Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway.
Adenine deoxynucleosides, such as 2-chlorodeoxyadenosine (2CdA) and fludarabine, induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. We previously demonstrated that that the 5'-triphosphate metabolite of 2CdA (2CdATP), similar to dATP, can cooperate with cytochrome c and apoptosis protein-activating factor-1 (APAF-1) to trigger a caspase pathway in a HeLa cell-free system. We used a fluorometry-based assay of caspase activation to extend the analysis to several other clinically relevant adenine deoxynucleotides in B-chronic lymphocytic leukemia extracts. The nucleotide-induced caspase activation displayed typical Michaelis-Menten kinetics. As estimated by the V(max)/K(m) ratios, the relative efficiencies of different nucleotides were Ara-ATP > 9-fluoro-9-beta-D-arabinofuranosyladenine 5'-triphosphate > dATP > 2CdATP > 9-beta-D-arabinofuranosylguanine 5'-triphosphate > dADP > ATP. In contrast to dADP, both ADP and its nonhydrolyzable alpha, beta-methylphosphonate analog were strong inhibitors of APAF-1-dependent caspase activation. The hierarchy of nucleotide activation was confirmed in a fully reconstituted system using recombinant APAF-1 and recombinant procaspase-9. These results suggest that the potency of adenine deoxynucleotides as co-factors for APAF-1-dependent caspase activation is due both to stimulation by the 5'-triphosphates and lack of inhibition by the 5'-diphosphates. The capacity of adenine deoxynucleoside metabolites to activate the apoptosome pathway may be an additional biochemical mechanism that plays a role in the chemotherapy of indolent lymphoproliferative diseases.
Genini
D
D
Department of Medicine and the Sam and Rose Stein Institute for Research on Aging, University of California San Diego, La Jolla, California 92093, USA.
Budihardjo
I
I
Plunkett
W
W
Wang
X
X
Carrera
C J
CJ
Cottam
H B
HB
Carson
D A
DA
Leoni
L M
LM
D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D003574:Cytochrome c Group / Q000494:pharmacology; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003839:Deoxyadenosines / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011684:Purine Nucleosides / Q000494:pharmacology*; D013329:Structure-Activity Relationship
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D003839:Deoxyadenosines; D004792:Enzyme Precursors; D011506:Proteins; D011684:Purine Nucleosides; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases