We are continuing to investigate the role of phospholipid and Ca?2+?-dependent protein kinase (protein kinase C) in the maturation of leukemia cells into macrophages. Data generated over the past year in many laboratories, including our own, confirm the hypothesis put forth in our original application: protein kinase C is the phorbol diester receptor. We have synthesized chemically defined diacylglycerols and shown them to be the """"""""endogenous phorbols"""""""" as evidenced by their ability to: (1) activate protein kinase C; (2) competitively inhibit radiolabeled phorbol diester binding to the soluble or cellular phorbol diester receptor; (3) induce leukemic cells to differentiate into macrophages; and (4) mimic phorbol diester-induced phosphoprotein changes that occur during macrophage differentiation. Biologically active phorbol diesters and cell-permeable diacylglycerols induce phosphorylation of proteins with Mr = 21, 52, 65, and 80 kilodaltons in ?32?P-loaded HL-60 cells. This year, emphasis will be placed on purification of protein kinase C from HL-60 cells and antibody production. The major phosphoproteins will be purified and ?32?P amino acids and peptide maps determined. The long-term objective of this project is to begin to understand myeloid maturation at a biochemical level. The major scientific disciplines involved are hematology, oncology, and cell biology. In vitro maturation of promyelocytic leukemia cells serves as a model for bone marrow differentiation. Studies such as these may define the molecular defect which leads to cessation of myeloid maturation and hence, leukemia. The investigation is health-related because it explores a molecular pathway of differentiation which is blocked in leukemia. Agents active as inducers of cell maturation will soon find clinical application in the experimental treatment of leukemia and other malignancies. (B)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA035680-02
Application #
3173277
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-08-01
Project End
1987-01-31
Budget Start
1985-02-01
Budget End
1986-01-31
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
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Salehi, Z; Taylor, J D; Niedel, J E (1988) Dioctanoylglycerol and phorbol esters regulate transcription of c-myc in human promyelocytic leukemia cells. J Biol Chem 263:1898-903
Rider, L G; Dougherty, R W; Niedel, J E (1988) Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils. J Immunol 140:200-7
McCachren Jr, S S; Salehi, Z; Weinberg, J B et al. (1988) Transcription interruption may be a common mechanism of c-myc regulation during HL-60 differentiation. Biochem Biophys Res Commun 151:574-82
Preiss, J E; Bell, R M; Niedel, J E (1987) Diacylglycerol mass measurements in stimulated HL-60 phagocytes. J Immunol 138:1542-5
Brandt, S J; Niedel, J E; Bell, R M et al. (1987) Distinct patterns of expression of different protein kinase C mRNAs in rat tissues. Cell 49:57-63
Rider, L G; Niedel, J E (1987) Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils. J Biol Chem 262:5603-8
Preiss, J E; Loomis, C R; Bell, R M et al. (1987) Quantitative measurement of sn-1,2-diacylglycerols. Methods Enzymol 141:294-300
Dougherty, R W; Niedel, J E (1986) Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes. J Biol Chem 261:4097-100
Conn, P M; Ganong, B R; Ebeling, J et al. (1986) Synthesis and use of diacylglycerols as activators of protein kinase C in neuroendocrine tissue. Methods Enzymol 124:57-63

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