The objective of this proposal is to utilize a macrophage-like cell line, J774, to develop a bank of variants with which to study drugs that interact with the cell membrane and certain components of the cytoskeleton. Variants, resistant to the growth inhibitory effects of trifluoperazine, taxol, vinblastine, colchicine and cytochalasin D, have been obtained and will be analyzed from the following points of view: cytology, biochemical abnormalities conferring resistance, expression of macrophage-specific functions. The hope is that information will be obtained about specific mechanisms of drug interaction and the acquisition of drug resistance as well as fundamental information about cell regulation and macrophage function. Two new leads have been uncovered during the past two years that form a major focus of the renewal application: 1) Synthesis of a 17 Kd protein in cells resistant to taxol and vinblastine. Synthesis appears to correlate with resistance and decreases as resistance is lost; 2) Presence of a heretofore undescribed calmodulin binding protein that is present in parental J774 and in activated murine peritoneal macrophages but is absent in the J774 variants resistant to trifluoperazine. Studies on the biosynthesis, purification, localization and function of these two proteins are proposed. In addition to studies related to microtubules and calmodulin, changes in membrane function, specifically protein kinase activation and tyrosine phosphorylation will be examined in wild type and variant cells exposed to insulin, colony stimulatory factor, interferon, immunoglobulin and other ligands for which J774 has specific cell membrane receptors.
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