Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4).

GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C.

O-Glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line.

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performing in vitro reactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype.

Regulation of cathepsin D dependent on the phenotype of colon carcinoma cells.

We have studied the intracellular trafficking of cathepsin D in different colon carcinoma cell populations: the HT-29 cell line, composed of >95% undifferentiated cells; 2 subpopulations derived from this cell line, containing cells committed to differentiation into mucin-secreting cells (HT-29 MTX) or enterocyte-like cells (HT-29 G-) after confluence; and the Caco-2 cell line, which spontaneously differentiates into enterocyte-like cells after confluence. Post-confluent undifferentiated HT-29 cells and differentiated enterocyte-like HT-29 G- and Caco-2 cells secrete significant levels of cathepsin D in culture medium, in contrast to post-confluent differentiated mucin-secreting HT-29 MTX cells, which secrete this enzyme at a very low level. The intracellular content and the mRNA level of cathepsin D increase after confluence in the different cell types, particularly in Caco-2 cells, which intensify the secretion of cathepsin D along with the differentiation process post-confluence.