Caco-2 cells differentiate spontaneously when cultured in confluence and on exposure to the physiologically relevant short-chain fatty acid, butyrate. This study aimed to compare the phenotype induced by these pathways and their relations to cell turnover. Caco-2 cells were treated with butyrate at a nontoxic concentration of 2 mM for 3 days, or allowed to spontaneously differentiate for 0-21 days. Brush border hydrolase activities and carcinoembryonic antigen (CEA) expression, transepithelial resistance and dome formation, expression of components of the urokinase system, and cell turnover by flow cytometry, and the degree of DNA fragmentation were quantified. Butyrate induced increases in alkaline phosphatase activity and CEA expression but not the activities of other hydrolases, while culture alone induced progressive increases in the activities/expression of all markers. Butyrate induced a significantly greater increase in transepithelial resistance (TER) than occurred during culture alone but the densities of domes were similar. Butyrate induced a ninefold increase in urokinase receptor expression and twofold increase in urokinase activity, while culture alone induced a significantly smaller increase in receptor expression, an increase in plasminogen activator inhibitor-1 but no change in activity. While both stimuli induced cell cycle arrest, only butyrate increased the proportion of cells undergoing apoptosis. In conclusion, differentiation of Caco-2 cells can proceed along multiple pathways but does not necessarily lead to apoptosis. The phenotypic changes during spontaneous differentiation mimic those that occur in normal colonic epithelial cells in vivo during their migration from the crypt base to neck, while butyrate-induced effects more closely follow those occurring when normal colonic epithelial cells migrate from crypt neck to the surface compartment.

Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism that dictates a cell's response to butyrate is its state of activation. First, the responses to 24 h exposure to butyrate (1-2 mM) of normal and neoplastic human colonic epithelial cells activated by their isolation and primary culture, and of colon cancer cell lines, LIM1215 and Caco-2, were examined. In primary cultures of normal and cancer cells, butyrate had no effect on alkaline phosphatase activities but significantly suppressed urokinase receptor expression by a mean +/- SEM of 30 +/- 12% and 36 +/- 9%, respectively. Interleukin-8 secretion was suppressed by 44 +/- 7% in normal cells (P < 0.05) but was unchanged in cancer cells. In contrast, the cell lines significantly increased alkaline phosphatase activities by >50%, urokinase receptor expression >2-fold and interleukin-8 secretion >3-fold in response to butyrate. Secondly, the effect of butyrate on Caco-2 cells was examined with or without prior exposure to a specific activating stimulus [tumour necrosis factor alpha (TNF alpha)]. Interleukin-8 secretion increased by 145 +/- 23% and 132 +/- 17% on 24 h exposure to 2 mM butyrate or 0.1 microM TNF alpha alone, respectively. However, in cells pre-treated with TNF alpha, butyrate significantly inhibited secretion by 34 +/- 7% below unstimulated levels. The response to butyrate of urokinase receptor, whose expression was not stimulated by TNF alpha, was unchanged. These effects were mimicked by trichostatin A, an inhibitor of histone deacetylase, suggesting that butyrate's paradoxical effects may have been operating by the same mechanism. In conclusion, some of the paradoxical effects of butyrate do not appear to represent inherent differences between normal and transformed cells. Rather, the response may be determined by the state of activation of the cells.

As the colonic epithelium is physiologically exposed to butyrate and to activators of protein kinase C, we examined the effect of the protein kinase C signalling pathway on butyrate-induced expression of markers of differentiation. Activators and inhibitors of protein kinase C were used in combination with butyrate and effects on the expression of markers of differentiation examined in colon cancer cell lines. When the protein kinase C activator phorbol myristate acetate (100 nM) was added for 24 h prior to the addition of 2 mM butyrate, there was a synergistic increase in alkaline phosphatase activity (154 +/- 11% above that for butyrate alone, P = 0.003) in a concentration- and time-dependent manner. Butyrate-induced expression of carcinoembryonic antigen and interleukin-8, dome formation and cell turnover were also markedly augmented by pre-treatment with phorbol myristate acetate. A similar effect was observed with propionate or acetate (but not other differentiating agents), when phorbol myristate acetate and butyrate were added concurrently, or when other protein kinase C activators were used. Pharmacological inhibition of protein kinase C activity did not alter butyrate-induced alkaline phosphatase activity, but abrogated the augmentation induced by phorbol myristate acetate. We conclude that protein kinase C does not mediate the differentiating effects of butyrate on colon cancer cells, but its activation regulates butyrate-induced cellular differentiation.