Effect of colonic luminal components on induction of apoptosis in human colonic cell lines

Apoptosis is central to cell number regulation in the colonic epithelium, a nd interest in its role in colon carcinogenesis has been growing rapidly. I t thus becomes of interest to characterize luminal components, possibly of dietary origin, that may influence this process. We have investigated the s ensitivity of two human colonic cell lines, the human adenocarcinoma cell l ine (HT-29) and the human fetal colonic mucosa cell line (FHC), to inductio n of apoptosis by sodium butyrate, bile acids, and human fecal water fracti ons. The apoptotic effect has been studied by 1) morphological changes in c ells examined by fluorescence microscopy, 2) DNA fragmentation analysis by gel electrophoresis, 3) flow cytometry analysis of DNA strand breaks assess ed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labe ling assay (TUNEL), and 4) poly(ADP-ribose) polymerase cleavage by Western blot. Sodium butyrate and bile acids induced a time- and concentration-depe ndent apoptosis in both cell lines, Quantitation of this effect, by use of the TUNEL assay, indicated that deoxycholic acid was most effective in indu cing this effect at lower concentrations and at shorter times. Apoptotic ef fects were also observed, in both cell lines, when the cells were exposed t o intact human fecal waters (the fecal fraction in direct contact with the epithelium) and their lipid extracts, with the intact samples being more ef fective. Although all fecal waters examined induced apoptosis, quantitation of the effect by the TUNEL assay indicated that the ability to induce apop tosis differed markedly between samples. Induction of apoptosis by the feca l waters was not correlated to cytotoxicity but was negatively correlated t o the pH of the samples. Interestingly, the cells derived from the fetal mu cosa (FHC) were consistently less sensitive to apoptotic effects of the lum inal components than the tumor-derived cells (HT-29). Thus human fecal wate r fractions induce apoptosis in colonic cells, and this effect is not due t o lipid components alone.