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

H Chabanon, L Persson, H M Wallace, M Ferrara, P Brachet

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with isoosmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effecters 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 cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [S-35]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or 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 inactivation and/or degradation.

Original languageEnglish
Pages (from-to)401-408
Number of pages8
JournalBiochemical Journal
Volume348
Publication statusPublished - 2000

Keywords

  • glycine
  • polyamine biosynthesis
  • nutrient
  • ODC
  • post-translational regulation
  • RAT-LIVER
  • INDUCTION
  • PROTEIN
  • EXPRESSION
  • TRANSPORT
  • ASPARAGINE
  • MECHANISMS
  • RNA
  • DIFFERENTIATION
  • STIMULATION

Cite this

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2. / Chabanon, H ; Persson, L ; Wallace, H M ; Ferrara, M ; Brachet, P .

In: Biochemical Journal, Vol. 348, 2000, p. 401-408.

Research output: Contribution to journalArticle

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abstract = "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) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with isoosmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effecters 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 cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [S-35]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or 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 inactivation and/or degradation.",
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AU - Persson, L

AU - Wallace, H M

AU - Ferrara, M

AU - Brachet, P

PY - 2000

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N2 - 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) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with isoosmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effecters 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 cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [S-35]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or 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 inactivation and/or degradation.

AB - 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) resulted in increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with isoosmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effecters 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 cells confirmed a requirement for protein synthesis. Pulse-labelling of cells with [S-35]methionine showed a 3-fold increase in the synthesis of ODC protein after 4 h of supplementation with glycine or 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 inactivation and/or degradation.

KW - glycine

KW - polyamine biosynthesis

KW - nutrient

KW - ODC

KW - post-translational regulation

KW - RAT-LIVER

KW - INDUCTION

KW - PROTEIN

KW - EXPRESSION

KW - TRANSPORT

KW - ASPARAGINE

KW - MECHANISMS

KW - RNA

KW - DIFFERENTIATION

KW - STIMULATION

M3 - Article

VL - 348

SP - 401

EP - 408

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

ER -