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

为了分离与骨肉瘤发生和/或进展有关的基因，进行了体外转化实验，将HPV16E7基因引入到P53-/-小鼠成骨细胞系MMC2中，使Rb蛋白失活。本实验建立了MMC2TC转化细胞系，并分离到两个新基因作为MMC2和MMC2TC的差异表达基因，其中DDM23是MMC2TC上调基因，编码710个氨基酸的蛋白质产物。同源性搜索表明，DDM23是Ral GDS家族的成员，由ras信号通路上的效应蛋白组成，并与该家族的一个新成员RGL3相同。克隆了人的同源基因，并利用人骨肉瘤细胞系和肿瘤标本分析了DDM23/RGL3的表达，其中一些表达水平较高，提示DDM23/RGL3参与了人骨肉瘤的发生。然而，…没有检测到致癌活性在进一步的转化实验中，DDM23/RGL3在人骨肉瘤发生发展中的作用仍有待研究。DDM36作为MMC2TC的下调基因，编码1,252个氨基酸的受体蛋白，属于Ig G超家族，包括新生素、DCC和PUNC。相同的基因最近被报道为Nope。克隆了人的同源基因，并确定了其基因组结构。使用人骨肉瘤样本进行了突变分析，尽管没有检测到明显的突变。用定量RT-PCR方法检测DDM36/NOPE的表达。一半的骨肉瘤样本没有表达或表达减弱，在骨和软组织肿瘤中，滑膜肉瘤的表达最低。在与相邻细胞接触的细胞膜上发现GFP标记的蛋白表达，提示DDM36/NOPE可能涉及细胞间识别机制，这种识别机制可能决定具有特定谱系的间充质细胞的命运。较少

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