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Extent and limitation of the control of nuclear apoptosis by DNA-fragmenting factor
1Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Trogerstrasse 9, Munich, 81675, Germany.
Abstract:
During apoptosis, changes to the nucleus of the dying cell include DNA degradation and structural collapse. These changes are accomplished by caspase-mediated cleavage of DNA-fragmenting factor DFF45, an inhibitor of the effector molecule DFF40. DFF45 and, more efficiently, a mutant lacking one caspase-cleavage site (DFF45m) inhibited nuclear changes in a cell-free system when apoptosis was initiated by adding caspase-3 to cell extracts. In primary tissues from several mammalian species, human caspase-3 activated and human DFF45m blocked nuclear apoptosis demonstrating evolutionary conservation of this step. However, DFF45m did not significantly inhibit DNA-fragmenting activity in extracts from staurosporine-treated cells from the human cell line Jurkat. In extracts from normal Jurkat cells, DFF45m blocked caspase-triggered DNA cleavage efficiently only if added within a short time of the addition of the caspase. At later time points, this inhibition by DFF45m was strongly reduced in efficiency while Zn2+ still completely blocked DNA fragmentation. These results demonstrate the evolutionary conservation of a linear pathway in apoptosis and suggest the existence of more complex events as final effector machinery.
Insights
This study shows that the inhibition of DNA fragmentation during apoptosis is conserved across species. However, the efficiency of this inhibition decreases over time, suggesting complex cellular events.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis involves nuclear DNA degradation and structural collapse.
- Caspase-mediated cleavage of DNA-fragmenting factor 45 (DFF45) activates the effector molecule DFF40.
- DFF45 regulates nuclear changes during programmed cell death.
Purpose of the Study:
- To investigate the role of DFF45 in apoptosis.
- To determine the evolutionary conservation of caspase-mediated nuclear apoptosis.
- To explore the kinetics of DFF45 inhibition in different cellular contexts.
Main Methods:
- Utilized a cell-free system with cell extracts.
- Initiated apoptosis by adding caspase-3.
- Employed a DFF45 mutant (DFF45m) lacking a caspase-cleavage site.
- Tested inhibition in primary mammalian tissues and Jurkat cell lines.
- Assessed DNA-fragmenting activity and inhibition over time.
Main Results:
- DFF45 and DFF45m inhibited nuclear changes in cell-free extracts.
- Human caspase-3 activated and human DFF45m blocked apoptosis in mammalian tissues, showing evolutionary conservation.
- DFF45m showed reduced inhibition in staurosporine-treated Jurkat cells.
- In normal Jurkat cells, DFF45m inhibition was time-dependent and less effective at later time points compared to Zn2+.
Conclusions:
- A conserved linear pathway exists for apoptosis.
- The final effector machinery of apoptosis may involve more complex events.
- The efficiency of DFF45-mediated inhibition is influenced by time and cellular conditions.