Cellular metabolism studies had demonstrated previously that low cellular concentrations of
2',2'-difluorodeoxycytidine (dFdC)
nucleotides are eliminated by
deoxycytidylate deaminase (dCMPD), whereas dCMPD activity is inhibited at high cellular dFdC
nucleotide levels (Heinemann et al.,
Cancer Res 52: 533-539, 1992). An assay for measuring dCMPD activity in intact human
leukemia cells has now been developed to permit investigations of the interactions of dFdC
nucleotides with dCMPD in intact cells in which the regulated nature of this
enzyme was not disrupted. Using [14C]dCyd as the substrate, radioactivity that accumulated in
dTTP was quantitated after high-pressure liquid chromotography by a radioactive flow detector. The assay was first characterized using either the dCMPD inhibitor tetrahydrodeoxyuridine (H4dUrd) which directly inhibits dCMPD, or
thymidine and
5-fluoro-2'-deoxyuridine (FdUrd) which indirectly inhibit and activate dCMPD, respectively, by affecting the cellular
dCTP:
dTTP value. Measured by this in situ assay, there was a strong correlation between dCMPD activity and
dCTP:
dTTP levels. Consistent with previous studies using partially purified
enzyme, incubation of cells with dFdC resulted in a concentration-dependent inhibition of dCMPD in situ. The mechanism of modulation of dCMPD by dFdC, however, was clearly different from that of
thymidine and FdUrd. In addition to the effect of dFdC on cellular
dCTP:
dTTP, our findings also suggested an additional inhibitory mechanism, possibly a direct interaction between dCMPD and dFdC 5'-triphosphate. Thus, results obtained using this direct assay of dCMPD in intact cells support the hypothesis that dCMPD is inhibited by
nucleotides of dFdC.