These studies are designed to investigate a cytolytic factor from human macrophage cultures and include purification and physiocochemical characterization. Continuous lines of promyelocytic cells are available that can be differentiated into macrophages by appropriate treatment. These macrophages can then be made to secrete a cytolytic factor when stimulated with bacterial lipopolysaccharide. Studies also include attempts to enhance and prolong production of the factor and to define other appropriate stimuli. Studies are also designed to investigate the mechanism of action of these factors on transformed and tumor cells. We hope to show how the cytolytic factor discriminates transformed from normal cells and how specific killing is achieved. Comparisons will be made with rabbit tumor necrosis factor and cytolytic factors obtained from rabbit macrophages of various sources, since we have had considerable experience with these factors. Cytolytic factors (from human and rabbit) are purified by a series of salt precipitations, gel filtration, ion exchange chromatography, and affinity chromatography. Greater than 2000-fold purification has been achieved. The human and rabbit factors have molecular weights of approximately 43 kilodaltons and 50 kilodaltons reapectively on gel filtration. Our plans include studying the interaction of these factors with transformed and normal cells in order to identify and characterize a receptor or substrate on susceptible cells which presumably is missing on normal resistant cells. If a receptor is identified, then we will determine if endocytosis of the factors results. Actinomycin D treatment of cells causes a marked enhancement of target cell killing, both in time of killing and dosage required. This finding has led to a hypothesis that cells resist destruction by having a repair mechanism that is impaired in the presence of inhibitors of macromolecular aynthesis. The eventual goal is to determine the mechanism of action; this achievement should reveal fundamental differences between tumor and normal cells and permit an exploitation of these differences in cancer control, treatment, and prevention. (HF)
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