Cloning of a novel tumor suppressor gene in Wilms' tumor associated with chromosome instability

与染色体不稳定相关的肾母细胞瘤中新型抑癌基因的克隆

• 批准号: 14570774
• 负责人: SUZUKI Hideaki
• 金额: $ 1.73万
• 依托单位: JIKEI UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570774/
• 关键词: aneuploidy; chromosome instability; Wilms'tumor; checkpoint; centromere; mitosis; 紡錘糸チェックポイント

Mosaic variegated aneuploidy type 1A (MVA1A) is a rare autosomal recessive chromosome instability syndrome associated with Wilms'tumor. We designed this study to identify the responsible gene of this syndrome.For this purpose, first, we used complementation cloning strategy. The retroviral library derived human mammary gland was divided into 60 small pool libraries that each including 3000 independent clones. All of the small pool libraries were transfected to immortalized MVA1A cells. Transfected cells were exposed with monastrol, one of anaphase inhibitor, for 24 hours ten times every three days. We obtained 16 candidate genes in. the first screening, but each of them didn't show no clear complementation against the MVA1A phenotype.We also underwent detailed studies about the progression of mitosis in MVA1A cells. We found next two new findings ; (1) When MVA1A cells were exposed with spindle.d epolymerizing agents ; the cells transiently arrested in mitosis by spindle checkpoint, and then overrided this checkpoint. (2) The sister centromeres were dissociated before anaphase. These results suggested that the primary defects of MVA1A were existed in centromere-kinetocore function. Then, we underwent mutation analysis of several known centromere-kinetocore genes, such as CENP-A, CENP-C, HP-1β, Ndc8O, Nuf2, AuroraB, Survivin, INCENP, in MVA1A. No responsible mutation have detected until now.

镶嵌杂色非整倍体1A型（MVA 1A）是一种罕见的常染色体隐性染色体不稳定综合征与肾母细胞瘤。本研究首先采用互补克隆的方法，寻找与该综合征相关的基因。将来源于人乳腺的逆转录病毒文库分成60个小库，每个库包括3000个独立克隆。将所有小库文库转染至永生化MVA 1A细胞。将转染的细胞用一种后期抑制剂monastrol处理24小时，每三天处理10次。共获得16个候选基因。结果表明，在第一次筛选时，它们都没有表现出与MVA 1A表型互补的特性，我们还对MVA 1A细胞的有丝分裂过程进行了详细的研究。我们还发现了以下两个新的发现：（1）当MVA 1A细胞暴露于纺锤体解聚剂时，细胞被纺锤体检查点短暂地阻滞在有丝分裂中，然后越过该检查点。(2)姐妹着丝粒在分裂后期前分离。这些结果提示MVA 1A基因的主要缺陷在于着丝粒-动核功能。在此基础上，我们对MVA 1A中的CENP-A、CENP-C、HP-1β、Ndc 8 O、Nuf 2、AuroraB、Survivin、INCENP等几个已知的着丝粒-动粒核心基因进行了突变分析。到目前为止，还没有发现任何负责任的突变。

项目成果

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