5-Aza-2′-deoxycytidine (5-AZA-CdR) is a potent antileukemic agent whose inhibitory effects are blocked by deoxycytidine (1-3). In order to be active, 5-AZA-CdR must first be phosphorylated by the enzyme deoxycytidine kinase (4). Leukemic cells deficient in the enzyme are resistant to 5-AZA-CdR (5). The lethal action of 5-AZA-CdR is related to its incorporation into DNA (6,7). The incorporation of 5-azacytosine analogs into DNA has been shown to induce the expression of differentiated phenotypes (8). This induction of differentiation by 5-AZA-CdR appears to be related to the inhibition of DNA methylation produced by these analogs (8, 9).

5-AZA-CdR, an analogue of deoxycytidine, is an effective antileukemic agent. The antineoplastic action of 5-AZA-CdR appears to be related to the inhibition of DNA methylation. This inhibition activates gene expression and induces cellular differentiaion. The metabolism of 5-AZA-CdR is important because changes in the intracellular pool of deoxynucleotides may modulate the action of nucleoside analogs. In fact Grant et al., (Cancer Res. 1982) have reported drug synergism between thymidine and 5-AZA-CdR in a human premyelocytic cell line and in our experiments on L1210 cells, a correlation between 5-AZA-CdR effect and DNA methylation has been found. We have investigated the kinetic interactions of 5-AZA-dCMP and its triphosphate with mammalian dCMP deaminase, an enzyme that plays an important role in deoxynucleotide metabolism by modulating the intracellular pool size of dCTP and dTTP. 5-AZA-dCMP is a substrate of the enzyme and is deaminated at a rate of about 100-fold lower than the natural substrate, dCMP. 5-AZA-dCTP is an allosteric activator which reverts the inhibitory effect of dTTP. The mechanism by which the antileukemic action of 5-AZA-CdR can be enchanced by thymidine is the inhibition of 5-AZA-dCMP deamination by dTTP.

5-AZA-2'-deoxycytidine-5'-monophosphate (5-AZA-dCMP) was tested as a substrate, and 5-aza-2'-deoxycytidine-5'-triphosphate (5-AZA-dCTP) was tested as an allosteric effector of purified spleen dCMP deaminase. Graphic analysis of the velocity of deamination of 5-AZA-dCMP versus its concentration gave a hyperbolic curve in which the estimated apparent Km was 0.1 mM. Since this curve was not sigmoidal and 5-AZA-dCMP at low concentrations stimulated the rate of deamination of the natural substrate, dCMP, it was proposed that the binding of 5-AZA-dCMP to the allosteric enzyme dCMP deaminase induced the R form. At substrate saturation, the rate of deamination of dCMP was 100-fold greater than that of 5-AZA-dCMP. dTTP inhibited the deamination of 5-AZA-dCMP with first-order kinetics. This inhibition was reversed by either 5-AZA-dCTP or dCTP. However, dCTP alone produced only a weak activation of the deamination of 5-AZA-dCMP in comparison to the potent activation when dCMP was the substrate. 5-AZA-dCTP was just as effective as dCTP for the allosteric activation of the deamination of dCMP. These results indicate that dCMP deaminase can play an important role in the metabolism 5-aza-2'-deoxycytidine nucleotides and may possibly modulate some of the pharmacological activity of this antimetabolite.