Induction of an apoptotic program in cell-free extracts by 2-chloro-2′-deoxyadenosine 5′-triphosphate and cytochrome c

Abstract

Adenine deoxynucleosides, such as 2-chloro-2-deoxyadenosine (2CdA) induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. However, it has remained puzzling why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis. The present experiments demonstrate that the 5-triphosphate metabolite of 2CdA (2CdA-5-triphosphate), similar to dATP, can cooperate with cytochrome c and Apaf-1 to activate caspase-3 in a cell free system. Chronic lymphocytic leukemia cells and normal peripheral blood lymphocytes expressed both caspase-3 and apoptotic protease activating factor 1. Incubation of the lymphocytes with 2CdA induced caspase-3 activation prior to DNA degradation and cell death. Stimulation of the caspase proteolytic cascade by 2CdA-5-triphosphate, in the context of DNA strand break formation, may provide an explanation for the potent cytotoxic effects of 2CdA toward nondividing lymphocytes.The effectiveness of cancer chemotherapy often depends upon the induction of apoptosis in malignant cells. Among antimetabolites, the 2Ј-deoxyadenosine congeners 2-chloro-2Ј-deoxyadenosine (2CdA, cladribine) and 9-␤-D-arabinofuranosyl-2-fluoroadenine (fludarabine) have the ability to induce apoptosis in nondividing lymphocytes, at concentrations that spare other cell types (1). For this reason, the deoxyadenosine analogs have achieved an important place in the treatment of indolent lymphoid malignancies, including hairy cell leukemia, chronic lymphocytic leukemia (CLL), and low grade lymphoma (2, 3).The cytotoxicity of 2CdA depends mainly upon the selective and progressive accumulation of its 5Ј-triphosphate metabolite (2CdATP) in lymphocytes that have a high ratio of deoxycytidine kinase (EC 2.7.1.74) to cytosolic 5Ј-nucleotidase (EC 3.1.3.5), compared with other cell types (1, 4). However, why 2CdATP triggers apoptosis in non-dividing cells is unclear.Various stimuli of apoptosis lead to the activation in the cytoplasm of cysteine proteases with specificity for aspartic acid residues, referred to as caspases. The activated caspases can cleave structural proteins and enzymes necessary for the survival of both proliferating and resting cells (reviewed in refs. 5-7). In addition, caspases have been shown to activate the endonuclease responsible for the internucleosomal cleavage of genomic DNA, a hallmark of apoptosis (8,9).One important component of the caspase cascade is caspase-3, which is activated by two sequential proteolytic events that cleave the 32-kDa precursor at aspartic acid residues to generate an active heterodimer of 20-and 12-kDa subunits (10). The activation can either be autocatalytic, or occur via a caspase cascade, similar to the serine protease cascade in the blood clotting process (7). In susceptible cells, caspase activation might amplify preexisting but sublethal apoptotic signals, leading to rapid and irreversible proteolysis.Recently, Wang and coworkers established a cell free system in ...

Nucleoside analogs: molecular mechanisms signaling cell death

“…Cytochrome c binds with Apaf-1, pro-caspase-9 and dATP to form the apoptosome, which activates caspase-9 to initiate the intrinsic cell death program (Riedl and Salvesen, 2007). In addition, the triphosphates of the deoxyadenosine nucleoside analogs can substitute for dATP and thus further tip the balance toward apoptosis by promoting apoptosome formation (Leoni et al, 1998;Genini et al, 2000b). Caspase-9 activation leads to the activating cleavage of executioner caspases, such as caspase-3 and caspase-7, an irreversible event leading to DNA endonuclease activation, DNA fragmentation and eventual cell death by apoptosis.…”

2-Chloro-2′-deoxyadenosine induces apoptosis through the Fas/Fas ligand pathway in human leukemia cell line MOLT-4

“…From these results, it was concluded that the expression of Fas/Fas-L contributed to the activation of caspases-8 and -3 in 2CdA-treated MOLT-4 cells, although Fas/Fas-L/caspase-8-independent activation of caspase-3 might exist. 31 …”

“…These two fragments then form a heterotetramer, which comprises the active enzyme. 29,30 Leoni et al 31 proposed that 2CdA-induced apoptosis was mediated by this Apaf-related pathway because they demonstrated that 2CdATP, instead of dATP, could act as an activator of caspase-3 in a cell-free system. In contrast, our preliminary experiments revealed that the protein synthesis inhibitor cycloheximide (CHX) significantly inhibited 2CdA-induced apoptosis in human leukemia cell line, MOLT-4.…”

Mechanisms of anti-cancer action and pharmacology of clofarabine

“…The damage caused by incorporation of clofarabine monophosphate into DNA initiates a chain of events that result in activation of pro-apoptotic pathways. Clofarabine can replace dATP and directly affects cytosolic apoptotic protease-activating factor-1 (APAF1), thus causing caspase activation [28,29]. Clofarabine also directly affects mitochondria by altering the transmembrane potential and, as a result, releasing cytochrome c and apoptosis-inducing factor (AIF) [30] …”