This revised small grant application concerns the regulation of bone-specific genes (i.e., osteocalcin (OC) and alkaline phosphatase (AP) genes) and molecular biological mechanisms responsible for bone-specific gene activation necessary for cell transformation in tumor cells. The bone- specific genes are expressed by osteoblasts after the completion of cell proliferation. Thus, these genes may be associated with the maturation and mineralization of bone matrix. The expression of these genes is up regulated by c-fos and c-jun protooncogenes in normal osteoblasts, but is deregulated in malignant cells. Since the mode of regulation of bone-specific gene expression by normal human osteoblasts remains unknown, the principal investigator plans to study the role of c-fos, c-jun, and c-myc protooncogenes on regulation. Furthermore, the project will study the regulatory differences between normal osteoblastic and osteosarcoma cells as a means of examining the pathogenesis of osteosarcoma. The overall hypothesis of the project is that normal regulatory mechanisms of AP and OC by protooncogene products are either altered or lost in osteosarcoma cells. The rationale of the hypothesis is that 1) osteosarcoma cells show abnormal expression of bone-specific genes, and 2) some cellular proto- oncogenes regulate the expression of bone-specific genes in osteoblastic cells. To test this hypothesis, the project aims: 1) to determine whether the osteosarcoma cells lose their normal expression pattern of protooncogenes (c-myc, c-fos and c-jun) using Northern blot hybridizations; 2) to investigate the deregulation of regulatory functions of protooncogenes on bone-specific gene expression in osteosarcoma cells after blocking the expression of protooncogenes with antisense oligonucleotides or after overexpressing the genes by the transfection of cells with protooncogene plasmids; and 3) to determine the regulatory mechanisms of bone-specific gene expression by protooncogene products in human primary osteoblasts and osteosarcoma cells using the CAT assay.
