Overexpression of Bcl-xL inhibits ARA-C-induced mitochondrial perturbations that activate the molecular cascade of apoptosis

C. N. Kim, X. Wang, Y. Huana, A. M. Ibrado, L. Liu, G. Fang, K. Bhalla

Research output: Contribution to journalArticlepeer-review


High-dose Ara-C (HIDAC) induces the cleavage and activity of caspase-3 (CPP32β/Yama/apopain), resulting in the degradation of specific substrates including lamins and poly (ADP-ribose) polymerase (PARP), producing the morphologic and biochemical features of apoptosis We have previously demonstrated that high levels of the anuapoptotic Bcl-xL or Bcl-2, relative to the proapoptotic Bax, inhibit HIDACinduced activity of caspase-3 and apoptosis in the control human AML HL-60/neo bui not in HL-60/Bcl-2 or HL-60/Bcl-xL cells, which possess enforced overexpression of Bcl-2 or Bcl-xL respectively, and display significantly lower ratio of free to bound Bax We also demonstrated that apoptotic stimuli cause the release of cytochrome c (cyt c) from the mitochondria into cytosol where, in the presence of dATP, it promotes the cleavage and activity of caspase-3 Overexpression of Bcl-2 or Bcl-xL was shown to block the mitcchondrial release of cyt c and apoptosis of HL-60/Bcl-2 orHL-60/Bcl-xL cells hi the present studies we determined the effect of 10 or 100 μM Ara-C for 4 hours on mitochondrial release of cyt c and other mitochondnal perturbations m HL-60/neo versus HL-60/Bcl-xL cells. The mitochondrial and cytosolic levels of cyt c were quantitated by immunoblot analysis. HIDAC-mduced loss of inner mitochondrial membrane potential (AYm) and the increase m reactive oxygen species (ROS) were determined by flow cytometnc analysis of the intracellular uptake of cationic tipophilic fluorochromes DiOC6(3) and hydroethidine (for ATM) or DCFH-DA (for ROS). Our results demonstrate that, in the initiation phase of apoptosis of HL-60/neo cells due to HIDAC there is, first, release of cyt c from the mitochondria to the cytosol (8-fold increment in the basal cytosolic levels), followed by the loss of ΔφPm and increase in the ROS, and these precede and trigger the cleavage and activity of caspase-3. These HIDAC-induced early mitochondrial and cytosolic perturbations were inhibited in HL60/Bcl-xL cells HODAC treatment for 4 hours also modestly increased the intracellular levels of free Bax relative to Bax bound to Bcl-2 and Bcl-xL in HL-60/neo but not in HL60/Bcl-xL cells. In HL-60/neo cells, HIDAC-induced progressive accumulation of cyt c in the cytosol as well as the decrease in ATm and increase in ROS were not inhibited by co-culture with the caspase inhibitor YVAD-cmk, which inhibits Ara-C-induced apoptosis These findings elucidate the molecular mechanism by which Bcl-xL inhibits HIDAC-induced mitochondrial and cytosolic perturbations, thereby preserving caspase3 in the inactive zymogen state and checking the molecular cascade of apoptosis.

Original languageEnglish (US)
Pages (from-to)783
Number of pages1
JournalExperimental Hematology
Issue number8
StatePublished - 1997

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research


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