Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC).

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.

The influence of N- and O-glycosylation inhibitors on the glycosylation profile of cellular membrane proteins and adhesive properties of carcinoma cell lines.

The effects of N- and O-glycosylation inhibitors on the expression of membrane proteins (MUC1 and some integrins) were evaluated in human endometrial (Ishikawa) and breast (MCF-7) cancer cells. Subconfluent cells were treated with 1-3 mg% concentration of tunicamycin and 2-10 mM of benzyl-N-acetyl-alpha-galactosaminide for 1-2 days, and used for flow cytometry, immunohistochemical staining, adhesion test and Western blotting. Benzyl-N-acetyl-alpha-galactosaminide inhibits MUC1 expression on the surface of breast more than endometrial cancer cells. Tunicamycin reduces MUC1 concentration on the cellular surface more than benzylglycoside, and greatly reduces glycosylation of glycoproteins, causing an increase in cell adhesion in both types of cancer cells. The expression of alpha2beta1 integrins on the surface of these cells was weak and decreased after treatment with inhibitors. Two different glycoforms of MUC1 proteins in endometrial cells and three in breast cancer cells were expressed and their molecular weights were reduced after treatment with glycosylation inhibitors. It was confirmed with lectin detection of carbohydrate epitopes (Tn and T) in MUC1 proteins. These observations show that glycosylation inhibitors altered the N- and O-glycan patterns in a sufficient manner, and positively modified the biological features of cancer cells.

Effect of estradiol and raloxifene on MUC1 expression and adhesive properties of Ishikawa cells.

The MUC1 is a transmembrane protein with a large mucin-like extracellular domain protruding high above the cell surface. Steroid regulation of MUC1 gene expression is essential, since overexpression of MUC1 may influence the metastatic potential of cancer cells. Our earlier results demonstrated that tamoxifen, alone and combined with estradiol, inhibits MUC1 biosynthesis in endometrial adenocarcinoma cells, in contrast to estradiol. In the present study, we examine the effect of administering raloxifene or estradiol at concentrations of 1 x 10(-8)-5 x 10(-7) M, and both drugs together, on the expression of MUC1 protein, its incorporation into the cell membrane, shedding to culture medium and adhesive properties of cancer cells to the extracellular matrix (ECM). The obtained results demonstrate that raloxifene, to a lesser degree than estradiol, stimulates [3)H]Thr incorporation to the cellular, as well as the extracellular MUC1 protein. Raloxifene-treated cells show a higher cell adhesion to collagen than estradiol-treated cells, especially at lower concentrations of these drugs, probably the result of smaller amounts of sialic acid residues in the terminal glycan chains with T and Tn antigens. Sole administration of raloxifene has a lesser effect on the expression of alpha(2)beta(1) integrin than estradiol, which is in contrast to the combined action of estradiol and raloxifene. Therefore, raloxifene, which stimulates MUC1 expression in cancer cells and inhibits their adhesion to collagen to a lesser degree than estradiol, may be a clinically safe treatment for the endometrium.

Inhibition of the O-glycan elongation limits MUC1 incorporation to cell membrane of human endometrial carcinoma cells.

We have studied how benzyl-N-acetyl-alpha-D-galactosaminide, O-glycosylation inhibitor, affects the polymorphism and shedding of membrane-bound MUC1 mucin, and change in adhesive properties of cancer cells. In endometrial adenocarcinoma cells (Ishikawa line), high molecular weight MUC1 mucin was shed from cellular membrane and could be detected in culture medium 24 h after [14C]threonine labelling. Short-time (2 days) exposure of these cells to benzyl-N-acetyl-alpha-D-galactosaminide was associated with a reduction in sialic acid level and increase in T antigen content in cellular MUC1 mucin. These changes could be inverted after removal of the inhibitor. A longer, 6-day action of the inhibitor induced a decrease in sialic acid and T antigen levels in cellular MUC1 mucin. Benzyl-N-acetyl-alpha-D-galactosaminide treatment caused the occurrence of a few incompletely glycosylated glycoforms of MUC1 in cells, but not in culture medium. Adhesion of endometrial cells to ECM compounds (type I collagen) was increased by benzyl-N-acetyl-alpha-D-galactosaminide treatment, indicating that glycosylation of extracellular domain of MUC1 can modulate adhesive properties of cells.