The leukemic cell is characterized by maturational arrest at a stage of hematopoietic development where the ability to proliferate is maintained. Human leukenic cells cultured in vitro can be stimulated to develop into a non-proliferating cell. Thus, the leukemias can be viewed, in part, as a disorder of differentiation as well as proliferation. One of the most potent agents stimulating differentiation of leukemic cells is 120tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester. By substituting for diacylglycerol, TPA activates the CA2+- phospholipiddependent kinase, protein kinase C. The ability of diacyglycerol derivatives to mimic TPA stimulated cellular responses indicates that protein kinase C activation may mediate a portion of the cellular effects exerted by TPA. However, diacylglycerol derivatives are unable to stimulate differentiation of the human leukemic cell line U937. In our laboratory, a non- protein kinase C, TPA stimulated kinase activity has been identified. Protein kinase C and the TPA stimulated kinase activity can be distinguished by differences in substrate specificity. The inability of diacylglycerol derivatives to stimulate differentiation or to activate this kinase suggests that activation of the TPA stimulated kinase may be important in mediating TPA stimulated differentiation. The goal of this proposal is to characterize the nonprotein kinase C, TPA stimulated kinase and to determine if activation of this kinase is crucial in mediating the signal stimulating differentiation of the leukemic cell. Using column chromatography, this kinase will be characterized and compared to other non-protein kinase C phosphotransferase activities stimulated by TPA. Using 32P-labeled intact cells and two dimensional electrophoretic analysis, endogenous substrates phosphorylated by this kinase will be defined. The effects of other agents inducing U937 differentiation (lymphokines, dibutryl cAMP, etc.) will be evaluated for effects on this kinase activity. A better understanding of the mechanism controlling differentiation of human leukemic cells would aid in developing therapeutic modalities specifically designed to stimulate the in vivo differentiation of leukemic cells. Such a therapy, in addition to providing an alternative form of treatment, would theoretically avoid many of the toxic side effects inherent to the currently used antineoplastic therapy. The proposed research could also provide insight into the mechanisms mediating the many diverse effects exerted by phorbol esters in other biological systems.
