Molecular cloning and mutation analyses of novel genes involving the development and progression of osteosarcoma

为了分离涉及骨肉瘤的发展和/或进展的基因，进行体外转化实验，其中将HPV 16 E7基因引入P53-/-鼠成骨细胞系MMC 2中以表达Rb蛋白。本实验建立了MMC 2 TC转化细胞系，并分离到两个差异表达的新基因，其中DDM 23是MMC 2 TC中的一个上调基因，编码710个氨基酸的蛋白产物。同源性分析表明，DDM 23是Ras信号通路上的效应蛋白Ral GDS家族的一员，与该家族的新成员RGL 3相同。克隆了人同源基因DDM 23/RGL 3，并使用人骨肉瘤细胞系和肿瘤样品分析了DDM 23/RGL 3的表达，其中一些显示出高表达，表明DDM 23/RGL 3参与人骨肉瘤。然而，致癌活性未被检测到， ...更多信息 DDM 23/RGL 3在人骨肉瘤发生发展中的意义尚待进一步研究。DDM 36是在MMC 2 TC中表达下调的基因，编码一个1,252个氨基酸的受体蛋白，属于IgG超家族，包括Neogenin、DCC和Punc。相同的基因最近被报道为Nope。克隆了人同源基因，并确定了基因组结构。使用人骨肉瘤样品进行突变分析，尽管没有检测到明显的突变。用定量RT-PCR分析DDM 36/Nope的表达。一半的骨肉瘤样本显示无表达或表达减少，在骨和软组织肿瘤中，滑膜肉瘤显示最低表达。GFP标记的蛋白质在与相邻细胞接触的细胞膜中表达，提示DDM 36/Nope可能参与了细胞间识别机制，该机制可能决定了特定谱系间充质细胞的命运。少

To isolate the genes involving the development and/or progression of osteosarcoma, in vitro transformation experiments were performed, in which the HPV16E7 gene was introduced to inactivate the Rb protein in MMC2, a p53-/- murine osteoblast cell line. MMC2TC was established as a transformed cell line in this experiment, and two novel genes were isolated as a gene that expressed differentially between MMC2 and MMC2TC.DDM23 was isolated as an up-regulated gene in MMC2TC and encoded a protein product with 710 amino acids. Homology search revealed that DDM23 is a member of the Ral GDS family consisting of the effector proteins on the ras signaling pathway, and identical with a new member of this family, RGL3. Human orthologue was cloned, and the expression of DDM23/RGL3 was analyzed using human osteosarcoma cell lines and tumors samples, of which some showed a high expression, suggesting the involvement of DDM23/RGL3 in human osteosarcomas. Oncogenic activity, however, was not detected by … More the transformation experiments, and the ignificance of DDM23/RGL3 in the development of human osteosarcoma was still to be investigated.DDM36 was isolated as a down-regulated gene in MMC2TC and encodes a receptor protein with 1,252 amino acids belonging with the IgG superfamily including Neogenin, DCC, and Punc. Identical gene was recently reported as Nope. Human orthologue was cloned and the genomic structure of was determined.Mutation analysis was performed using human osteosarcoma samples, although no apparent mutations were detected. Expression of DDM36/Nope was analyzed by quantitative RT-PCR. A half of osteosarcoma samples showed no or reduced expression, and among the bone and soft tissue tumors, synovial sarcoma showed the lowest expression. GFP-labeled protein expression was found in cell membrane contacting with adjacent cells, suggesting that DDM36/Nope may involve the cell-to-cell recognizing mechanisms which may determine the fate of mesenchymal cells with a specific lineage. Less