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 (DeltaPsim). 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.
Adenosylhomocysteinase ; Amino Acid Chloromethyl Ketones ; Apoptosis ; bcl-2-Associated X Protein ; bcl-X Protein ; Cell Death ; Cytochromes c ; Cytosol ; HL-60 Cells ; Humans ; Membrane Potential, Mitochondrial ; Mitochondria ; Tubercidin

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.
Amino Acid Chloromethyl Ketones/pharmacology ; Apoptosis/*drug effects ; Bongkrekic Acid/pharmacology ; Caspases/*metabolism ; Cell Line ; Cyclosporine/pharmacology ; Cytochrome c/drug effects/metabolism ; Enzyme Activation ; Human ; Leukocytes, Mononuclear/cytology ; Ligands ; Membrane Glycoproteins/metabolism ; Tubercidin/*pharmacology ; U937 Cells

3-Deazaadenosine (DZA), one of the potent inhibitors of S-adenosylhomocysteine hydrolase, is known to possess several biological properties including an induction of apoptosis. To evaluate a possibility that DZA may be utilized for the treatment of human leukemia, we studied molecular events of cell death induced by DZA in human leukemia HL-60 and U-937 cells. DZA induced a specific cleavage of poly ADP-ribose polymerase (PARP) and an activation of the cysteine protease caspase-3/CPP32 which is known to cleave PARP. DZA-mediated nuclear DNA-fragmentation was completely blocked in the presence of a universal inhibitor of caspases (z-VAD-fmk) or the specific inhibitor of caspase-3 (z-DEVD-fmk) unlike of cycloheximide (CHX). DNA fragmentation was preceded by the lowering of c-myc mRNA in the DZA treated cells. In addition, DZA-induced apoptosis was blocked by pretreatment with adenosine transporter inhibitors such as nitrobenzylthioinosine (NBTI) and dipyridamole (DPD). Taken together, these results demonstrate that DZA-induced apoptosis initiated through an active transport of DZA into human leukemia cells, is dependent on the caspase-3-like activity without de novo synthesis of proteins and possibly involves c-myc down-regulation.
Adenosine/metabolism ; *Apoptosis ; Biological Transport, Active ; Carrier Proteins/metabolism ; Caspases/*metabolism ; Down-Regulation ; Enzyme Activation ; Genes, myc ; HL-60 Cells ; Human ; Leukemia, Promyelocytic, Acute/*drug therapy ; Thioinosine/*analogs & derivatives/pharmacology ; Transcription Factors/genetics ; Tubercidin/*pharmacology ; U937 Cells