Modulation of Arabinosylnucleoside Metabolism by Arabinosylnucleotides in Human Leukemia Cells

Previous studies have indicated that deoxycytidine kinase (dCK) is requisite and rate limiting in the phosphorylation of 1-β-D-arabinofuranosylcytosine (ara-C) and 9-β-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) on the pathway to their respective cytotoxic 5’-triphosphates. In K562 cells, the rate of triphosphate accumulation was maximal during incubation with 10 μM ara-C (35 μM/h) and 300 μm F-ara-A (102 μM/h). Under these conditions, accumulation of cellular ara-CTP plateaued at about 110 μm after 3 h, whereas in separate cultures, F-ara-ATP continued to accumulate at a linear rate to cellular concentrations greater than 500 μm after 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 μM ara-CTP. After washing into medium containing F-ara-A, the rate of F-ara-ATP accumulation was significantly decreased (37 μM/h). However, cells loaded with F-ara-ATP exhibited an increased rate of ara-CTP accumulation (110 μM/h) that resulted in cellular ara-CTP concentrations in excess of 400 μM after 5 h. This stimulation was proportional to the cellular concentration of F-ara-ATP, achieving a maximum effect between 75 and 100 μM. Phosphorylation of ara-C by cell-free extracts supplemented with physiological levels of ribo- and 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 μM ara-ctp suggests that this active triphosphate may limit its own synthesis 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.