The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology.

The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of highdose ara-C clinical protocols. In this study, the incorporation of [3H]-dThd and [3H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [3H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [3H]-dThd incorpration, DNA synthesis was not completely inhibited since [3H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [3H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [3H]-dThd to [3H]-dTTP. Within 30 min exposure to 10 μM ara-C, the rate of cellular [3H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [3H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [3H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5′-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [3H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [3H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 μM ara-C (60 μM and 200 μM, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP. The ara-C-induced decline in dCTP noted above was apparently a secondary effect resulting from the inhibition of ribonucleotide reductase by the elevated dTTP and dATP. CDP reductase activity in the presence of dATP and dTTP at the concentrations found in ara-C-treated cells was 58% of the activity observed in the presence of nucleotide levels found in control cells. The decrease in dCTP levels was associated with a reciprocal increase in the rate of [3H]-ara-C phosphorylation following subsequent exposure to unlabeled ara-C. Thus, ara-C self-potentiated its own uptake in these cells. These observations of the self-limiting and self-potentiating effects of high concentrations of ara-C may be relevant to the selection of the optimal dose and the duration of exposure in the clinical use of high-dose ara-C infusions.