Interferons (IFNs) are cytokines that have documented efficacy in the treatment of hairy cell leukemia, and show promising results in the treatment of renal cell carcinoma, chronic myelocytic leukemia, hepatitis and acquired immune deficiency syndrome. The goal of the proposed research program is to define the molecular basis of the antiproliferative action of IFNs, focussing on characterization of the IFN alpha receptor; the antagonism of IFN alpha with growth factors at the level of signal transduction pathways; and IFN alpha-induced down-regulation of growth factor receptor expression. Studies of the basis for the antiproliferative action of IFN are critically aided by comparative analysis of the effects of IFN on sensitive and resistant Daudi and renal carcinoma cell lines all of which express IFN receptors. Such cell lines permit critical experiments to identify specific mechanisms underlying the inhibitory action of IFN alpha on cell proliferation. Using such cell lines, we will define the roles of IFN alpha-induced receptor dimerization, the high and low affinity binding components, and the glycosylated portion of the IFN alpha receptor in IFN-induced expression of IFN-stimulated genes (ISGs) and the induction of antiproliferative and antiviral activities. Based on our recent findings that IFN alpha stimulates GTP binding to membranes and depresses arachidonic acid (AA) release in IFN- sensitive but not in resistant cells, we hypothesize that these may be early and perhaps obligatory steps in the antiproliferative action of IFN alpha. The role of GTP-binding proteins in IFN action will be investigated in the activation of ISGs and the antiproliferative action of IFN. We propose that IFN blocks signal transduction pathways activated by relevant growth factors (EGF, PDGF, insulin, etc.). We will directly examine the effects of IFN on arachidonic acid (AA) release, protein kinase C activity, and membrane GTPase activity in the presence and absence of added growth factors. Some important effects of IFN on cell function become manifest only after a period of several hours. Based on our findings that down-regulation of growth factor receptors by IFN alpha treatment attenuates the ability of cells to respond to such factors, we will determine whether IFN modulates growth factor receptor expression at both transcriptional and post- transcriptional levels. We will define post-transcriptional effects of IFN alpha by measuring the phosphorylation and recycling of insulin, transferrin and EGF receptors, and the stability of the mRNA for these receptors. We plan to define the molecular basis of transcriptional regulation by determining whether there are specific IFN-responsive elements in the 5' region upstream from the start site of the EGF receptor gene. This will be done using cloned segments of this gene and transient expression assays. We will also determine whether there are transcription factors that bind such elements.
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