Growth Inhibition of Granulocyte-Macrophage Colony-Forming Cells by
Human Cytidine Deaminase Requires the Catalytic Function of the Protein
Christine Gran,
Arne Bøyum,
Rune F. Johansen,
Dagfinn Løvhaug, and
Erling C. Seeberg
From the Institute of Medical Microbiology, Department of Molecular
Biology, University of Oslo, The National Hospital, Oslo; the Norwegian
Defense Research Establishment, Division for Environmental Toxicology,
Kjeller; and NYCOMED Imaging A/S, Oslo, Norway.
Previous studies have indicated that cytidine deaminase (CDD) is a
potent growth inhibitor of granulocyte-macrophage colony-forming cells
(GM-CFC). In this study, we have undertaken molecular
cloning and purification of recombinant human CDD to elucidate the
growth regulatory potential and mechanism behind the growth suppressive effect. The purified protein had a specific activity of 1.35 × 105 U/mg and a Km value of 30 µmol/L. In the
GM-CFC assay, the recombinant protein was shown to reduce colony
formation to 50% at 16 pmol/L concentration. Similarly, as was
observed with CDD derived from granulocyte extract, the effect depended
on the presence of thymidine (
4 × 10-5 mol/L). These
results imply that CDD is an extremely potent inhibitor of GM-CFC and
that no additional factor from the granulocyte extract is required for
the growth inhibitory effect. Modification of CDD by truncation from
the C-terminal end, or by amino acid substitution of an
active site glutamate residue, eliminated both the enzyme activity and
the growth regulatory potential of CDD. Furthermore, CDD from
Escherichia coli was found to be even more effective than human
CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory
activity corresponding to a 10-fold higher enzymatic activity. Taken
together, these results show that the catalytic nucleoside deaminating
function of the protein is essential for the growth suppressive effect
of CDD. Most probably, CDD exerts growth inhibition by depleting the
cytidine and deoxycytidine pool required for DNA synthesis, as addition
of deoxycytidine monophosphate, which is not a substrate for CDD,
neutralizes the inhibiting effect.
Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4127-4135
© 1998 by The American Society of Hematology.
