Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway

Adenine deoxynucleosides, such as 2-chloradeoxyadenosine (2CdA) and fludara bine, induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases, We previously d emonstrated that that the 5'-triphosphate metabolite of 2CdA (2CdATP), simi lar to dATP, can co-operate with cytochrome c and apoptosis protein-activat ing factor-1 (APAF-1) to trigger a caspase pathway in a HeLa cell-free syst em. We used a fluorometry-based assay of caspase activation to extend the a nalysis to several other clinically relevant adenine deoxynucleotides in B- chronic lymphocytic leukemia extracts. The nucleotide-induced caspase activ ation displayed typical Michaelis-Menten kinetics. As estimated by the V-ma x/K-m ratios, the relative efficiencies of different nucleotides were Ara-A TP > 9-fluoro-9-beta-D-arabinofuranosyladenine 5'-triphosphate > dATP > SCd ATP > 9-beta-D-arabinofuranosylguanine 5'-triphosphate > dADP > ATP. In con trast to dADP, both ADP and its nonhydrolyzable alpha,beta-methylphosphonat e 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 APA F-1-dependent caspase activation is due bath to stimulation by the 5'-triph osphates and lack of inhibition by the 5'-diphosphates, The capacity of ade nine deoxynucleoside metabolites to activate the apoptosome pathway may be an additional biochemical mechanism that plays a role in the chemotherapy o f indolent lymphoproliferative diseases.