Butyrate-induced differentiation of Caco-2 cells is associated with apoptosis and early induction of p21Waf1/Cip1 and p27kip1

Background. Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefined. The purpose of our study was to (1) assess the relationship between apoptosis and enterocyte differentiation and (2) determine whether the cell-cycle inhibitors, p21(Waf1/Cip1) and p27(Kip1), the apoptosis inhibitors, Bcl-2 and Bcl-X-L, may be involved. Methods. Gut-derived Caco-2 cells were treated with sodium butyrate. Apoptosis was assessed by Hoechst stain, DIVA laddering, and annexin V assay; differentiation was determined by alkaline phosphatase and sucrase activity. RNA and protein were analyzed for expression of p21(Waf1/Cip1), p27(Kip1), and members of the Bcl-2 family. Results, Treatment of Caco-2 cells with sodium butyrate resulted in the concomitant induction of both differentiation (increased alkaline phosphatase and sucrase activity) and apoptosis. Increased level of p21(Waf1/Cip1) and p27(KiP1) mRNA and protein were detected at 24 hours, occurring before apoptosis or differentiation; decreased mRNA level of Bcl-2 and Bcl-X-L were noted at 24 hours. Conclusions. Differentiation and apoptosis occurred simultaneously in Caco-2 cells, suggesting that apoptosis may be linked to enterocyte differentiation. The induction of p21(Waf1/Cip1) and p27(Kip1) and the down-regulation of Bcl-2 and Bcl-X-L further suggest a link between the cell-cycle mechanisms regulating enterocyte differentiation and apoptosis.