The apoptotic cascade is regulated by both upstream Bcl-2 and the distal IAP family proteins. Mcl-1, a member of antiapoptotic Bcl-2 family proteins is highly expressed in various cancer cells. It was reported that its expression is elevated at the time of leukemic relapse (

Blood 1998;19:991–1000
). XIAP, the most potent cellular caspase inhibitor, inhibits both the initiator caspase-9 and the effector caspase-3 and suppresses apoptosis. Our previous study showed that decrease of XIAP by its antisense oligonucleotide enhances Ara-C-induced apoptosis (
Leukemia 2003;17:2081–2089
). In this study, we investigated the regulation of Mcl-1 expression, its potential as a therapeutic target, and apoptosis induction by simultaneous inhibition of Mcl-1 and XIAP in AML cells. We found that like survivin, Mcl-1 protein level is decreased by MEK inhibition in HL-60 and OCI-AML2 cells suggesting that Mcl-1 expression is regulated through the MAPK signaling pathway. However, in contrast to survivin levels, no significant differences in Mcl-1 expression in G0/G1, S, and G2M cells were observed in FACS-sorted HL-60 cells. Downregulation of Mcl-1 by its antisense oligonucleotide (Mcl-1-AS, 20408, ISIS Pharmaceuticals) induced cell death accompanied by decrease in mitochondrial membrane potential (MMP), caspase activation and annexin V positivity in HL-60 cells, while control oligonucleotide was not toxic. By itself, MEK inhibitor primarily induced G1/G0 cell cycle block and no apoptosis. When combined with Mcl-1-AS, MEK inhibition further decreased Mcl-1 protein levels and significantly increased Mcl-1-AS induced cell death (from 25.8±1.3% to 40±3.8%, p=0.02). Inhibition of XIAP by its antisense oligonucleotide (XIAP-AS, 102369, ISIS Pharmaceuticals) did not induce significant loss of MMP at 24 hrs and slightly increased annexin V positive cells at 48 hrs (14.5%±2.9% dead cells vs. 6.2%±0.72% in XIAP-NS treated cells). However, when combined with Mcl-1 inhibition, significant increases in loss of MMP at 24 hrs and annexin V positive cells at 48 hrs (p=0.003), more than inhibition of each protein alone, was observed indicating that activating the upstream and downstream apoptotic cascade will amplify the caspase activation loop and more efficiently induce cell death. Our study suggests that Mcl-1 is regulated by the MAPK signaling pathway. Its expression is cell cycle independent. Mcl-1 is essential for HL-60 cell survival and simultaneously downregulation of the upstream anti-apoptotic protein Mcl-1 and the downstream caspase inhibitor XIAP significantly enhances leukemia cell apoptosis.

Topics:
annexin a5, antiapoptotic agents, antisense oligonucleotides, apoptosis, birc4 gene, caspase inhibitors, caspase-3, caspase-9, caspases, leukemia

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2005, The American Society of Hematology
2005
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