PHOSPHOLIPID-CA2+ AND PHOSPHOPROTEINS IN LEUKEMIC CELLS

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The specific aims of this research cover four major areas of the newly-identified phospholipid/Ca2+-stimulated protein phosphorylation system in human leukemic cells. (1)\Phospholipid-sensitive Ca2+-dependent protein kinase will be purified to homogeneity from leukemic cells from patients, and its properties and mechanism of activation by phospholipid and Ca2+ will be studied. Immunocytochemistry of the enzyme in normal neutrophils, leukemic cells from patients, and HL60 and K562 cells in culture will be studied using polyclonal antibodies to the enzyme already developed in this lab. (2)\Endogenous substrate proteins for the enzyme will be searched for, identified, and characterized. Attention will be paid to the variation in the substrates so that their pathophysiologic relevance can be suggested. Monoclonal antibodies to the substrates "specific" to leukemic cells will be developed; and their ability to inhibit certain leukocyte functions, as well as their diagnostic and therapeutic usefulness in leukemias, will be explored. Endogenous protein phosphorylation stimulated by calmodulin/Ca2+, cAMP, and cGMP also will be carried out so that the interplays among the cellular mediators at the level of protein phosphorylation in the biology and pathology of human leukocytes can be studied. (3)\Alterations in the enzyme (activity level and subcellular distribution) and substrates will be investigated in HL-60 stimulated to terminally differentiate by dimethylsulfoxide and phorbol ester. (4)\Structure-activity relationship of alkyl-lysophospholipid analogs to inhibit the enzyme system will be examined, and the results will be compared with their clinical efficacy (if known) and their ability to affect leukemic cell growth in culture. (B)