REGULATION OF 1-BETA-DEUTERIUM-ARABINOFURANOSYLCYTOSINE 5'-TRIPHOSPHATE ACCUMULATION IN HUMAN-LEUKEMIA CELLS BY DEOXYCYTIDINE 5'-TRIPHOSPHATE

  • 1 March 1986
    • journal article
    • research article
    • Vol. 46  (3) , 1079-1083
Abstract
Cell cycle-specific fluctuations in the ability of human leukemic cells to phosphorylate 1-.beta.-D-arabinofuranosylcytosine (ara-C) to the toxic metabolite 1-.beta.-D-arabinofuranosylcytosine 5''-triphosphate (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 accululation 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''-triphosphate (dCTP) also potently inhibited ara-C phosphorylation in CCRF-CEM cell extracts (Ki = 5.9 .mu.M). Deoxynucleotide pool levels determined by high pressure liquid chromatography showed a 5 .mu.M dCTP concentration in G1-enriched cells, whereas S phase-enriched cells contained 15 .mu.M 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 phosphorylating 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.