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

Recombinant tumor necrosis factor alpha (rTNF- a )-induced release of endogenous fatty acids was examined in WEHI 164 clone 13 fibrosarcoma cells using a highly sen- sitive HPLC method. The initial rTNF- a -induced extracellu- lar 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 en- dogenous AA and DNA fragmentation occurred simulta- neously and preceded cell death by approx. 2 h. Methyl arachidonoyl fluorophosphonate and LY311727, specific in- hibitors of Ca 2 1 -dependent cytosolic PLA 2 (cPLA 2 ) and secretory PLA 2 (sPLA 2 ), respectively, neither blocked rTNF- a -induced cytotoxicity or endogenous AA release. However, both inhibitors reduced rTNF- a -induced release of other endogenous fatty acids. In comparison, the antioxidant bu- tylated hydroxyanisole (BHA) completely inhibited the rTNF- a -induced cytotoxicity as well as AA release mediated through the TNF receptor p55, while the very similar anti- oxidant 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- a for 4 h did not increase cPLA 2 enzyme activity. The data indicate that neither cPLA 2 or sPLA 2 mediate rTNF- a -induced apo- ptosis and extracellular AA release in WEHI cells. The re- sults 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. Spec- ificity of endogenous fatty acid release during tumor necro- sis factor-induced apoptosis in WEHI 164 fibrosarcoma cells. J. Lipid Res. 1999. 40: 2223-2233.