Mechanisms causing chromosomal translocation and its biological consequence in multiple myeloma

1.MUM1转基因小鼠的建立：采用Eμ-SV-MUM1-myc tag-hGH构建物在成熟b细胞中建立转基因小鼠。在长期随访中，该实验系统未发生b细胞恶性肿瘤。鉴定在多发性骨髓瘤中不受MUM1/IRF4和MAFB调控的靶基因：a)MUM1：表达MUM1的小鼠prob细胞系BaF3的生长速度比亲本细胞快，这表明MUM1通过调控其下游靶基因为b细胞提供了生长优势。通过cDNA阵列分析，我们发现IFNγ(Mig)诱导的单因子是MUM1直接调控的基因之一。使用Mig基因启动子序列的报告基因实验表明，当MUM1和PU.1一起转染时，荧光素酶活性升高，表明它们在Mig转录方面的合作。经染色体免疫沉淀试验证实，MUM1也能直接与Mig启动子序列结合。有趣的是，mum1阳性的b细胞慢性淋巴细胞白血病细胞也表达Mig及其受体CXCR3，中和的抗CXCR3或抗Mig抗体部分抑制了它们的增殖，这表明Mig和CXCR3是磷脂酰肌醇3激酶- akt信号的下游执行子，受到MAFB的正调控。ARK5的异位表达导致了对低营养条件的抵抗，并促进了对纤维连接蛋白的粘附。与双链DNA断裂修复相关的非同源末端连接（NHEJ）蛋白突变分析：在14个多发性骨髓瘤细胞系中，尽管Ku70和XRCC4基因没有突变，但有1个、1个和2个存在Ku80、连接酶4和DNA- pkcs的错义突变。由于剩余的等位基因保持了野生型序列，突变蛋白在骨髓瘤细胞中的显性负作用有待进一步阐明。

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.