Volume 120, Issue 4, Pages 889-899 (March 2001) Resistance to butyrate-induced cell differentiation and apoptosis during spontaneous Caco-2 cell differentiation  John M. Mariadason, Anna Velcich, Andrew J. Wilson, Leonard H. Augenlicht, Peter R. Gibson  Gastroenterology  Volume 120, Issue 4, Pages 889-899 (March 2001) DOI: 10.1053/gast.2001.22472 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of butyrate on cell cycle arrest at various stages of Caco-2 cell differentiation. Caco-2 cells precultured 0–21 days after confluence were treated with butyrate (2 mmol/L) for 72 hours, and effects on the cell cycle were determined by PI staining and fluorescence-activated cell sorter analysis. Results are the mean ± SE of 4 independent experiments. For each phase of the cell cycle, the changes induced by butyrate differed significantly across the times of preculture of the cells (P < 0.005, ANOVA). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of butyrate on apoptosis at various stages of Caco-2 cell differentiation. Caco-2 cells precultured for 0–21 days after confluence were treated with (A) 2–10 mmol/L butyrate for 72 hours, and apoptosis was determined by PI staining and fluorescence-activated cell sorter analysis; or (B) with butyrate (2 mmol/L) for 72 hours, and apoptosis was determined by ELISA. Results are the mean ± SE of 4 and 3 independent experiments for A and B, respectively. The changes induced by butyrate at all concentrations differed significantly across the times of preculture of the cells (P < 0.005, ANOVA). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of butyrate on ALP activity at various stages of Caco-2 cell differentiation. Caco-2 cells precultured 0–21 days in confluence were treated with butyrate (2 mmol/L) for 72 hours, and ALP activity was determined in cell homogenates. Results are the mean ± SE of 4 independent experiments. The change induced by butyrate differed significantly across the times of preculture of the cells (P < 0.001, ANOVA). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of butyrate on TER and cell migration in undifferentiated and differentiated Caco-2 cells. (A) Caco-2 cells precultured for 1-day (undifferentiated) or 7-day (differentiated) postconfluence were treated with 2 mmol/L butyrate for 72 hours, and TER was measured. (B) Caco-2 cell monolayers precultured for 1-day (undifferentiated) or 14-days (differentiated) postconfluence were treated with 2 mmol/L butyrate for 24 hours, and cell migration after wounding was determined. For both A and B, results are the mean ± SE of 4 independent experiments. The change induced by butyrate differed significantly across the times of preculture of the cells (P = 0.024 and P = 0.001, ANOVA, for A and B, respectively). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of butyrate on (A) cell-associated uPAR expression and (B) IL-8 secretion at various stages of Caco-2 cell differentiation. Caco-2 cell monolayers precultured 0–21 days in confluence were treated with 2 mmol/L butyrate for 72 hours; (A) uPAR expression was measured in cell homogenates, and (B) IL-8 secretion was measured in supernatants. For both A and B, results are the mean ± SE of 4 independent experiments. The change induced by butyrate differed significantly across the times of preculture of the cells (P < 0.001, ANOVA, for A and B). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 (A and B) Effect of butyrate on apoptosis and ALP expression during HT29cl.19A cell differentiation. (A) HT29cl.19A cells, 2, 6, 14, or 25 days after seeding, were treated with butyrate for 72 hours and apoptosis determined by flow cytometry. (B) HT29 cl.19A cells, 3 or 10 days after seeding, were treated with 5 mmol/L butyrate, and ALP expression was measured by Northern blot. As a control for loading, blots were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase. (C) SW620 cells grown to confluence (day 0) or 2–21 days' postconfluence were treated with butyrate for 72 hours and apoptosis determined by flow cytometry. Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of butyrate on histone acetylation at various stages of Caco-2 cell differentiation, and expression of HDAC-1 during spontaneous Caco-2 cell differentiation. (A) Caco-2 cells precultured 0–21 days in confluence were treated with butyrate (2–5 mmol/L) for 24 hours, and the degree of histone acetylation was measured by Western blot. (B) Expression of HDAC-1 protein in Caco-2 cells cultured to 0–21 days' postconfluence. Blots were reprobed for actin to correct for equivalent loading in all wells. Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 Consumption of SCFAs by Caco-2 cells. Caco-2 cells at confluence were treated with 2 mmol/L of a panel of SCFAs, 2–8 carbon atoms in length (straight lines). SCFA levels remaining in the medium were quantified by gas chromatography at various time points. In each case, SCFA levels in medium not exposed to cells were measured in parallel as a control for volatility (dotted line). Values are the mean ± SE of the percentage of SCFA remaining relative to the starting amount of 4 separate experiments. In each case the change in SCFA concentration differed significantly across the culture period (P < 0.001, ANOVA). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 9 Butyrate consumption at various stages of Caco-2 cell differentiation. Caco-2 cells precultured 0–21 days after confluence were treated with butyrate (2 mmol/L) for 72 hours, and the amount of butyrate remaining in the culture medium was determined by gas chromatography. Values are the mean ± SE of 3 separate experiments. The change in the amount of butyrate remaining differed significantly across the times of preculture of the cells (P < 0.001, ANOVA). Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 10 Effect of SCFAs (C2-C8) on (A) ALP activity and (B) TER. Caco-2 cells at confluence were treated with 2 mmol/L (■) or 8 mmol/L (○) of a panel of SCFAs for 72 hours, and (A) ALP activity or (B) TER was determined. For both A and B, values are the mean ± SE of the increase relative to control of 4 separate experiments. Gastroenterology 2001 120, 889-899DOI: (10.1053/gast.2001.22472) Copyright © 2001 American Gastroenterological Association Terms and Conditions