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Abstract
3-Deazaadenosine (DZA), a potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential (ΔΨm). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.
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Fig. 1.
Induction of caspase-dependent apoptosis in HL-60 cells by DZA. (A) HL-60 cells treated with the indicated micromolar concentrations of DZA for 6 h were fixed, stained with PI and then analyzed by flow cytometry as described in METHODS. (B) HL-60 cells treated the same as described in (A) were subjected to immunoblot analysis against the indicated procaspases. GAPDH was used for equal loading of proteins. (C) After incubating HL-60 cells in the absence or presence of 30μM z-VAD-Fmk for 1 h, they were treated with the indicated micromolar concentrations of DZA for 6 h further. The cells then were subjected to PI staining and flow cytometric analysis. The figures are representative of three independent experiments.
Fig. 2.
DZA-induced cyt c release in a PT- and caspase-independent manner. (A) Cytosolic and mitochondrial fractions of HL-60 cells treated with the indicated concentrations of DZA for 6 h were subjected to immunoblot analysis against cyt c. Porin and GAPDH were used for the equal loading of mitochondrial and cytosolic proteins, respectively. (B) HL-60 cells incubated in the absence or presence of 30μM z-VAD-Fmk for 1 h were treated with vehicle or 100μM DZA. Six hours later, cytosolic and mitochondrial fractions were prepared and subjected to immunoblot analysis against cyt c. Cyt c oxidase and GAPDH were used for equal loading of mitochondrial and cytosolic proteins, respectively. (C) HL-60 cells treated with vehicle, the indicated micromolar concentrations of DZA for 6 h or 50μM CCCP for 5 min were incubated with 2μM JC-1. After 15 min, cells were harvested, washed and analyzed on FACSCalibur with emission filters of FL1 and FL2 for green and red fluorescences, respectively. CCCP was used to confirm that JC-1 adequately reacted to MMP dissipation. The results are representative of three independent experiments.
Fig. 3.
DZA-induced BAX activation in HL-60 cells. (A) Subcellular fractions of HL-60 cells treated with indicated concentrations of DZA were prepared and subjected to immunoblot analysis against the indicated proteins. GAPDH and cyt c oxidase were used for equal loading of cytosolic and mitochondrial proteins, respectively. (B) Cytosolic and mitochondrial fractions of HL-60 cells incubated with vehicle or 30μM z-VAD-Fmk for 1 h and then 100μM DZA for 6 h further were prepared and subjected to immunoblot analysis. Cyt c oxidase and GAPDH were used for equal loading of mitochondrial and cytosolic fractions, respectively. (C) Left, HL-60 cells were treated with vehicle or 100μM DZA. Right, HL-60 cells incubated in the absence or presence of 30μM z-VAD-Fmk for 1 h were treated with DZA. After 6 h, cytosolic and mitochondrial fractions were incubated in the presence of 2 mM BMH as described in METHODS. Each fraction was subjected to immunoblot analysis against BAX and cyt c oxidase. The results are representative of three independent experiments.
Fig. 4.
Inhibition of DZA-induced apoptosis by BCL-XL. (A) HL-60 cells transiently transfected with empty pcDNA3.1(–) vector (EV) or plasmid expressing BCL-XL (pcDNA3.1-/BCL-XL) for 48 h were treated with vehicle or 100μM DZA. After 6 h, cytosolic fractions were subjected to immunoblot analysis against cyt c. GAPDH were used for equal loading of cytosolic proteins. (B) HL-60 cells transfected the same as in experiment (A) were treated with vehicle or 100μM DZA for 6 h. Mitochondrial fractions were then treated with 2 mM BMH as described in METHODS and subjected to immunoblot analysis against BAX. Cyt c oxidase was used for equal loading of mitochondrial proteins. (C) HL-60 cells transfected as indicated were treated with vehicle or 100μM DZA for 6 h and subjected to immunoblot analysis. GAPDH were used for equal loading of cell lysates. (D) HL-60 cells transiently transfected as indicated were treated with vehicle or 100μM DZA for 6 h. Cells were then fixed and subjected to PI staining and flow cytometric analysis. The figures are representative of three independent experiments.
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