Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2

• Published in: Biochemical journal Vol. 348 Pt 2; no. 2; pp. 401 - 408
• Main Authors: Chabanon, H, Persson, L, Wallace, H M, Ferrara, M, Brachet, P
• Format: Journal Article
• Language: English
• Published: England Portland Press 01.06.2000
• Subjects: Amino Acids - metabolism; Amino Acids - pharmacology; Biochemistry, Molecular Biology; Caco-2 Cells; Culture Media, Serum-Free; Enzyme Inhibitors - metabolism; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Neoplastic - drug effects; Humans; Kinetics; Life Sciences; Methionine - metabolism; Ornithine Decarboxylase - biosynthesis; Ornithine Decarboxylase - genetics; Ornithine Decarboxylase - metabolism; Protein Biosynthesis; Proteins - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3480401

The mechanisms of the response of ornithine decarboxylase(ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resultedin increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA,respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cellsconfirmed a requirement for protein synthesis. Pulse-labelling of cellswith [(35)S]methionine showed a 3-fold increase in thesynthesis of ODC protein after 4 h of supplementation with glycineor L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme in activation and/or degradation.