/胃癌中该突变的发生率约为40%。接下来，我们发现突变ap ...更多信息 梨是hMSH3基因失活移码突变的结果，hMSH3基因是一种DNA错配修复基因，参与2至4个核苷酸的错配环的修复过程。这两种突变之间的相关性大于80%（通过Fisher精确检验，P = 0.0015）。已知E2F4基因在体内和体外活细胞中过表达时表现为活性癌基因。进一步的，我们得到了一个初步的结果，改变版本的E2F4基因能够刺激啮齿类细胞系统中的细胞增殖。因此，E2F4基因首次被鉴定为在实际人类恶性肿瘤中涉及hMSH3蛋白的错配修复功能缺陷的靶基因。作为相关研究结果，我们发现了与肿瘤抑制基因RB密切相关的人类p107基因的新的遗传改变。该基因遗传改变的最明确的情况是，在弥漫性大B细胞淋巴瘤细胞系中检测到的是含有该基因5个编码外显子的15 kbp区域的间质缺失。与RB基因相反，迄今为止从未鉴定出p107基因的肿瘤特异性改变。我们在人类恶性血液病中的发现是此类病例的首次发现。我们分析了p107基因的全基因组结构，发现该基因跨越约90 kbp的区域，由总共22个编码外显子组成。我们的其他研究项目包括：1）建立了竞争性PCR分析基因表达的通用策略，2）鉴定了人结肠癌中K-ras基因第22位密码子的新激活突变，3）发现了一个位于染色体3p21上的新的肿瘤抑制基因候选基因，该基因可能与多种实体瘤相关。少

As a prerequisite of this research, we established a tumor bank containing about 800 pairs of the specimens from a wide variety of human tumor patients during the term of this project. Using these materials, we have developed two novel approaches for detecting tumor-specific genetic alterations, one is the comprehensive genomic scanning method with inter-Alu long PCR amplification, and the other is the multiple SSCP analysis of the whole region of a gene.On application of these novel techniques, we found a novel tumor-specific mutation in the gene encoding E2F4 transcription regulator in a subset of genetically unstable (MI+) colorectal cancers. The mutation was decrease in CAG repeat number within a coding exon of the gene, giving rise to reduction of one or two amino acids of 13 consecutive serine residues of the E2F4 protein. The incidence of this mutation in MI+ colorectal/gastric cancers was approximately 40% in both Japan and American patients. Next, we found that the mutation ap … More pears to be a result of the inactivating frame-shift mutation of the hMSH3 gene, a DNA mismatch-repair gene implicated in the repair process of mismatch-loops of two to four nucleotides. The correlation between these two mutations was above 80 % (P = 0.0015 by Fisher's exact test). The E2F4 gene is known to behave as an active oncogene when it is overexpressed in living cells both in vivo and in vitro. Further, we got a preliminary result that the altered version of E2F4 gene is capable of stimulating cellular proliferation in rodent cell system. Thus, the E2F4 gene is identified, for the first time, as a target of the defective mismatch repair function involving hMSH3 protein, in actual human malignancies.As a relevant research result, we have found novel genetic alterations of the human p107 gene, a close relative of the tumor suppressor gene RB.The most clear case of the genetic alteration of the gene, detected in a diffuse-large B cell lymphoma cell line, was an interstitial deletion of a 15 kbp region containing 5 coding exons of the gene. In contrast to the RB gene, tumor-specific alteration of the p107 gene has never been identified as far. Our finding in human hematologic malignancies is the first discovery of such cases. We analyzed the entire genomic structure of the p107 gene and found that the gene spans about 90 kbp region and is composed of total 22 coding exons.Other results of our research projects include, 1) establishment of a general strategy for measuring gene expression by competitive PCR analysis, 2) identification of a novel activating mutation at 22nd codon of the K-ras gene in a human colon cancer, 3) discovery of a novel candidate of the tumor suppressor gene residing on chromosome 3p21, which may be related to many types of solid tumors. Less