Cell surface expression of urokinase receptor in normal mammary epithelial cells and breast cancer cell lines

Background: The urokinase receptor (uPAR) is important in the process of extracellular matrix degradation occurring during cancer cell invasion and metastasis. We wished to quantify uPAR on the surfaces of normal mammary epithelial cells (HMEC) and 6 well-known breast cancer cell lines using flow cytometry. Materials and Methods: Cell surface uPAR was labelled with a monoclonal antibody, and this was detected with a florescent-labelled second antibody and accurately measured using flow cytometry. The measured fluorescent signals of the stained cells were interpolated with those of Quantum Simply Cellular bead standards to determine the number of uPAR sites per cell. Results: The breast cancer cell lines ranged from 13,700 to 50,800 uPAR sites per cell, whilst HMEC cells had only 2,500 sites. Conclusions: This simple and reliable method showed that the expression of cell surface uPAR is higher in the breast cancer cell lines than in the normal mammary cells.

Epithelial transport and deamidation of gliadin peptides:a role for coeliac disease patient immunoglobulin A

In coeliac disease, the intake of dietary gluten induces small-bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical-to-basal passage of gluten-derived gliadin peptides p31-43 and p57-68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation-dependent coeliac patient-derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated.

A novel role for retromer in the control of epithelial cell polarity

The establishment and maintenance of epithelial cell polarity is essential throughout the development and adult life of all multicellular organisms. A key player in maintaining epithelial polarity is Crumbs (Crb), an evolutionarily conserved type-I transmembrane protein initially identified in Drosophila. Correct Crb levels and apical localization are imperative for its function. However, as is the case for many polarized proteins, the mechanisms of its trafficking and strict apical localization are poorly understood. To address these questions, we developed a liposome-based assay to identify trafficking coats and interaction partners of Crb in a native-like environment. Thereby, we demonstrated that Crb is a cargo for Retromer, a trafficking complex required for transport from endosomes to the trans-Golgi-network. The functional importance of this interaction was revealed by studies in Drosophila epithelia, which established Retromer as a novel regulator of epithelial cell polarity and verified the vast potential of this technique.