cladribine-triphosphate and 2--deoxyadenosine-triphosphate

The purine nucleoside 2-chlorodeoxyadenosine (CdA) is often used in leukemia therapy. Its efficacy, however, is compromised by the emergence of resistant cells. In the present study, 3 CdA-resistant cell lines were generated and characterized. Their ability to accumulate 2-chloroadenosine triphosphate (CdATP) varied, reflecting differences in activities of deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Nonetheless, the selected lines were uniformly resistant to CdA-induced apoptosis, as assessed by caspase activation and DNA fragmentation. In contrast, cytosols from resistant cells were capable of robust caspase activation when incubated in the presence of cytochrome c and dATP. Moreover, replacement of dATP with CdATP also resulted in caspase activation in the parental and some of the resistant cell lines. Strikingly, CdA-induced decreases in mitochondrial transmembrane potential and release of cytochrome c from mitochondria were observed in the parental cells but not in any resistant lines. The lack of cytochrome c release correlated with an increased ability of mitochondria from resistant cells to sequester free Ca2+. Consistent with this enhanced Ca2+ buffering capacity, an early increase in cytosolic Ca2+ after CdA treatment of parental cells but not resistant cells was detected. Furthermore, CdA-resistant cells were selectively cross-resistant to thapsigargin but not to staurosporine- or Fas-induced apoptosis. In addition, CdA-induced caspase-3 activation and DNA fragmentation were inhibited by the Ca2+ chelator BAPTA-AM in sensitive cells. Taken together, the data indicate that the mechanism of resistance to CdA may be dictated by changes in Ca2+-sensitive mitochondrial events.

Topics: Adenosine Triphosphate; Antimetabolites, Antineoplastic; Apoptosis; Calcium Signaling; Caspases; Chelating Agents; Chromatography, High Pressure Liquid; Cladribine; Cytochrome c Group; Deoxyadenine Nucleotides; Deoxycytidine Kinase; DNA Fragmentation; Drug Resistance, Neoplasm; Egtazic Acid; Enzyme Activation; Intracellular Membranes; Membrane Potentials; Mitochondria; Neoplasm Proteins; Neoplastic Stem Cells; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-bcl-2; Staurosporine; Thapsigargin