1,25-DIHYDROXYVITAMIN D-3 PROTECTS HUMAN LEUKEMIC-CELLS FROM TUMOR NECROSIS FACTOR-INDUCED APOPTOSIS VIA INACTIVATION OF CYTOSOLIC PHOSPHOLIPASE A(2)

The mechanism by which tumor necrosis factor (TNF) induces death of ca ncer cells appears to involve the activation of cytosolic phospholipas e A(2) (cPLA(2)). U937 human leukemic cells treated with 1,25-dihydrox yvitamin D-3 [1,25(OH)(2)D-3; 10(-8) M] become resistant to TNF, an ef fect that is independent of cell cycle status and expression of TNF re ceptors or BCL-2, In this study, TNF produced a dose-and time-dependen t enhancement of [H-3]arachidonic acid release in U937 cells. The amou nt of [H-3]arachidonic acid release was positively associated with TNF -induced apoptosis, Both immunofluorescence microscopy and Western blo tting of cell subcompartments demonstrated translocation of cPLA(2) fr om the cytosol to the cell membrane in response to TNF, In addition, T NF up-regulated expression of cPLA(2) mRNA. An antisense oligonucleoti de to cPLA(2) and the cPLA(2) inhibitor 4-bromophenacyl bromide signif icantly inhibited TNF-induced cytotoxicity. Prior incubation of cells with 1,25(OH)(2)D-3 significantly inhibited (a) TNF-induced [H-3]arach idonic acid release and apoptosis, (b) TNF-induced translocation of cP LA(2) to the membrane, and (c) the up-regulation of cPLA(2) mRNA with TNF. Furthermore, the inhibitory effect of 1,25(OH)(2)D-3 was not reve rsed by inhibitors of transcription or translation, The data suggest t hat activation of cPLA(2) is involved in TNF-induced apoptosis of leuk emic cells, 1,25(OH)(2)D-3 directly inhibits cPLA(2) translocation and mRNA up-regulation induced by TNF, Disruption of cPLA(2) activation m ay represent a possible mechanism whereby leukemic cells can become re sistant to TNF-mediated killing.