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Regulation of 1-ß-D-Arabinofuranosylcytosine 5'-Triphosphate Accumulation in Human Leukemia Cells by Deoxycytidine 5'-Triphosphate¹

Department of Chemotherapy Research, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030

Cell cycle-specific fluctuations in the ability of human leukemic cells to phosphorylate 1-ß-D-arabinofuranosylcytosine (ara-C) to the toxic metabolite 1-ß-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 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'-triphosphate (dCTP) also potently inhibited ara-C phosphorylation in CCRF-CEM cell extracts (K_i = 5.9 µM). Deoxynucleotide pool levels determined by high pressure liquid chromatography showed a 5 µM dCTP concentration in G₁-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 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.

¹ Supported in part by Grant CA 28596 from the National Cancer Institute and Grant CH-130 from the American Cancer Society.

² To whom requests for reprints should be addressed, at Division of Clinical Hematology and Oncology, Department of Medicine, Huddinge Hospital and Karolinska Institutet, S-141 86 Huddinge, Sweden.

Received 6/ 3/85. Revised 10/24/85. Accepted 11/ 7/85.

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