We recently identified phosphatidylcholine as a novel source of diglyceride (DG) mediators; the long-term goal of the proposed project is to define the role of these species of DG in the stimulus-response coupling of normal and neoplastic cells. The proposed investigations will focus on the specificity and regulation of the phospholipase C that degrades choline phosphoglycerides (PC) and on the role of PC-derived DG species as intracellular second-messengers. We will identify the molecular species of DG formed by this pathway and determine the ability of different molecular species to activate protein kinase C. In related studies we will investigate the role of the PC-specific phospholipase C in the turnover of cellular phospholipids.
The specific aims of this proposal are: 1) To determine the specificity of phospholipase C for different subclasses and molecular species of PC. The PC fraction of many cell types contains a mixture of alkyl-PC and acyl-PC. We have shown that degradation of PC results in a mixture of acyl-DG and alkyl-DG. The relative amounts of these subclasses of DG will be determined after cell stimulation. 2) To continue studies on the activity of phospholipase C by in vitro assay. These studies will determine the substrate specificity, cofactor requirements and subcellular localization of the PC degrading phospholipase C. 3) To determine the structural requirements for protein kinase C activation or inhibition by DG. We have shown that alkyl-DGs are inhibitors of PKC activation by, acyl-DG. We will continue these studies using the molecular species of DG observed after cell stimulation and separate the isozymes of PKC to test the effects of alkyl- and acyl-DG on each isoenzyme. 4) To determine the role of phospholipase C in the conversion of PC to ethanolamine phosphoglycerides (PE). In preliminary studies we found that alkyl-PC can be converted to alkenyl-PE by a previously undefined pathway; further studies will be performed to determine the pathway of-conversion and investigate the role of phospholipase C. The combination of the proposed experiments will further define the role of PC-derived DG in cellular regulation. The experiments will also provide further insight into the mechanism of action of phorbol diester tumor promoters and the cellular response to growth factors. It is anticipated that these studies will lead to a better understanding of the role of PC turnover in growth control.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA048995-02
Application #
3192919
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-09-01
Project End
1994-05-31
Budget Start
1990-06-01
Budget End
1991-05-31
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
Civoli, F; Daniel, L W (1998) Quaternary ammonium analogs of ether lipids inhibit the activation of protein kinase C and the growth of human leukemia cell lines. Cancer Chemother Pharmacol 42:319-26
Whitman, S P; Civoli, F; Daniel, L W (1997) Protein kinase CbetaII activation by 1-beta-D-arabinofuranosylcytosine is antagonistic to stimulation of apoptosis and Bcl-2alpha down-regulation. J Biol Chem 272:23481-4
Small, G W; Strum, J C; Daniel, L W (1997) Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. Lipids 32:715-23
Civoli, F; Pauig, S B; Daniel, L W (1996) Differentiation of HL-60 cells distinguishes between cytostatic and cytotoxic effects of the alkylphospholipid ET-18-OCH3. Cancer Chemother Pharmacol 38:269-72
Ghosh, S; Strum, J C; Sciorra, V A et al. (1996) Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells. J Biol Chem 271:8472-80
Daniel, L W; Civoli, F; Rogers, M A et al. (1995) ET-18-OCH3 inhibits nuclear factor-kappa B activation by 12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosis factor-alpha or interleukin 1 alpha. Cancer Res 55:4844-9
Huang, C; Wykle, R L; Daniel, L W (1995) Phospholipase D hydrolyzes ether- and ester-linked glycerophospholipids by different pathways in MDCK cells. Biochem Biophys Res Commun 213:950-7
Strum, J C; Small, G W; Pauig, S B et al. (1994) 1-beta-D-Arabinofuranosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells. J Biol Chem 269:15493-7
Huang, C; Wykle, R L; Cabot, M C (1993) Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts. FEBS Lett 319:141-4
Strum, J C; Daniel, L W (1993) Identification of a lysophospholipase C that may be responsible for the biosynthesis of choline plasmalogens by Madin-Darby canine kidney cells. J Biol Chem 268:25500-8

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