The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 muM), PD98059 (40 muM), or PD184352 (10 muM) with 10-100 muM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (Delta psi (m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and procaspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of Delta psi (m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.