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Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3
T Miyashita1, K Nagao, S Krajewski
1Department of Genetics, National Children's Medical Research Center, Tokyo, Japan. tmiyashita@nch.go.jp
Abstract:
Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.
Insights
Glucocorticoids induce apoptosis in lymphoid cells via mitochondrial damage. Bcl-2 and caspase inhibitors prevent this, suggesting caspase-3 activation is downstream of mitochondrial events in glucocorticoid-induced cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Glucocorticoids (GCs) are vital for treating lymphomas and leukemias.
- GCs induce apoptosis, a programmed cell death, in specific lymphoid cells.
- Bcl-2 protein completely inhibits GC-induced apoptosis.
Purpose of the Study:
- To investigate the role of Bcl-2 and caspases in GC-mediated apoptosis.
- To determine the sequence of events involving mitochondrial potential, reactive oxygen species (ROS), and caspase activation.
- To elucidate the specific caspases involved in GC-induced lymphoid cell death.
Main Methods:
- Utilized broad-spectrum caspase inhibitor (zVAD-fmk) and caspase-3 specific inhibitor (Ac-DEVD-CHO).
- Assessed mitochondrial membrane potential (Δψm) and ROS production.
- Analyzed caspase cleavage/activation, PARP proteolysis, and DNA fragmentation.
Main Results:
- Bcl-2 and zVAD-fmk prevented loss of Δψm and ROS production induced by GCs.
- Ac-DEVD-CHO did not inhibit Δψm loss or ROS production.
- Caspase-6, but not caspase-3, was activated; PARP was proteolyzed.
- DNA fragmentation was inhibited by Ac-DEVD-CHO, but plasma membrane damage (cell death) was only marginally affected.
Conclusions:
- Bcl-2 inhibition and initiator caspase activation precede mitochondrial damage in GC-induced apoptosis.
- Caspase-3 activation occurs downstream of mitochondrial events.
- Caspase-6 plays a role in GC-mediated apoptosis, while caspase-3's role in overall cell death is limited.
- GC-induced apoptosis involves complex signaling pathways impacting mitochondria and downstream caspase activation.