Identification of the mechanism of activation of 9-beta-D-arabinofuranosyladenine in human lymphoid cells using mutants deficient in nucleoside kinases.
The biochemical basis of cellular resistance to 9-beta-D-arabinofuranosyladenine (ara-A) and its natural purine derivative, deoxyadenosine, was investigated with two mutants of cultured human T-lymphoblastoid CCRF-CEM cells. One mutant that lacked deoxycytidine kinase activity, designated CEM/ara-C, retained about 10% of wild-type deoxyadenosine kinase and deoxyguanosine kinase activity each but maintained normal adenosine kinase or thymidine kinase activity. This suggested that in these human T-lymphoblastoid cells, as in other previously studied mammalian cells, deoxycytidine and purine deoxyribonucleosides are phosphorylated by the same enzyme. Despite this extensive reduction of purine nucleoside kinase activities, the cytotoxicity of ara-A or deoxyadenosine was not appreciably affected, decreasing by only 2.5- and 6-fold, respectively. A second mutant, isolated by selecting CEM/ara-C mutants that were resistant to ara-A, showed a 100-fold increase in resistance to ara-A cytotoxicity. This ara-A-resistant subline was deficient in the activities of two enzymes, deoxycytidine kinase and adenosine kinase, and showed a high degree of resistance to deoxyadenosine, adenosine, and pyrazofurin but not to pyrimidine analogs, such as 5-fluorodeoxyuridine or 5-fluorouridine. Further studies of ara-A and deoxyadenosine phosphorylation in wild-type and resistant cell lines disclosed that, although deoxycytidine kinase is the principal enzyme for their phosphorylation in vitro, their intracellular conversion to cytotoxic nucleotides depends on the joint action of deoxycytidine kinase and adenosine kinase rather than purine-specific deoxynucleoside kinase, as previously thought.
D000248:Adenosine Kinase / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003839:Deoxyadenosines / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000378:metabolism; D004351:Drug Resistance; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D014740:Vidarabine / Q000378:metabolism* / Q000494:pharmacology
D003839:Deoxyadenosines; D010770:Phosphotransferases; D000248:Adenosine Kinase; D003842:Deoxycytidine Kinase; D014740:Vidarabine