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 journalArticle

Abstract

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)
Number of pages1
JournalExperimental Hematology
Volume25
Issue number8
StatePublished - Dec 1 1997

ASJC Scopus subject areas

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

Fingerprint Dive into the research topics of 'Overexpression of Bcl-x<sub>L</sub> inhibits ARA-C-induced mitochondrial perturbations that activate the molecular cascade of apoptosis'. Together they form a unique fingerprint.

Cite this