A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins

Abstract

A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.

Characterization of a sialic‐acid‐rich mucus glycoprotein secreted by a premalignant human colorectal adenoma cell line

“…The material purified from the PC/AA cell secretion shows all of these properties typical of muicus glycoproteins. Thus the general characteristics are similar to those described for colonic mucins isolated from mouse xenogpfts of colonic adenocarcinoma cells (Boland et al, 1987;Byrd et al, 1988), a mucussecreting clonal derivative (C1.14E) of human HT29 cells (Maoret et al, 1989) and from normal colon (Podolsky and Isselbacher, 1983, and Table I ) . The pre-malignant PC/ AA-insoluble gel isolated directly from the cultures shows a ratio of carbohydrate to protein of 2.7: 1, high N-acetylgalactosamine and sialic acid with low mannose.…”

Expression of selenium‐binding protein 1 characterizes intestinal cell maturation and predicts survival for patients with colorectal cancer

“…HT29 cl.16E cells are a well-characterized model of secretory cell differentiation. Upon contact inhibition the cells spontaneously undergo growth arrest and differentiate into goblet cells that synthesize and secrete mucin, CEA, and alkaline phosphatase, which are maximal 21 days postconfluence [31,32]. Parental HT29 cells do not undergo this differentiation program.…”

HT29 Cell Line

“…growth in glucose-free medium, cell differentiation is very similar to that observed in Caco-2 cells but in HT29 the time course of the differentiation process is longer (30 days vs. 15-20 days in Caco-2); the levels of enzymatic activities are lower than in Caco-2; lactase is absent; and only 40-50 % of HT29 cells express sucrose-isomaltase (Zweibaum 1986 ;Zweibaum et al 2011 ). However, one of the main differences between this cell line and Caco-2 is that HT29 cells can produce mucin at a relatively high level (Huet et al 1987 ;Maoret et al 1989 ). Stepwise adaptation of exponentially growing HT29 cells to increasing concentrations of methotrexate (MTX) (up to 10 −5 mol) resulted in their transformation into mucus-secreting differentiated cells (Lesuffl eur et al 1990 ).…”