Calcium-activated DNA fragmentation kills immature thymocytes

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.