1,25-Dihydroxyvitamin D₃ Protects Human Leukemic Cells from Tumor Necrosis Factor-induced Apoptosis via Inactivation of Cytosolic Phospholipase A₂

The mechanism by which tumor necrosis factor (TNF) induces death of cancer cells appears to involve the activation of cytosolic phospholipase A₂ (cPLA₂). U937 human leukemic cells treated with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃; 10^-8 m] become resistant to TNF, an effect that is independent of cell cycle status and expression of TNF receptors or BCL-2. In this study, TNF produced a dose- and time-dependent enhancement of [³H]arachidonic acid release in U937 cells. The amount of [³H]arachidonic acid release was positively associated with TNF-induced apoptosis. Both immunofluorescence microscopy and Western blotting of cell subcompartments demonstrated translocation of cPLA₂ from the cytosol to the cell membrane in response to TNF. In addition, TNF up-regulated expression of cPLA₂ mRNA. An antisense oligonucleotide to cPLA₂ and the cPLA₂ inhibitor 4-bromophenacyl bromide significantly inhibited TNF-induced cytotoxicity. Prior incubation of cells with 1,25(OH)₂D₃ significantly inhibited (a) TNF-induced [³H]arachidonic acid release and apoptosis, (b) TNF-induced translocation of cPLA₂ to the thermore, the inhibitory effect of 1,25(OH)₂D₃ was not reversed by inhibitors of transcription or translation. The data suggest that activation of cPLA₂ is involved in TNF-induced apoptosis of leukemic cells. 1,25(OH)₂D₃ directly inhibits cPLA₂ translocation and mRNA up-regulation induced by TNF. Disruption of cPLA₂ activation may represent a possible mechanism whereby leukemic cells can become resistant to TNF-mediated killing.