Research Papersα-Difluoromethylornithine (DFMO) as a Potent Arginase Activity Inhibitor in Human Colon Carcinoma Cells
Section snippets
Reagents
All the amino acids used in this study were purchased from Sigma. Radiochemicals were obtained from Amersham or DuPont/NEN. Fetal calf serum was purchased from Boehringer and Dulbecco’s modified Eagle’s medium from Life Technologies. DFMO was kindly provided by the Marion Merrell Dow Research Institute. Purified bovine liver arginase was from Sigma. The silicon oil used for the measurement of net uptake was purchased from TAI Lubricants.
Cell Culture and Protein Content
The human adenocarcinoma cell line HT-29 was established
Effect of DFMO on l-Arginine Flux through Arginase
When 10 mM of DFMO was tested on 1 mM of l-[guanido-14C] arginine flux through arginase in intact HT29 cells after a 90–min incubation at 37°, it induced 51% inhibition of the basal flux value (Table 1). In comparison, l-valine induced 73% inhibition of the basal value when tested at the same concentration (Table 1). However, since the l-arginine flux through arginase resulted from l-arginine net uptake across the plasma membrane and the activity of arginase, we first measured l-[U-14C]
Discussion
Our results clearly demonstrated that DFMO was able to severely decrease the flux of l-arginine through arginase in isolated HT-29 cells. DFMO is therefore almost as potent as l-valine in inhibiting this flux. This latter amino acid is considered to be a very potent arginase inhibitor 21, 22. The observed inhibitory effect of DFMO on the l-arginine flux through arginase was not due to a decrease in exogenous l-arginine availability inside HT-29 proliferative cells, because DFMO did not affect l
Acknowledgements
The authors wish to thank Mrs. M. Defrance for secretarial help and Mrs. K. Archbold–Zenon from the Translation Department at INRA for revising the manuscript.
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