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ATP-dependent steps in apoptotic signal transduction
Y Eguchi1, A Srinivasan, K J Tomaselli
1Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, Suita, Japan.
Abstract:
Apoptotic changes of the nucleus induced by Fas (Apo1/CD95) stimulation are completely blocked by reducing intracellular ATP level. In this study, we examined the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction using two cell lines. In SKW6.4 (type I) cells characterized by rapid formation of the death-inducing signaling complex on Fas treatment, the activation of caspases 8, 9, and 3, cleavage of DFF45 (ICAD), and release of cytochrome c from the mitochondria to the cytoplasm were not affected by reduction of intracellular ATP, although chromatin condensation and nuclear fragmentation were inhibited. On the other hand, in the Fas-mediated apoptosis of Jurkat (type II) cells, which is characterized by involvement of mitochondria and, thus, shares signal transduction mechanisms with apoptosis induced by other stimuli such as genotoxins, activation of the three caspases, cleavage of DFF45 (ICAD), and nuclear changes were blocked by reduction of intracellular ATP, whereas release of cytochrome c was not affected. These results suggested that the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction in type I cells are only located downstream of caspase 3 activation, whereas the activation of caspase 9 by released cytochrome c is the most upstream ATP-dependent step in type II cells. These observations also confirm the existence of two pathways for Fas-mediated apoptotic signal transduction and suggest that the Apaf-1 (Ced-4 homologue) system for caspase 9 activation operates in an ATP-dependent manner in vivo.
Insights
Adenosine triphosphate (ATP) levels are critical for Fas-mediated apoptosis. This study reveals distinct ATP-dependent steps in type I and type II cells, impacting caspase activation and nuclear changes during apoptosis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Fas (Apo1/CD95) stimulation triggers nuclear apoptotic changes.
- Intracellular adenosine triphosphate (ATP) levels are essential for these nuclear events.
- Understanding the ATP-dependent steps in Fas-mediated apoptosis is crucial.
Purpose of the Study:
- To investigate the ATP-dependent steps in Fas-mediated apoptotic signal transduction.
- To compare these ATP-dependent mechanisms in type I (SKW6.4) and type II (Jurkat) cells.
Main Methods:
- Utilized two cell lines (SKW6.4 and Jurkat) to study Fas-mediated apoptosis.
- Manipulated intracellular ATP levels to assess their impact on apoptotic signaling pathways.
- Monitored caspase activation (caspases 8, 9, and 3), cytochrome c release, DFF45 (ICAD) cleavage, and nuclear fragmentation.
Main Results:
- In type I cells, ATP reduction inhibited nuclear changes but not caspase activation or cytochrome c release.
- In type II cells, ATP reduction blocked caspase activation and nuclear changes, but not cytochrome c release.
- Identified distinct ATP-dependent steps downstream of caspase 3 in type I cells and upstream of caspase 9 activation in type II cells.
Conclusions:
- Fas-mediated apoptosis involves at least two distinct signaling pathways.
- Type I cells have ATP-dependent steps only after caspase 3 activation.
- Type II cells rely on ATP for caspase 9 activation, indicating the Apaf-1 system's ATP dependence in vivo.