We propose experiments to define the mechanism by which interferon (IFN) treatment blocks the response of quiescent fibroblasts to mitogenic stimulation. We have determined that IFN blocks or delays cell cycle activation in a concentration-dependent manner by acting during the period in which platelet-derived growth factor (PDGF) establishes replicative competence in BALB/c 3T3 cells. IFN treatment inhibits the PDGF-induced synthesis of a prominent nuclear 29 kDa protein (pI) thought to be involved in the establishment of competence, and induces the synthesis of a number of proteins both in quiescent and PDGF-stimulated BALB/c 3T3 cells. We propose to test the hypothesis that IFNs inhibit the calcium messenger system in growth factor-induced cell cycle activation of quiescent fibroblasts. We will define the role of the calcium messenger system in: (a) the synthesis of distinctive proteins induced by PDGF or IFN; (b) the PDGF-induced phosphorylation of the myosin light chain and of an 80 kDa protein, and in the possible inhibition by IFN; (c) the PDGF-induced cytoskeletal and plasma membrane changes and the possible inhibition by IFN. In addition, we will determine the state of the calcium messenger system in control and IFN-treated mitogenically stimulated cells by measuring: (a) free cytosolic Ca2+ concentration; (b) Ca2+ uptake and efflux; (c) calmodulin level; (d) inositol phospholipid turnover and C kinase function. To gain understanding of the mechanism by which IFN signals the cell, we will examine (2',5')A3 cores for their ability to reproduce the inhibitory effects of IFN on events in cell cycle activation by PDGF. PCNA (cyclin) and statin are nuclear proteins that are uniquely associated with proliferating vs. nonproliferating normal cells. We will use antibodies against these proteins to investigate their occurrence in control and IFN-treated, quiescent and PDGF-stimulated human fibroblasts, with the aim of defining further PDGF and IFN actions at the level of specific cellular proteins. For these studies we will use mouse BALB/c 3T3 (clone A31) cells and purified mouse Alpha/Beta or Beta IFNs, and human diploid skin fibroblasts (0038A cells) and human Beta IFN.
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