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

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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