|Budget Amount *help
¥10,400,000 (Direct Cost : ¥10,400,000)
Fiscal Year 1998 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 1997 : ¥6,400,000 (Direct Cost : ¥6,400,000)
1. Mutation analyses of molecules on the signal transduction pathway of TGF-beta super family.
PCR-SSCP and direct sequencing analyses of type II receptor of TGF-beta (TGF_R II) and CBFA1 gene were performed using genomic DNA from 60 primary osteosarcoma tumors. No apparent mutation was found in the hot spot regions of TGF_R II, which include the poly (A) stretch in exon 3, and exon 6 and 7 encoding the kinase domain, In the case of CBFA1 gene, an insertion of nine bases was found in one case, which will add three alanine residues in the stretch of 17 alanines in exon 4. Although the significance of this insertion was not clear, this may be a rare polymorphism, because the identical change was found in DNA from normal tissue in the patient, who did not manifest any skeletal anormalies. Entire coding sequence of Smad 1 gene was RT-PCR amplified from eight osteosarcoma cell lines. Sequence analyses of amplified fragment showed no apparent mutations. These results suggest that mutations of these molecules may be a rare event in osteosarcomas.
2. Expression of molecules on BMP signals in osteosarcoma cell lines.
RNA expression of genes on the BMP signal pathway, which include BMP2 and 4 as ligands, type I and type II receptors, Smad 1, and CBFA1 gene, was analyzed in eight osteosarcoma cell lines. Summarized results suggested that there are some osteosarcomas in which the BMP signal may be transducted as an autocrine signal. Among analyzed molecules, the expression of CBFA1 gene was well correlated with the osteoblastic phenotypes of each osteosarcoma cells. To further investigate the role of CBFA1 gene in osteosarcomas, we established the inducible system of CBFA 1 gene by the tetracycline inducible system.