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. 2000 Apr;46(4):507–514. doi: 10.1136/gut.46.4.507

Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression

S Siavoshian 1, J Segain 1, M Kornprobst 1, C Bonnet 1, C Cherbut 1, J Galmiche 1, H Blottiere 1
PMCID: PMC1727889  PMID: 10716680

Abstract

BACKGROUND—Sodium butyrate, a product of colonic bacterial fermentation, is able to inhibit cell proliferation and to stimulate cell differentiation of colonic epithelial cell lines. It has been proposed that these cellular effects could be linked to its ability to cause hyperacetylation of histone through the inhibition of histone deacetylase.
AIM—To analyse the molecular mechanisms of butyrate action on cell proliferation/differentiation and to compare them with those of trichostatin A, a well known inhibitor of histone deacetylase.
METHODS—HT-29 cells were grown in the absence or presence of butyrate or trichostatin A. Cell proliferation and cell cycle distribution were studied after DNA staining by crystal violet and propidium iodide respectively. Cell cycle regulatory proteins were studied by western blot and reverse transcription-polymerase chain reaction. Cell differentiation was followed by measuring brush border enzyme activities. Histone acetylation was studied by acid/urea/Triton acrylamide gel electrophoresis.
RESULTS—Butyrate blocked cells mainly in the G1 phase of the cell cycle, whereas trichostatin A was inhibitory in both G1 and G2 phases. Butyrate inhibited the mRNA expression of cyclin D1 without affecting its protein expression and stimulated the protein expression of cyclin D3 without affecting its mRNA expression. Trichostatin A showed similar effects on cyclin D1 and D3. Butyrate and trichostatin A stimulated p21 expression both at the mRNA and protein levels, whereas their effects on the expression of cyclin dependent kinases were slightly different. Moreover, butyrate strongly stimulated the activity of alkaline phosphatase and dipeptidyl peptidase IV, whereas trichostatin A had no effect. Finally, a six hour exposure to butyrate or trichostatin A induced histone H4 hyperacetylation. At 15 and 24 hours, histone H4 remained hyperacetylated in the presence of butyrate, whereas it returned to control levels in the presence of trichostatin A.
CONCLUSIONS—The data may explain how butyrate acts on cell proliferation/differentiation, and they show that trichostatin A does not reproduce every effect of butyrate, mainly because of its shorter half life.


Keywords: butyrate; cyclin D; p21; trichostatin A; colonic epithelial cells; histone acetylation

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Figure 1  .

Figure 1  

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Growth curve of HT-29 cells estimated by the crystal violet staining method. Each point corresponds to the mean and SEM of experiments carried out in quadruplicate. The results presented are from one experiment representative of the four performed. Cells were cultured without or with increasing concentrations of butyrate (A) and trichostatin A (B), as indicated.

Figure 2  .

Figure 2  

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Histograms of DNA content of untreated HT-29 cells (A) and cells treated with 5 mM butyrate (B) or 0.5 µM trichostatin A (C). Cells were treated with each substance for 24 hours, and their DNA content was determined as described in Materials and methods. The cell cycle phase distributions (%) for each treatment are indicated within each panel.

Figure 3  .

Figure 3  

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Western blot analysis of cell cycle regulatory protein expression in HT-29 cells treated for 24 hours in the presence of the indicated substances. Equal volumes of whole cell extracts containing 15 µg proteins were separated and electrophoretically blotted. Proteins were probed with antibodies to cyclin D1 and D3, as indicated. TSA, trichostatin A. 

Figure 4  .

Figure 4  

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Comparison of the effect of trichostatin A (TSA) and butyrate on cyclin D1 and D3 mRNA as studied by reverse transcription-polymerase chain reaction (RT-PCR). PCR products were analysed on a 1.5% agarose gel stained with ethidium bromide. Primers and conditions are specified in Materials and methods. β-Actin is shown as a control. The sizes of the PCR products were 620 bp for β-actin, 577 bp for cyclin D1, and 264 bp for cyclin D3.

Figure 5  .

Figure 5  

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Kinetic analysis of the effect of butyrate on p21 mRNA (A) and protein (B) expression. HT-29 cells were either exposed to medium alone or to 5 mM butyrate, and total mRNA and proteins were extracted at the indicated time after stimulation. In (A), polymerase chain reaction (PCR) products were analysed on a 1.5% agarose gel stained with ethidium bromide. Primers and conditions are specified in Materials and methods. In (B), equal volumes of whole cell extracts containing 15 µg proteins were separated and electrophoretically blotted. Proteins was probed with antibody to p21.

Figure 6  .

Figure 6  

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Comparison of the effect of trichostatin A (TSA) at increasing concentrations and butyrate (at 5 mM) on the expression of p21 mRNA (A) and protein (B) in HT-29 cells. In (A), polymerase chain reaction (PCR) products were analysed on a 1.5% agarose gel stained with ethidium bromide. Primers and conditions are specified in Materials and methods. The sizes of the PCR products were 620 bp for β-actin and 449 bp for p21. In (B), equal volumes of whole cell extracts containing 15 µg proteins were separated and electrophoretically blotted. Proteins were probed with antibody to p21.

Figure 7  .

Figure 7  

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Effect of trichostatin A (TSA) and butyrate on the expression of cdk2, cdk4, and cdk6 proteins in HT-29 cells. Equal volumes of whole cell extracts containing 15 µg proteins were separated and electrophoretically blotted. Proteins were probed with antibodies to the indicated proteins.

Figure 8  .

Figure 8  

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Effect of butyrate and trichostatin A (TSA) on histone H4 acetylation. Cells were cultured for six hours and 24 hours in the absence (control) or presence of 5 mM butyrate or 1 µM trichostatin A. Histones were then separated by acid/urea/Triton acrylamide gel electrophoresis, and stained with Coomassie blue.

Figure 9  .

Figure 9  

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Western blot analysis of cell cycle regulatory protein expression in IEC-6 cells treated for 24 hours in the presence of increasing concentrations of butyrate. Equal volumes of whole cell extracts containing 15 µg proteins were separated and electrophoretically blotted. Proteins were probed with antibodies to cdk2, cdk4, cdk6, cyclins D, D1 and D3, as indicated.

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