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

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 Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)) and secretory PLA(2) (sPLA(2)), 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 cPLA(2) or sPLA(2) enzyme activity in vitro. Furthermore, stimulation of cells with rTNF-alpha for 4 h did not increase cPLA(2) enzyme activity. The data indicate that neither cPLA(2) or sPLA(2) 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.