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Potentiation of apoptosis by mitochondria in a cell-free system
P Juin1, K Tremblais, M T LeCabellec
1Unité INSERM 419, Nantes, France.
Abstract:
Using a cell-free system, we show that rat liver mitochondria, but not mitochondrial extracts, potentiated apoptosis triggered by cytosols derived from apoptotic cells. Apoptosis potentiated by mitochondria appeared to be inhibited by caspase 3 but not by caspase 1 inhibitors. A cytosolic caspase-3-like activity was increased by the addition of mitochondria to apoptotic cytosols; the latter activation was inhibited by the addition of bcl-2. Chelation of calcium by EGTA significantly and specifically inhibited the apoptosis potentiated by mitochondria as well as the increase of caspase-3-like activity. The incubation of mitochondria with apoptotic cytosols led to the release of cytochrome c, this latter phenomenon being inhibited by EGTA. Calcium or cytochrome c and dATP, however, did not reproduce the mitochondrial potentiation in the absence of the organelle. Thus, mitochondria can initiate and potentiate apoptosis through similar but not identical mechanisms.
Insights
Rat liver mitochondria potentiate apoptosis in a cell-free system. This process involves calcium, caspase-3-like activity, and cytochrome c release, highlighting mitochondria's role in programmed cell death.
Area of Science:
- Cell biology
- Biochemistry
- Molecular biology
Background:
- Mitochondria play a crucial role in cellular processes, including programmed cell death (apoptosis).
- The precise mechanisms by which mitochondria influence apoptosis, particularly in cell-free systems, require further elucidation.
Purpose of the Study:
- To investigate the role of intact rat liver mitochondria in potentiating apoptosis using a cell-free system.
- To identify key molecular players and signaling pathways involved in mitochondrial-mediated apoptosis potentiation.
Main Methods:
- Utilized a cell-free system with cytosols from apoptotic cells and intact rat liver mitochondria.
- Assessed apoptosis potentiation using caspase inhibitors (caspase 3, caspase 1) and bcl-2.
- Measured caspase-3-like activity and monitored cytochrome c release.
- Investigated the role of calcium using EGTA chelation.
- Tested the effects of calcium, cytochrome c, and dATP in the absence of intact mitochondria.
Main Results:
- Intact mitochondria, but not mitochondrial extracts, potentiated apoptosis.
- Mitochondrial potentiation of apoptosis was inhibited by caspase 3 inhibitors and bcl-2.
- Addition of mitochondria increased cytosolic caspase-3-like activity, which was calcium-dependent.
- EGTA inhibited mitochondrial potentiation of apoptosis and caspase-3-like activity.
- Mitochondria incubation with apoptotic cytosols led to EGTA-inhibitable cytochrome c release.
Conclusions:
- Rat liver mitochondria can initiate and potentiate apoptosis in a cell-free system.
- Calcium and cytochrome c release are critical mediators of mitochondrial-driven apoptosis.
- Mitochondria utilize distinct but overlapping mechanisms to initiate and potentiate apoptosis.