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Transport and Metabolism of Deoxycytidine and 1-ß-D-Arabinofuranosylcytosine into Cultured Novikoff Rat Hepatoma Cells, Relationship to Phosphorylation, and Regulation of Triphosphate Synthesis¹

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

The zero-trans transport of deoxycytidine and 1-ß-D-arabinofuranosylcytosine was determined in cultured Novikoff rat hepatoma cells that had been depleted of adenosine 5'-triphosphate by preincubation in glucose-free medium containing KCN and lodoacetate and thus did not phosphorylate the substrates. Transport of both nucleosides was so rapid that the intracellular concentration approached that in the extracellular fluid within less than 1 min. Initial transport velocities were computed from pseudo-first-order time courses of intracellular substrate accumulation as determined by a rapid mixing-sampling technique. The zero-trans K_m was similar for both nucleosides, between 250 and 500 µM. The rates of transport of deoxycytidine and 1-ß-D-arabinofuranosylcytosine into the cells were 10 to 100 times higher than their rates of intracellular phosphorylation in untreated cells in which phosphorylation was not prevented by adenosine 5'-triphosphate depletion. Thus phosphorylation rather than transport was the rate-determining step in their incorporation into the nucleotide pool in these cells. The intracellular phosphorylation of the nucleosides, however, became enhanced 5 to 10 times within minutes of addition of 0.1 mM thymidine or 0.5 to 1 mM hydroxyurea or of appropriate concentrations of other inhibitors of ribonucleotide reductase, imidazopyrazole, and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone to the medium. Pyrazofurin, an inhibitor of de novo pyrimidine synthesis, 3-deazauridine, an inhibitor of cytidine 5'-triphosphate synthestase, and alanosine, an inhibitor of the conversion of inosine 5'-phosphate to adenosine 5'-phosphate, had a similar but more delayed effect. The stimulation of deoxycytidine and 1-ß-D-arabinofuranosylcytosine incorporation into the nucleotide pool resulted to varying extents in their enhanced incorporation into DNA depending on the degree of inhibition of DNA synthesis caused by the various treatments.

The results indicate that the intracellular deoxycytidine 5'-triphosphate concentration is normally high enough to cause severe feedback inhibition of deoxycytidine kinase. A decrease in 5'-triphosphate concentration due to inhibition of its de novo synthesis caused by thymidine, 3-deazauridine, or pyrazofurin results in enhancement of the salvage pathway. The effect of ribonucleotide reductase inhibitors and of alanosine, on the other hand, may be related to the depletion of the cells of deoxyadenosine 5'-triphosphate.

¹ This work was supported by USPHS Research Grant CA 16228 and by Contract NOI-CM-43788 from the Division of Cancer Treatment, National Cancer Institute, NIH, Department of Health, Education, and Welfare.

² To whom requests for reprints should be addressed.

³ Recipient of USPHS Research Fellowship CA 00800. Present address: Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse II, 5020 Saizburg, Austria.

Received 9/12/77. Accepted 1/ 3/78.

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