MITOGENIC SIGNALS IN OSTEOGENIC SARCOMAS

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This project characterizes osteosarcomas by theirs response to growth factors and defines early events regulated by growth factors in osteosarcoma cells. Many osteosarcoma cell lines produce PDGF, a potent mitogen. Antisera to PDGF and to PDGF receptors will be used to define tumors which produce PDGF and may have the capacity to respond to it. Production of PDGF by tumor cells in vivo may affect prognosis. PDGF production either directly stimulates growth of the tumor cells (autocrine) or growth of surrounding stromal cells (paracrine). To differentiate between these two mechanisms, production of endogenous PDGF will be inhibited by use of either antisense oligonucleotides or conditionally active antisense constructs. An autocrine mechanism of stimulation would be suggested if: 1. growth is restricted by inhibition of endogenous PDGF synthesis; and 2. addition of exogenous PDGF to cells with inhibited endogenous PDGF production restores growth. Osteosarcomas which do not produce PDGF will also be investigated. One, the MG-63 cell line, does not grow in response to PDGF but does express c- myc RNA. Unlike other cell lines, however, PDGF stimulates c-myc expression in a protein kinase C- independent fashion. It will be learned if: 1. resistance to PDGF-induced growth and protein kinase C-independent c-myc expression is a general property of non-PDGF-producing osteogenic sarcoma cells; and 2. exogenous PDGF stimulates protein kinase C- independent c-myc expression by PDGF producing osteosarcoma cells when endogenous PDGF synthesis is inhibited. Because PDGF stimulates c-myc RNA expression independently of protein kinase C in MG-63 cells, there is likely to be a defect in phosphoinositide metabolism. To characterize this defect PDGF stimulated production of inositol phosphates and diacylglycerol will be characterized as will complex formation between the activated PDGF- receptors and phospholipase C-gamma. Growth factor regulated early gene expression will be examined in MG-63 cells. Particular attention will be paid to IGF-1 because IGF-1 stimulates growth of these cells. A cDNA library will be prepared from IGF-1 treated cells to identify and characterize IGF-1 regulated genes that potentially control growth. In summary, this project characterizes osteogenic tumor cells by their response to growth factors and defines early events regulated by growth factors in these cells.