Interferons are a family of peptide hormones which elicit a variety of important biological responses from cells. These include antiviral and antiproliferative effects, complex immunologic responses and modulation of differentiation. The ability of interferons to modulate cell proliferation is undoubtedly related to their antitumor effects. While clinical trials are being carried out to evaluate the efficacy of interferon treatment on a variety of cancers, immunologic disorders and viral disease, the biochemical events underlying the effects of interferon on growth and differentiation are complex and largely unexplored. The overall objective of the proposed study is to utilize mouse 3T3-F442A preadipocyte fibroblasts to (i) investigate the mechanisms by which the interferon- induced dsRNA-dependent eIF-2alpha; kinase (dsI) is regulated at the post translational level by two novel cellular factors, a regulatory RNA responsible for the activation of dsI and a protein factor which inhibits its activation and (ii) define the role of these cellular factors and dsI in growth and differentiation.
The specific aims i nclude, cloning and determining the identity and structure of the regulatory RNA, and the cloning and characterizing of the inhibitor of dsI activation, which is observed in 3T3-F442A cells cultured under conditions which suppress adipose differentiation. 3T3-F442A cells will be cultured under conditions which support or suppress adipose differentiation and the regulatory RNA and the inhibitor will be prepared from these cells. The activity of the regulatory RNA and the inhibitor will generally be determined by monitoring the phosphorylation and activation of dsI. Recombinant DNA methods, sequencing, site-directed mutagenesis and RNA-band shift gels will be utilized for the analysis of the regulatory RNA. The inhibitor will be purified from cell cultures in sufficient amounts to prepare antibodies and obtain amino acid sequence information to facilitate its cloning and sequencing. Recombinant DNA methods will be used to alter the expression of the inhibitor in 3T3-F442A cells to delineate its physiologic role. It is anticipated that the proposed studies will increase our understanding of the physiologic role of interferon and dsl during growth and differentiation, and cellular regulation in general. These studies will also define an important model system for the analysis of RNA-protein interaction and their cellular effects.
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