ALPHA-DIFLUOROMETHYLORNITHINE (DFMO) AS A POTENT ARGINASE ACTIVITY INHIBITOR IN HUMAN COLON-CARCINOMA CELLS

alpha-Difluoromethylornithine (DFMO) is commonly used as a specific or nithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been sho wn to be strictly necessary for cell proliferation. In HT-29 Glc(+/-) cells, L-arginine is the major precursor of these molecules through th e sequential actions of arginase, which leads to L-ornithine generatio n and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. S ince DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51 +/- 11% by 10 mM of DFMO whereas 10 m M of L-valine, a known potent arginase inhibitor, inhibited this flux by 73 +/- 6%. DFMO equilibrated between extracellular and intercellula r spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated K-i (inhibitory constant) equal to 3.9 +/- 1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of t he enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the me tabolic conversion of L-arginine to polyamines, is of potential physio logical importance, since it could alter the production of ornithine a nd thus its metabolism in pathways other than the ODC pathway. (C) 199 5 Elsevier Science Inc.