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Mechanisms causing chromosomal translocation and its biological consequence in multiple myeloma

多发性骨髓瘤中染色体易位的机制及其生物学后果

基本信息

批准号：
13671072
负责人：
IIDA Shinsuke
金额：
$ 2.62万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671072/
关键词：
multiple myeloma chromosomal translocation MUM1 IRF4 MAFB transcription factor target gene MIG non-homologous end joining BOB.1 Pou2AF1 Non homologens end joining Multiple Myeloma Double strand break repair gene Pou2A1 Ku80 /

项目摘要

1.Establishment of the MUM1 transgenic mice : An Eμ-SV-MUM1-myc tag-hGH construct was used for establishing transgenic mice in mature B-cells. During a long-term follow-up, no B-cell malignancy has developed in this experimental system.2.Identification of the target genes regulated by MUM1/IRF4 and by MAFB, which are deregulated in multiple myeloma : a)MUM1 : MUM1-expressing mouse proB-cell line, BaF3, grew faster than its parent cells, suggesting that MUM1 provides the B-cells with a growth advantage by regulating its downstream target genes. By means of cDNA array analysis, we identified monokine induced by IFNγ(Mig) as one of the directly regulated genes by MUM1. A reporter assay using Mig gene promoter sequences has resulted in the elevated luciferase activity when transfected with MUM1 together with PU.1, indicating their cooperation in terms of Mig transcription. MUM1 also directly binds to Mig promoter sequences, proven by chromosome immunoprecipitation assay. Interestingly, MUM1-positive B-cell chronic lymphocytic leukemia cells also expressed Mig and its receptor, CXCR3, and neutralizing anti-CXCR3 or anti-Mig antibodies partially inhibited their proliferation, suggesting that Mig and CXCR3 constitute an which is a downstream executer of the Phosphatidyl inositol 3 kinase-Akt signaling, is positively regulated by MAFB. Ectopic expression of the ARK5 resulted in the resistance against hypo-nutritional condition and in the promoted adhesion to fibronectin.3.Mutation analysis of the non-homologous end joining (NHEJ) protein associated with the repair of the double strand DNA breaks : Out of 14 multiple myeloma cell lines, one, one, and two harbored missense mutations of the Ku80, Ligase 4 and DNA-PKcs, in spite of the absence of mutation in Ku70 and XRCC4 genes. Since the remaining alleles kept the wild-type seguences, dominant-negative effect of the mutated proteins remains to be clarified further in myeloma cells.

1. MUM 1转基因小鼠的建立：使用Eμ-SV-MUM 1-myc tag-hGH构建体在成熟B细胞中建立转基因小鼠。2. MUM 1/IRF 4和MAFB调控的靶基因在多发性骨髓瘤中的失调控：a）MUM 1：表达MUM 1的小鼠proB细胞系BaF 3的生长速度比其亲本细胞快，表明MUM 1通过调节其下游靶基因为B细胞提供生长优势。通过cDNA微阵列分析，我们确定了MUM 1直接调控的基因之一是IFNγ诱导的单核因子（Mig）。使用Mig基因启动子序列的报告基因测定在MUM 1与PU.1一起转染时导致荧光素酶活性升高，表明它们在Mig转录方面的合作。MUM 1也直接结合到Mig启动子序列，通过染色体免疫沉淀试验证明。有趣的是，MUM 1阳性B细胞慢性淋巴细胞白血病细胞也表达Mig及其受体CXCR 3，中和性抗CXCR 3或抗Mig抗体部分抑制其增殖，表明Mig和CXCR 3构成了一个磷脂酰肌醇3激酶-Akt信号传导的下游执行器，受到MAFB的正调控。ARK 5的异位表达导致了对低营养条件的抵抗和对纤连蛋白的促进粘附。3.与双链DNA断裂修复相关的非同源末端连接（NHEJ）蛋白的突变分析：在14个多发性骨髓瘤细胞系中，1个、1个和2个具有Ku 80、连接酶4和DNA-PKcs的错义突变，尽管Ku 70和XRCC 4基因没有突变。由于其余等位基因保持野生型序列，突变蛋白的显性负效应仍有待进一步阐明在骨髓瘤细胞。