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Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA degradation
1Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
Glucocorticoids are well known to stimulate apoptosis in immature thymocytes. Apoptosis in this and other cells is characterized by cell shrinkage, DNA fragmentation and activation of a class of proteases named caspases. We have utilized the flow cytometer to evaluate the coordinate regulation of cell shrinkage and DNA fragmentation in glucocorticoid-treated rat thymocytes and explore the role of caspases upstream of both changes. The results indicate that the activation of apoptosis by glucocorticoids in a cell population is an asynchronous event with only a percentage of the cells displaying apoptotic characteristics at any given time. Both cell shrinkage and chromatin degradation are tightly coupled with similar proportions of the cells displaying each characteristic. The coordinate appearance of these characteristics may suggest a similar mechanism of regulation. Incubation of thymocytes with the general caspase inhibitor Z-VAD-FMK completely blocked both cell shrinkage and DNA fragmentation in spontaneous and glucocorticoid-induced thymocyte apoptosis, implicating an early upstream role for proteases in the activation of thymocyte apoptosis.
Insights
Glucocorticoids induce apoptosis in thymocytes, marked by cell shrinkage and DNA fragmentation. Caspases play an early, crucial role in this process, as inhibiting them blocks these apoptotic signs.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Glucocorticoids are known to induce apoptosis (programmed cell death) in immature thymocytes.
- Apoptosis involves characteristic features like cell shrinkage and DNA fragmentation.
- Caspases, a class of proteases, are implicated in the apoptotic pathway.
Purpose of the Study:
- To investigate the coordinated regulation of cell shrinkage and DNA fragmentation in glucocorticoid-induced thymocyte apoptosis using flow cytometry.
- To explore the role of caspases as upstream regulators of these apoptotic events.
Main Methods:
- Utilized flow cytometry to assess cell shrinkage and DNA fragmentation in rat thymocytes.
- Treated thymocytes with glucocorticoids to induce apoptosis.
- Employed the general caspase inhibitor Z-VAD-FMK to evaluate the role of caspases.
Main Results:
- Glucocorticoid-induced apoptosis in thymocytes is an asynchronous process, with varying percentages of cells showing apoptotic characteristics over time.
- Cell shrinkage and DNA fragmentation were found to be tightly coupled, appearing in similar proportions of cells.
- Complete inhibition of both spontaneous and glucocorticoid-induced thymocyte apoptosis, including cell shrinkage and DNA fragmentation, was observed upon incubation with Z-VAD-FMK.
Conclusions:
- Caspases play an early, upstream role in the activation of thymocyte apoptosis induced by glucocorticoids.
- The coordinated regulation of cell shrinkage and DNA fragmentation suggests a shared underlying mechanism in glucocorticoid-induced apoptosis.