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Calcium-activated DNA fragmentation kills immature thymocytes
D J McConkey1, P Hartzell, P Nicotera
1Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Abstract:
Glucocorticoid hormones kill immature thymocytes by activating a self-destructive process that involves extensive DNA fragmentation. It has been demonstrated that thymocyte suicide is dependent on an early, sustained increase in cytosolic Ca2+ concentration, and new protein synthesis, but the biochemical lesion that leads to cell death has not been established. To determine whether endonuclease activation or activation of another Ca2+-dependent process could mediate cell killing, we treated thymocytes with the glucocorticoid methylprednisolone in the presence of inhibitors of various Ca2+-dependent degradative enzymes. The role of poly(ADP-ribose) polymerase, an enzyme known to be activated by DNA damage, was also assessed. Glucocorticoid-induced chromatin cleavage and cell killing were blocked by the endonuclease inhibitor aurintricarboxylic acid, whereas inhibitors of other Ca2+-dependent degradative processes or of poly(ADP-ribose) polymerase did not abrogate cell death. In addition, stimulation of thymocyte DNA fragmentation by the Ca2+ ionophore A23187 resulted in cell killing that could be blocked by the endonuclease inhibitor. Together, our results suggest that thymocyte suicide is caused by extensive Ca2+-stimulated DNA fragmentation.
Insights
Glucocorticoids induce thymocyte apoptosis via DNA fragmentation. This cell death pathway is mediated by calcium (Ca2+)-stimulated endonuclease activation, not other degradative enzymes.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Glucocorticoid hormones induce apoptosis in immature thymocytes.
- This process involves DNA fragmentation and requires increased cytosolic calcium (Ca2+) and protein synthesis.
- The precise biochemical mechanism leading to cell death remains unclear.
Purpose of the Study:
- To investigate the role of endonuclease activation in glucocorticoid-induced thymocyte apoptosis.
- To determine if Ca2+-dependent degradative enzymes mediate this cell death pathway.
- To assess the involvement of poly(ADP-ribose) polymerase.
Main Methods:
- Thymocytes were treated with methylprednisolone in the presence of specific enzyme inhibitors.
- The Ca2+ ionophore A23187 was used to stimulate DNA fragmentation.
- The effects of endonuclease inhibitors and other enzyme inhibitors on cell death and DNA fragmentation were evaluated.
Main Results:
- Glucocorticoid-induced chromatin cleavage and thymocyte killing were inhibited by aurintricarboxylic acid, an endonuclease inhibitor.
- Inhibitors of other Ca2+-dependent enzymes or poly(ADP-ribose) polymerase did not prevent cell death.
- Ca2+ ionophore A23187-induced DNA fragmentation and cell killing were also blocked by the endonuclease inhibitor.
Conclusions:
- Glucocorticoid-induced thymocyte apoptosis is mediated by Ca2+-stimulated DNA fragmentation.
- Endonuclease activation plays a critical role in this programmed cell death pathway.
- The findings elucidate a key mechanism in glucocorticoid-induced thymocyte elimination.