Regulation of 1-β-D-Arabinofuranosylcytosine 5–Triphosphate Accumulation in Human Leukemia Cells by Deoxycytidine 5–Triphosphate

Cell cycle-specific fluctuations in the ability of human leukemic cells to phosphorylate 1-β-D-arabinofuranosylcytosine (ara-C) to the toxic metabolite 1–β-D-arabinofuranosylcytosine 5–triphos–phate (ara-CTP) was investigated in whole cells and in cell extracts. Exponentially growing CCRF-CEM cells were fractionated into populations enriched for G1 phase cells and S phase cells by centrifugal elutriation. The accumulation of ara-CTP by S phase-enriched cells was 50% greater than in G1-enriched cells. However, the ability of extracts of S phase-enriched cells to phosphorylate ara-C was twice that of G1 phase-enriched cell extracts. As cells passed from G1 to S phase, this disproportionality was significant. As demonstrated in other cell types, deoxycytidine 5–triphos–phate (dCTP) also potently inhibited ara-C phosphorylation in CCRF-CEM cell extracts (K1 = 5.9 μm). deoxynucleotide pool levels determined by high pressure liquid chromatography showed a 5 μMdCTP concentration in Gi-enriched cells, whereas S phase-enriched cells contained 15 μm dCTP. These findings suggest that the lack of proportionality between the accumulation of ara-CTP in whole cells and the increase of ara-C phosphorylation in extracts during the G1 to S phase transition may be caused by more stringent regulation of ara-C phosphorylation in whole cells by the concomitant increase in cellular dCTP concentrations. Because such regulation is unlikely to be observed in cell extracts, these results indicated that assays of ara-C phosphorytating activity in cell extracts represent upper limits for that function in whole cells. Such determinations may not reflect the regulated nature of the metabolic pathway.