ABSTRACT
We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.
Subject(s)
Calcium-Binding Proteins/biosynthesis , Intestine, Small/cytology , Intestine, Small/metabolism , Nerve Tissue Proteins , Blotting, Western , Caco-2 Cells , Calcium-Binding Proteins/genetics , Cell Differentiation , Cell Division , Fluorescent Antibody Technique, Indirect , HT29 Cells , Humans , Lithostathine , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Pancreatic trypsin has been found to induce tight junction or dome formation in some colon cancer cell lines (HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type II, has been isolated from another colon cancer cell line (COLO 205). We have tried to determine if trypsinogen is present and how its expression varies during cell culture in HT-29 Glc+/- and Caco-2 cells, which exhibit enterocytic differentiation, and in HT-29 Glc+ cells, which never differentiate. Trypsinogen mRNA presence and expression were demonstrated in these cells by mRNA hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot analysis, and gel filtration. Trypsinogen was found to be trypsinogen type I and was mainly in zymogen form in culture media. Differentiating cells exhibited variations in trypsinogen I expression, but cells that remained undifferentiated did not. In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the first steps of differentiation.
Subject(s)
Adenocarcinoma/enzymology , Cell Differentiation , Cell Division , Colonic Neoplasms/enzymology , Pancreas/enzymology , Trypsinogen/genetics , Adenocarcinoma/pathology , Blotting, Western , Chromatography, Gel , Colonic Neoplasms/pathology , Fluorescent Antibody Technique , Gene Expression , Humans , Nucleic Acid Hybridization , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase , Tumor Cells, CulturedABSTRACT
Trophic effects of milk fermented with Lactobacillus helveticus, Lactobacillus paracasei ssp. paracasei, Bifidobacterium sp., or the combination of Lactobacillus bulgaricus and Streptococcus thermophilus (yogurt) were studied on the IEC-6 intestinal epithelial cell line. Incorporation of [methyl-3H]thymidine, mitochondrial dehydrogenase activities, cyclic AMP production, and differentiation of levels of the IEC-6 strain were evaluated between the 15th and 30th passage in culture. All fermented and unfermented milks enhanced trophic responses of IEC-6 cells in a dose-dependent manner. Compared with the corresponding milks, supernatant fractions were more effective in stimulating mitochondrial dehydrogenase response. Fermented milk supernatants were also more effective than the corresponding unfermented fractions. Increases in DNA synthesis and cyclic AMP confirmed the activation observed with mitochondrial dehydrogenase. Yogurt induced the more trophic response with an increased number of the more differentiated cell morphotype. Fermentation with L. casei also demonstrated an important trophic adaptation of IEC-6 cells. Milk processing by lactic acid bacteria enhanced trophic and proliferation responses of intestinal epithelial cell line IEC-6. These results suggested that IEC-6 cells could represent an accurate and easy in vitro model for testing the trophic quality of various nutrients and for an optimization of physiological digestive functions.