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

通过对每个基因的整个编码区域同时进行多重 SSCP 分析，对人类癌症中的遗传改变进行综合分析。

• 批准号: 08457049
• 负责人: SHIMIZU Kenji
• 金额: $ 4.86万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457049/
• 关键词: Colorectal cancer; Genetic instability; Mismatch-repair genes; Cell-cycle regulators; Transcription factors; E2F4; p107 gene; Tumor suppressor genes; がん遺伝子; hMSH3; PCR-SSCP解析; 点突然変異; ras活性化変異; マイクロサテライト; ゲノム不安定性; E2F-4遺伝子

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

作为这项研究的先决条件，我们建立了一个肿瘤库，该肿瘤库在该项目期限期间包含来自各种人类肿瘤患者的约800对规格。使用这些材料，我们开发了两种用于检测肿瘤特异性遗传变化的新型方法，一种是一种全面的基因组扫描方法，具有alu Inter-Alu长PCR扩增，另一种是对这些新技术的整个区域的多重SSCP分析，我们在这些新技术的应用中，我们建立了一种新型的肿瘤特异性统一的基因统一的eNee Endrobility in Corgifity in Corgifation in ecoder in ecoder in e2f4 E2F4 E2F4 FRSTARES contiques in e2f4 contrantique in apose。 （MI+）结直肠癌。该基因的编码外显子内CAG重复数的突变减少，从而导致E2F4蛋白连续13个连续的丝氨酸保留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+大肠癌/胃癌中的事件约为40％。接下来，我们发现突变AP…更多的梨是HMSH3基因灭活框架变速突变的结果，HMSH3基因是一种植入了两到四个核苷酸的不匹配环修复过程中的DNA失配基因。这两个突变之间的相关性高于80％（通过Fisher的精确测试，P = 0.0015）。当E2F4基因在体内和体外均过表达时，E2F4基因在活体细胞中过表达时表现为活性癌基因。此外，我们获得了一个初步的结果，即E2F4基因的改变版本能够刺激啮齿动物细胞系统中的细胞增殖。这是在实际人类恶性肿瘤中，第一次鉴定出E2F4基因是涉及HMSH3蛋白的缺陷不匹配修复功能的目标。作为一项相关的研究结果，我们发现了人类P107基因的新遗传变化，该基因的亲密关系是肿瘤抑制基因rb的最明显的基因clind cell的基因，属于基因的clent clind cell in gene cye，是基因的遗传，属于基因，是基因的遗传，是基因的遗传症状。线是15 kbp区域的间质缺失，该区域包含5个基因的编码外显子。与RB基因相比，p107基因的肿瘤特异性改变从未被鉴定出来。我们在人类血液学损害中的发现是此类病例的第一个发现。 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位于3P21染色体上的抑制基因，这可能与许多类型的实体瘤有关。

项目成果

Takimoto, H., Tsukuda, K., Ichimura, K., Hanafusa, H.Nakamura, A., Oda, M., Harada, M.and Shimizu, K.: "Genetic alterations in the retinoblastoma protein-related p107 gene in human hematologic malignancies." Biochem.Biophys.Res.Commun.251. 264-268 (1998)

Takimoto, H.、Tsukuda, K.、Ichimura, K.、Hanafusa, H.Nakamura, A.、Oda, M.、Harada, M. 和 Shimizu, K.：“视网膜母细胞瘤蛋白相关 p107 基因的遗传改变

清水憲二: "ヒトがんにおける遺伝子異常の総合的検索" 生化学. 69(5). 311-323 (1997)

Kenji Shimizu：“人类癌症遗传异常的综合研究”生物化学 69(5) (1997)。

Matsubara, N., Hiramatsu, M., Edamatsu, R., Mizukawa, K., Mori, A.and Orita, K.: "Possible involvement of free radical scavenging properties in the action of tumor necrosis factor-alpha." Free Radic.Biol.Med.22. 679-687 (1997)

Matsubara, N.、Hiramatsu, M.、Edamatsu, R.、Mizukawa, K.、Mori, A. 和 Orita, K.：“自由基清除特性可能参与肿瘤坏死因子-α 的作用。”

Zheng, P.-S., Iwasaka, T., Yamasaki, F., Ouchida, M., Yokoyama, M., Nakao, Y., Fukuda, K., Matsuyama, T.and Sugimori, H.: "Telomerase activity in gynecologic tumors." Gynecol.Oncol.64. 171-175 (1997)

郑 P.-S.、岩坂 T.、山崎 F.、大内田 M.、横山 M.、中尾 Y.、福田 K.、松山 T. 和杉森 H.：“端粒酶

清水憲二(分担執筆): "がん研究最前線 研究と臨床の現場から" 第11回「大学と科学」公開シンポジウム組織委員会、クバプロ, 211 (1997)

Kenji Shimizu（合著者）：“癌症研究的前沿：来自研究和临床环境”，第 11 届“大学与科学”公共研讨会组委会，Kubapro，211（1997）