Abstract
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.
Supported in part by Grant CA 28596 from the National Cancer Institute, Department of Health and Human Services, and Grant CH-130 from the American Cancer Society.
