Modulation of Arabinosy Inucleoside Metabolism by Arabinosylnucleotides in Human Leukemia Cells1

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-0-D-arabinofuranosylcytosine (ara-C) and 9-/8-D-arabinofuranosyl-2-fluoroadenine (Fara-A) on the pathway to their respective cytotoxic 5'-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 //M ara-C (35 ¿(M/h) and 300 MMF-ara-A (102 fiM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 /IM after 3 h, whereas in separate cultures, F-araATP continued to accumulate at a linear rate to cellular concentrations greater than 500 »IMafter 5 h. Other laboratories have demonstrated that dCK activity in cell-free extracts was inhibited by ara-CTP. To determine whether ara-CTP exhibited the same activity in whole cells, K562 cells were preincubated with ara-C to accumulate 110 MMara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 fiM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumu lation (110 fiM/h) that resulted in cellular ara-CTP concentrations in excess of 400 JIM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 MM.Phosphorylation of ara-C by cell-free extracts supple mented with physiological levels of riboand deoxyribonucleoside 5'triphosphates was stimulated by addition of F-ara-ATP. The decreased rate of accumulation of products of dCK in intact cells containing 110 MM ara-CTP suggests that this active triphosphate may limit its own synthe sis and phosphorylation of other substrates. In contrast, stimulation of the accumulation of ara-CTP in cells containing F-ara-ATP suggests new possibilities for the design of combination chemotherapy regimens.