FAILURE TO ACTIVATE CYTOSOLIC PHOSPHOLIPASE A(2) CAUSES TNF RESISTANCE IN HUMAN LEUKEMIC-CELLS

Activation of cytosolic phospholipase A(2) (cPLA(2)) by TNF has been s hown to be an important component of the signaling pathway leading to cell death. The role of cPLA(2) in the cytotoxic action of TNF was inv estigated in a panel of human leukemic cell lines, TNF could activate cPLA(2) only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, GEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA(2) inhibitor, rende red U937 and HL60 cell lines resistant to the cytotoxic effect of TNF, Immunoblot and reverse-transcriptase PCR demonstrated that cPLA(2) ex pression was detectable at both transcriptional and translational leve ls in all leukemic cell lines studied, although CEM and CEM/VLB100 cel ls expressed cPLA(2) mRNA and protein at lower levels. The protein syn thesis inhibitor, cycloheximide, increased TNF-induced cPLA(2) activit y and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels o f cPLA(2) activity in the KG1a cell line could be activated by the cPL A(2) activator mellitin, or the calcium ionophore A23187, The data sug gest that cPLA(2) activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve eit her protein inhibitors that act upstream of cPLA(2) in the TNF-signali ng pathway or constitutive defects of cPLA(2) itself, possibly involvi ng calcium utilization.