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Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas
1Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan.
Abstract:
CPP32/apopain (Caspase-3), a protease of the Ced-3/ICE family, is a central mediator in the apoptosis induced by TNF or anti-Fas. In this study we demonstrate that wortmannin, an inhibitor of PI-3K, enhances the activation of CPP32 (Caspase-3) and DNA fragmentation in TNF-treated U937 cells and anti-Fas-treated Jurkat cells. Caspase-3-like activity, Ac-DEVD-MCA cleavage activity, is enhanced by wortmannin in the range of the concentration (1 - 100 nM) specifically inhibiting PI-3K. LY294002, another PI-3K inhibitor, also enhances Caspase-3-like activity, but inhibitors for myosin light chain kinase and calmodulin dependent kinase do not have any effect on the Caspase-3-like activity. Wortmannin (1 - 100 nM) enhances the processing of Caspase-3 (32K) into active form (17K) in TNF- or anti-Fas-treated cells, but not in untreated cells. These observations suggest that inhibition of PI-3K induces the activation of processing enzyme of Caspase-3 or increases the susceptibility of Caspase-3 to the processing enzyme. PI-3K seems to protect the cells from apoptosis by suppressing the activation of Caspase-3.
Insights
Phosphoinositide 3-kinase (PI-3K) inhibition enhances apoptosis by increasing Caspase-3 activation. PI-3K appears to protect cells from programmed cell death by suppressing Caspase-3 processing.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspase-3 (CPP32/apopain), a key protease in the Ced-3/ICE family, mediates apoptosis induced by tumor necrosis factor (TNF) or anti-Fas receptor activation.
- Phosphoinositide 3-kinase (PI-3K) is a signaling enzyme involved in various cellular processes, including cell survival and proliferation.
Purpose of the Study:
- To investigate the role of PI-3K in the regulation of Caspase-3 activation and apoptosis.
- To determine if PI-3K inhibition affects the processing and activity of Caspase-3.
Main Methods:
- Treatment of U937 and Jurkat cells with TNF or anti-Fas, respectively.
- Application of wortmannin and LY294002, specific inhibitors of PI-3K, at concentrations of 1-100 nM.
- Assay of Caspase-3-like activity (Ac-DEVD-MCA cleavage) and analysis of Caspase-3 processing (32 kDa to 17 kDa conversion).
Main Results:
- Wortmannin significantly enhanced Caspase-3 activation and DNA fragmentation in TNF- or anti-Fas-treated cells.
- PI-3K inhibition by wortmannin and LY294002 increased Caspase-3-like activity in a dose-dependent manner.
- Wortmannin promoted the conversion of pro-Caspase-3 (32 kDa) to its active form (17 kDa) in treated cells.
Conclusions:
- Inhibition of PI-3K potentiates Caspase-3 activation and processing, suggesting a role in apoptosis induction.
- PI-3K activity appears to suppress Caspase-3 activation, thereby protecting cells from apoptosis.
- Targeting PI-3K may represent a therapeutic strategy to enhance apoptosis in certain cellular contexts.