Resistance to TNF-Induced Cytotoxicity Correlates with an Abnormal Cleavage of Cytosolic Phospholipase A2

Abstract

To investigate the mechanism underlying the absence of arachidonic acid (AA) release by TNF in TNF-resistant cells, we first performed comparative analysis of phospholipid pools in both TNF-sensitive (MCF7) and their equivalent resistant cells (C1001). Quantification and incorporation studies of [3H]AA indicated that TNF-resistant cells were not depleted in AA. Furthermore, distribution of this fatty acid in different phospholipid pools was similar in both sensitive cells and their resistant counterparts, ruling out a defect in phospholipid pools. Since phospholipase A2 (PLA2) are the main enzymes releasing free AA, we investigated their relative contribution in the acquisition of cell resistance to TNF-induced cell death and AA release. For this purpose, we used two PLA2 inhibitors, methylarachidonyl fluorophosphate (MAFP) and bromoenol lactone (BEL), which selectively and irreversibly inhibit the cytosolic PLA2 (cPLA2) and the Ca2+-independent PLA2, respectively. Although a significant inhibitory effect of MAFP on both TNF-induced AA release and PLA2 activity in MCF7 was observed, BEL had no effect. The inhibitory effect of MAFP on cPLA2 activity correlated with an inhibition of TNF-induced cell death. Western blot analysis revealed that TNF induced a differential cleavage of cPLA2 in TNF-sensitive vs TNF-resistant cells. Although the p70 (70-kDa) form of cPLA2 was specifically increased in TNF-sensitive cells, a cleaved form, p50 (50 kDa), was selectively observed in TNF-resistant C1001 cells in the presence or absence of TNF. These findings suggest that the acquisition of cell resistance to this cytokine may involve an abnormal cPLA2 cleavage.