Transport and metabolism of deoxycytidine and 1-beta-D-arabinofuranosylcytosine into cultured Novikoff rat hepatoma cells, relationship to phosphorylation, and regulation of triphosphate synthesis.

The zero-trans transport of deoxycytidine and 1-/3-Darabinofuranosylcytosine was determined in cultured No vikoff rat hepatoma cells that had been depleted of adenosine 5 -triphosphate by preincubation in glucose-free medium containing KCN and iodoacetate and thus did not phosphorylate the substrates. Transport of both nucleosides was so rapid that the ¡ntracellularconcentration approached that in the extracellular fluid within less than 1 min. Initial transport velocities were computed from pseudo-first-order time courses of ¡ntracellularsubstrate accumulation as determined by a rapid mixing-sampling technique. The zero-trans Kmwas similar for both nucleosides, between 250 and 500 /*M. The rates of transport of deoxycytidine and 1-/3-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 incorpo ration into the nucleotide pool in these cells. The intracel lular phosphorylation of the nucleosides, however, be came enhanced 5 to 10 times within minutes of addition of 0.1 rriM thymidine or 0.5 to 1 mw hydroxyurea or of appropriate concentrations of other inhibitors of ribonucleotide reducÃ-ase, imidazopyrazole, and 4-methyl-5amino-1-formylisoquinoline thiosemicarbazone to the me dium. Pyrazofurin, an inhibitor of de novo pyrimidine synthesis, 3-deazauridine, an inhibitor of cytidine 5 -tri phosphate synthetase, and alanosine, an inhibitor of the conversion of inosine 5'-phosphate to adenosine 5 -phos phate, had a similar but more delayed effect. The stimu lation of deoxycytidine and 1-/3-o-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 inhi bition of its de novo synthesis caused by thymidine, 3deazauridine, or pyrazofurin results in enhancement of the salvage pathway. The effect of ribonucleotide reduc Ã-aseinhibitors and of alanosine, on the other hand, may be related to the depletion of the cells of deoxyadenosine 5 -triphosphate.