Biochemical and kinetic characterization of the glucocorticoid-induced apoptosis of immature CD4+ CD8+ thymocytes

We characterized kinetic and biochemical changes during glucocorticoid (GC)-induced apoptosis of immature CD8(+)CD4(+) double-positive (DP) thymocytes, A GC analog dexamethasone (Dex) induced rapid apoptotic commitment and a transient up-regulation of the NF-kappa B/RelA-p50-binding activity in DP cells. This required an early activation of proteasome, as judged by the ability of a specific proteasomal inhibitor, lactacystine, to delay apoptosis and to suppress Dex-dependent NF-kappa B activation. Dex-induced apoptotic commitment was preceded by the rapid (3 h) cleavage of both a typical caspase substrate, poly(ADP-ribose) polymerase (PARP), and of nuclear transcription factors AP-1, NF-kappa B p50-p50 and NUR-77, By contrast, phorbol myristate acetate (PMA) and/or ionomycin-induced apoptosis had much slower kinetics, were preceded by an early increase of NF-kappa B/RelA-p50, AP-1 and NUR-77 activities, and were insensitive to proteasome inhibition. Both the transgenic Bcl-2 and zVAD-fmk, an inhibitor of caspases, affected all features of Dex-induced apoptosis in a similar fashion, by inhibiting cell death and PARP cleavage, and by stabilizing AP-1, NF-kappa B p50-p50 and NUR-77 levels. Furthermore, Bcl-2 prevented Dex-induced RelA-p50 activation. However, a higher gene dosage of the transgenic Bcl-2 was required for protection against Dex, compared to the PMA and/or ionomycin-induced apoptosis, These findings highlight the unique mechanistic features of GC-induced apoptosis.