4'-thio-beta-D-arabinofuranosylcytosine (T-araC) exhibits excellent in vivo antitumor activity against a variety of solid
tumors despite its structural similarity to beta-D-
arabinofuranosylcytosine (araC), an agent which is poorly active against solid
tumors in vivo. It is of great interest to elucidate why these compounds show a profound difference in antitumor activity. Because
deoxycytidine kinase (dCK) is the critical
enzyme in the activation of both compounds, here we report the differences in the substrate characteristics with human dCK between these compounds. The catalytic efficiency (V(max)/K(m)) of araC was 100-fold higher than that of T-araC using either
ATP or
UTP as the
phosphate donor. However, V(max) values of araC and T-araC were similar when
UTP was the
phosphate donor. Since
UTP is believed to be the true
phosphate donor for dCK in intact cells, these data indicated that the rates of phosphorylation of these two compounds at high pharmacologically relevant concentrations would be similar. This prediction was confirmed in intact cell experiments, which supported the hypothesis that
UTP is the physiological
phosphate donor for dCK phosphorylation in cells. The relative lack of importance of
phosphate donor to the phosphorylation of T-araC by dCK revealed important insights into the activation of this compound in human cells at pharmacological doses. These studies indicated that replacement of the 4'-oxygen with
sulfur significantly reduced the substrate activity of
nucleoside analogs with dCK and that the superior activity of T-araC with respect to araC against solid
tumors was not due to superior activity with dCK.