Resistance to TNF-induced cytotoxicity correlates with an abnormal cleavage of cytosolic phospholipase A(2)

To investigate the mechanism underlying the absence of arachidonic acid (AA ) release by TNF in TNF-resistant cells, we first performed comparative ana lysis of phospholipid pools in both TNF sensitive (MCF7) and their equivale nt resistant cells (C1001), Quantification and incorporation studies of [H- 3]AA indicated that TNF-resistant cells were not depleted in AA, Furthermor e, distribution of this fatty acid in different phospholipid pools was simi lar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A(2) (PLA(2)) are the mai n enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA releas e. For this purpose, we used two PLA(2) inhibitors, methylarachidonyl fluor ophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreve rsibly inhibit the cytosolic PLA(2) (cPLA(2)) and the Ca2+-independent PLA( 2), respectively. Although a significant inhibitory effect of IMAFP on both TNF-induced AA release and PLA, activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA(2) activity correlated with a n inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA(2) in TNF-sensitive vs TNF-res istant cells. Although the p70 (70-kDa) form of cPLA(2) was specifically in creased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selective ly observed in TNF-resistant C1001 tells in the presence or absence of TNF, These findings suggest that the acquisition of cell resistance to this cyt okine mag. involve an abnormal cPLA(2) cleavage.