Related Experiment Videos
Failure to activate cytosolic phospholipase A2 causes TNF resistance in human leukemic cells
Y L Wu1, X R Jiang, A C Newland
1Department of Hematology, St. Bartholomew's and Royal London School of Medicine and Dentistry, University of London, United Kingdom.
Abstract:
Activation of cytosolic phospholipase A2 (cPLA2) by TNF has been shown to be an important component of the signaling pathway leading to cell death. The role of cPLA2 in the cytotoxic action of TNF was investigated in a panel of human leukemic cell lines. TNF could activate cPLA2 only in U937 and HL60 TNF-sensitive leukemic cells, but not in KG1a, CEM, and CEM/VLB100 cells that are relatively resistant to TNF. Pretreatment with 4-bromophenacyl bromide, a cPLA2 inhibitor, rendered U937 and HL60 cell lines resistant to the cytotoxic effect of TNF. Immunoblot and reverse-transcriptase PCR demonstrated that cPLA2 expression was detectable at both transcriptional and translational levels in all leukemic cell lines studied, although CEM and CEM/VLB100 cells expressed cPLA2 mRNA and protein at lower levels. The protein synthesis inhibitor, cycloheximide, increased TNF-induced cPLA2 activity and cytotoxicity in both CEM and CEM/VLB100 cell lines. Low levels of cPLA2 activity in the KG1a cell line could be activated by the cPLA2 activator mellitin, or the calcium ionophore A23187. The data suggest that cPLA2 activity is involved in TNF-induced cytotoxicity in leukemic cells. Resistance to TNF-induced cytotoxicity may involve either protein inhibitors that act upstream of cPLA2 in the TNF-signaling pathway or constitutive defects of cPLA2 itself, possibly involving calcium utilization.
Insights
Cytosolic phospholipase A2 (cPLA2) activation is crucial for TNF-induced cell death in leukemia. Resistance to TNF cytotoxicity may stem from cPLA2 pathway defects or inhibitors.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Tumor necrosis factor (TNF) triggers cell death pathways.
- Cytosolic phospholipase A2 (cPLA2) activation is implicated in TNF-induced apoptosis.
Purpose of the Study:
- To investigate the role of cPLA2 in TNF-induced cytotoxicity in human leukemic cell lines.
- To explore mechanisms of resistance to TNF in leukemia.
Main Methods:
- Utilized human leukemic cell lines (U937, HL60, KG1a, CEM, CEM/VLB100).
- Assessed cPLA2 activation by TNF, its inhibition by 4-bromophenacyl bromide.
- Analyzed cPLA2 expression via immunoblot and RT-PCR.
- Investigated effects of cycloheximide, mellitin, and A23187 on cPLA2 activity and cytotoxicity.
Main Results:
- TNF activated cPLA2 in TNF-sensitive cells (U937, HL60) but not resistant ones (KG1a, CEM, CEM/VLB100).
- cPLA2 inhibition conferred resistance to TNF cytotoxicity.
- All cell lines expressed cPLA2, but resistant lines had lower levels.
- Cycloheximide enhanced TNF-induced cPLA2 activity and cytotoxicity in resistant cells.
- cPLA2 activity could be modulated by activators in resistant cells.
Conclusions:
- cPLA2 activity is essential for TNF-induced cytotoxicity in leukemic cells.
- Resistance mechanisms may involve upstream inhibitors or intrinsic cPLA2 defects, potentially related to calcium signaling.