IDENTIFICATION OF CYTIDINE DEAMINASE AS INHIBITOR OF GRANULOCYTE-MACROPHAGE COLONY FORMATION

Mature human blood granulocytes produce regulatory factors that inhibi t colony formation by human and murine granulocyte-macrophage colony-f orming cells (GM-CFC). The inhibition of GM-CFC by granulocyte extract (GRE) was strongly enhanced by the addition of thymidine (3 to 6x10(- 5) M for human cells) and by the presence of fetal calf serum (FCS) in the growth medium. Deoxycytidine and deoxyuridine produced effects si milar to those of thymidine, but at higher concentrations (2 to 4x10(- 4) M). It was further observed that GRE prevented the antiproliferativ e effects of cytosine arabinoside (Ara-C) and azadeoxycytidine, sugges ting that GRE contained cytidine deaminase (CDD) activity, since CDD i s known to abolish the effects of these nucleoside analogs. Accordingl y, the GRE was tested for and shown to contain an enzymatic activity t hat converted deoxycytidine to deoxyuridine, confirming the presence o f CDD activity in GRE. The GM-CFC inhibition factor was found to copur ify with CDD activity during three succeeding chromatographic separati ons, indicating that CDD was indeed the inhibiting factor itself. This conclusion was further substantiated by gel filtration experiments de monstrating a molecular weight (MW) of approximately 50 kd, which corr esponds to the MW previously published for CDD activity. Furthermore, addition of tetrahydrouridine (THU), a known specific inhibitor of CDD , abolished the suppressive effect of GRE on GM-CFC, which independent ly confirmed the identification of CDD as an inhibitor of GM-CFC. The growth-regulating property of CDD could be explained by depletion of d eoxycytidine nucleotides necessary for DNA synthesis or by a direct ef fect of CDD binding to specific receptors on progenitor cells.