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改变的新方法，一种是采用inter-Alu long PCR扩增的综合基因组扫描方法，另一种是对一个基因的整个区域进行多重SSCP分析。在这些新技术的应用中，我们在遗传不稳定（MI+）结直肠癌的一个亚群中发现了编码E2F4转录调节因子的基因中的一个新的肿瘤特异性突变。突变是基因编码外显子内CAG重复数减少，导致E2F4蛋白连续13个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，这种突变在MI+结直肠癌/胃癌中的发生率约为40%。接下来，我们发现这种突变可能是由于hMSH3基因的移框突变失活的结果，hMSH3基因是一种DNA错配修复基因，与2到4个核苷酸错配环的修复过程有关。这两个突变之间的相关性在80%以上（经Fisher精确检验P = 0.0015）。当E2F4基因在体内和体外活细胞中过度表达时，它的行为是一种活跃的致癌基因。进一步，我们得到了E2F4基因的改变版本能够刺激啮齿动物细胞系统的细胞增殖的初步结果。因此，在实际的人类恶性肿瘤中，E2F4基因首次被确定为涉及hMSH3蛋白的缺陷错配修复功能的靶标。作为相关的研究结果，我们发现了人类p107基因的新的遗传改变，p107基因是肿瘤抑制基因RB的近亲。在弥漫性大B细胞淋巴瘤细胞系中检测到的最明显的基因遗传改变病例是含有该基因5个编码外显子的15 kbp区域的间质性缺失。与RB基因相反，p107基因的肿瘤特异性改变迄今尚未被发现。我们在人类血液恶性肿瘤中的发现是此类病例的首次发现。我们分析了p107基因的整个基因组结构，发现该基因横跨约90kbp的区域，由22个编码外显子组成。我们的其他研究成果包括：1)建立了一种通过竞争性PCR分析来测量基因表达的通用策略；2)鉴定了人类结肠癌K-ras基因第22个密码子的一个新的激活突变；3)发现了位于3p21染色体上的一个新的肿瘤抑制基因候选基因，该基因可能与多种类型的实体肿瘤有关。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