Potentiation of 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial damage and apoptosis in human leukemia cells (U937) overexpressing Bcl-2 by the kinase inhibitor 7-hydroxystaurosporine (UCN-01)

Antileukemic interactions between the nucleoside analog 1-beta-D-arabinofur anosylcytosine (ara-C) and the kinase inhibitor 7-hydroxystaurosporine (UCN -01) have been examined in relation to Bcl-2 expression/phosphodilation, mi tochondrial damage, caspase activation, and loss of clonogenic potential. S ubsequent exposure of ara-C-pretreated U931 cells (1 mu M; 6 hr) to UCN-01 (300 nM; 24 kr) resulted in marked potentiation of pro-caspase-3 and -9 cle avage/activation, poly(ADP-ribose)polymerase degradation, diminished mitoch ondrial membrane potential (Delta psi(m)), enhanced cytochrome c release, r eduction in the S-phase fraction, and induction of classic apoptotic morpho logic features. Enforced expression of full-length Bcl-2 significantly prot ected cells (at 24 hr) from ara-C/UCN-01-induced caspase activation and apo ptosis, but was ineffective in preventing loss of Delta psi(m) and cytochro me c release. Ectopic expression of a Bcl-2 N-terminal phosphorylation loop -deleted protein (Bcl-2 Delta(32-80)) was more potent than its full-length counterpart in blocking drug-induced loss of Delta psi(m), caspase activati on, and apoptotic morphology, but not cytochrome c release. Examination of cells at later intervals revealed that ectopic expression of Bcl-2 or Bcl-2 Delta(32-80) could only delay, but not prevent, mitochondrial damage, casp ase activation, and cell death induced by ara-C/UCN-01 treatment. Despite t heir initial ability to inhibit apoptosis, neither full length nor truncate d Bcl-2 protein restored clonogenic potential to drug-treated cells. These findings indicate that subsequent exposure of ara-C pretreated human leukem ia cells to UCN-01 potently triggers mitochondrial damage and apoptosis, an d that these events are postponed but not prevented by ectopic expression o f Bcl-2 or its phosphorylation loop-deleted counterpart. (C) 2000 Elsevier Science Inc.