The long-term objective of this application is an understanding of the mechanism of action of interferons (IFNs) and tumor necrosis factor (TNF). IFNs are currently being evaluated as anticancer agents in clinical trials, wheras TNF is being considered for such use, but we do not yet understand the mechanism of action of these agents. IFNs bind to receptors on the plasmamembrane and the IFN/receptor complexes communicate signals which induce transcription of a set of genes and synthesis of several proteins. It is not known whether some of these proteins have antiproliferative activity. Studies on TNF will initially establish whether it binds to specific receptors on the plasmamembrane of sensitive cells. TNF has been labeled to high specific activity without loss of biological activity and its interaction with cells in culture will be studied by the same approach employed to characterize IFN receptors. Subsequent studies will investigate the mechanism of action of TNF, which is basically different from that of IFNs, since RNA synthesis is not required for the cytostatic or cytolytic activity of TNF. Experiments are proposed to examine whether protein synthesis is required for these effects of TNF. Other studies will investigate the synergism of action between TNF and IFNs to test a working hypothesis that IFNs induce synthesis of TNF receptors and augment in this way the cytotoxic response to TNF. Studies on the antiproliferative effect of IFNs will pursue two separate lines of research. Elevated levels of 5'-methylthioadenosine (MTA) have been detected in IFN-treated cells sensitive to growth inhibition by IFN, but not in resistant cells. MTA is formed from S-adenosylmethione (AdoMet) and it is an intermediate in the biosynthesis of polyamines. MTA is toxic for cells in culture and it seams possible that its accumulation in IFN-treated cells may contribute to growth inhibition. The cause for MTA accumulation will be examined by measuring enzymatic activities involved in MTA and AdoMet metabolism. Another line of research will examine the antiproliferative effects of IFNs on human osteosarcoma cells which are highly sensitive to growth inhibition. The synthesis of sis mRNA and the secretion of PDGF will be measured in cells adapted to growth in serum-free medium, which synthesize elevated levels of PDGF and may be dependent on autocrine stimulation for cell growth. The effect of IFNs on PDGF receptors will also be evaluated by binding assays with labeled PDGF.
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