J. Lipid Res.  Neurobiology of Lipids (ISSN1683-5506)
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Journal of Lipid Research, Vol. 40, 2223-2233, December 1999
Copyright © 1999 by Lipid Research, Inc.


Original Article

Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells

Ole-L. Brekkea, Erling Sagena, and Kristian S. Bjervea
a Department of Clinical Chemistry, University Hospital, Norwegian University of Science and Technology, N-7006 Trondheim, Norway

Correspondence to: Kristian S. Bjerve

Recombinant tumor necrosis factor alpha (rTNF-{alpha})-induced release of endogenous fatty acids was examined in WEHI 164 clone 13 fibrosarcoma cells using a highly sensitive HPLC method. The initial rTNF-{alpha}-induced extracellular release of endogenous fatty acids was dominated by 20:4n;–6, 22:4n;–6, 24:4n;–6, and 18:1n;–9 showing relative rates of 2.9, 0.9, 1.1, and 1.0, respectively. Release of endogenous AA and DNA fragmentation occurred simultaneously and preceded cell death by approx. 2 h. Methyl arachidonoyl fluorophosphonate and LY311727, specific inhibitors of Ca2+-dependent cytosolic PLA2 (cPLA2) and secretory PLA2 (sPLA2), respectively, neither blocked rTNF-{alpha}-induced cytotoxicity or endogenous AA release. However, both inhibitors reduced rTNF-{alpha}-induced release of other endogenous fatty acids. In comparison, the antioxidant butylated hydroxyanisole (BHA) completely inhibited the rTNF-{alpha}-induced cytotoxicity as well as AA release mediated through the TNF receptor p55, while the very similar antioxidant butylated hydroxytoluene had no effect. BHA did not inhibit recombinant cPLA2 or sPLA2 enzyme activity in vitro. Furthermore, stimulation of cells with rTNF-{alpha} for 4 h did not increase cPLA2 enzyme activity.

The data indicate that neither cPLA2 or sPLA2 mediate rTNF-{alpha}-induced apoptosis and extracellular AA release in WEHI cells. The results suggest that a BHA-sensitive signaling pathway coupled to AA release is a key event in TNF-induced cytotoxicity in these cells.—Brekke, O-L., E. Sagen, and K. S. Bjerve. Specificity of endogenous fatty acid release during tumor necrosis factor-induced apoptosis in WEHI 164 fibrosarcoma cells. J. Lipid Res. 1999. 40: 2223;–2233.

Supplementary key words: apoptosis, arachidonic acid, butylated hydroxyanisole, cell culture, fatty acids, phospholipase A2


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