BUTYRATE-INDUCED DIFFERENTIATION OF CACO-2 CELLS IS ASSOCIATED WITH APOPTOSIS AND EARLY INDUCTION OF P21(WAF1 CIP1) AND P27(KIP1)/

Background. Intestinal mucosal turnover is a process of proliferation, differentiation, and apoptosis; the mechanisms remain largely undefin ed. The purpose of our study was to (1) assess the relationship betwee n apoptosis and enterocyte differentiation and (2) determine whether t he cell-cycle inhibitors, p21(Waf1/Cip1) and p27(Kip1), the apoptosis inhibitors, Bcl-2 and Bcl-X-L, may be involved. Methods. Gut-derived C aco-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 differen tiation (increased alkaline phosphatase and sucrase activity) and apop tosis. Increased level of p21(Waf1/Cip1) and p27(KiP1) mRNA and protei n were detected at 24 hours, occurring before apoptosis or differentia tion; decreased mRNA level of Bcl-2 and Bcl-X-L were noted at 24 hours . Conclusions. Differentiation and apoptosis occurred simultaneously i n 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 t he cell-cycle mechanisms regulating enterocyte differentiation and apo ptosis.