Alteration of the glycocalyx of two blood-brain barrier mimicking cell lines is inducible by glioma conditioned media (original) (raw)
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Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and eoncanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalln A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated coneanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites (K d = 2 • 10 -7 M). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high aff'mity concanavalin A binding sites.
Glia, 2005
The communication between glial cells and brain capillary endothelial cells is crucial for a well-differentiated bloodbrain barrier (BBB). It has been suggested that in vitro primary glial cells (GCs) be replaced by the glial C6 cell line to standardise the model further. This study compares directly the structural and functional differentiation of bovine brain capillary endothelial cells (BBCECs) induced by co-culture with rat primary GCs or C6 cells, for the first time. Transendothelial electrical resistance (TEER) measurements showed that under no condition were C6 cells able to reproduce TEER values as high as in the presence of GCs. At the same time, permeability of the BBCECs to both radioactive sucrose and FITC-inulin was 2.5-fold higher when cells were co-cultured with C6 than with GCs. Furthermore, immunocytochemistry studies showed different cell morphology and less developed tight junction pattern of BBCECs co-cultured with C6 toward GCs. Additionally, studies on P-glycoprotein (P-gp) showed much lower P-gp presence and activity in BBCECs co-cultured with C6 than GCs. Both VEGF mRNA expression and protein content were dramatically increased when compared with GCs, suggesting that VEGF could be one of the factors responsible for higher permeability of BBB. Our results clearly indicate that, in the presence of the glial C6 cell line, BBCECs did not differentiate as well as in the coculture with primary GCs at both structural and functional levels.
C6 glioma cells express modified neural-cell adhesion molecule-like glycoproteins
Brain Research, 1987
We have studied the expression of neural-cell adhesion molecule (N-CAM)-Iike glycoproteins in a C6 glioma cell line. We found that: (a) C6 cells express N-CAM-like proteins on the cell surface, (b) the N-CAM-like proteins in C6 ceils have apparent molecular weights of 130,000 and 150,000 kDa which are not seen in rodent brain and (c) deglycosylation of (26 N-CAMs suggest that the modifications are both in the carbohydrate and protein parts of the N-CAM. The expression of modified N-CAM glycoproteins is of interest in relation to the regulation of N-CAM expression.
Endothelial glycoconjugates: a comparative lectin study of the brain, retina and myocardium
Journal of Anatomy, 2000
There is evidence that the endothelial cell (EC) glycocalyx is a significant determinant of vascular permeability, acting as a charge-size filter to permeant molecules. We have therefore examined its oligosaccharide composition in 3 classes of microvessel with differing permeabilities. EC in rat brain, retina and myocardium were labelled with a panel of lectins and subjected to a semiquantitative analysis. Surprisingly, no substantial differences were evident for any lectin labelling between the 3 microvessel types despite their marked morphophysiological diversity. In particular, all showed substantial sialic acid expression, with Maackia amurensis (MAA) labelling sialic acid in an α2-3 linkage to β-galactose and Sambucus nigra (SNA) recognising sialic acid in an α2-6 linkage to β-galactose. Arachis hypogaea (PNA) binding after neuraminidase digestion indicated the presence of Gal β1-3GalNAc attached to terminal sialic acid. The results therefore show that the sequences NeuNAc α2-3Gal β1-3GalNAc and NeuNAc α2-6Gal β1-3GalNAc are strongly expressed in the 3 microvessel types irrespective of their permeability properties. This homogeneity suggests that these lectin ligands may be involved in a common set of EC functions, e.g. cell :cell and cell :matrix interactions. However, we cannot rule out the possibility that glycocalyx differences may exist between vessels in the paracellular cleft which may alter its filtration properties.
Surface glycoproteins of normal and neoplastic glia cells in culture
International Journal of Cancer, 1980
Two normal and seven malignant human glia lines grown in vitro were labelled by lactoperoxidase catalysed iodination. The labelled cell surface glycoproteins were isolated by lectin affinity chromatography and compared by SDS gel electrophoresis. The glia and the glioma lines possess a common characteristic glycoprotein pattern. Seven glycoproteins in the molecular weight range between 70,000 and 220,000 daltons and several minor components of low molecular weight could be distinguished. The expression of the glycoproteins was independent of the passage number or the growth conditions although the expression of the different glycoproteins showed quantitative differences for the individual cell lines. The differences found in the tumor lines were either due to an amplification or decrease in the expression of the different glycoproteins and/or their accessibility to the lactoperoxidase-catalysed labelling.
Influence of Host Cell Infiltration on the Glycolipid Content of Mouse Brain Tumors
Journal of Neurochemistry, 2002
Previous studies showed that levels of some glycosphingolipids (GSL5) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT-2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro. The markers included ganglioside N-glycolylneuraminic acid (NeuGc), GAl (asialo-GM1), and Fc receptor-bearing cells. NeuGc-containing gangliosides, GAl, and Fc receptors are expressed by macrophages and lymphoid-type cells of the mouse host immune system but are not normally expressed by mouse neural cells. Differences in the relative content of Fc receptor-bearing cells in ependymoblastoma and CT-2A tumors grown in vivo (8.3 and 16.8%, respectively) were proportional to differences in the relative content of NeuGc-containing gangliosides (25.5 and 45.1%) and GAl (8.5 and 13.8%), respectively. Neither cultured tumor cell line expressed Fc receptors, GAl, or NeuGccontaining gangliosides. These findings suggest that nonneoplastic host infiltrating cells (macrophages) contribute significantly to the GSL composition of solid tumors growing in vivo.
Journal of Neurochemistry, 2005
One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 lg/mL puromycin for the first 2 days of culture or 3 lg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 · 10 )6 cm/s) and a high electrical resistance (up to 500 W · cm 2 ). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.
Immunodiagnosis of the primary brain tumor (glioma) by the endogenous lectin
Clinica Chimica Acta, 2002
Background: A sugar-binding protein, lectin, was identified in the cystic fluid of brain tumor (glioma). Methods: The protein was purified by high performance liquid chromatography. Results: The molecular weight of the lectin was 29 kDa. The activity of the purified protein was inhibited strongly by p-nitro-b-D-galactose and asialofetuin. The lectin activity was not Ca 2 +-or Mg 2 +-dependent. The antiserum of this pure lectin specifically cross-reacted in the cerebrospinal fluid (CSF) of glioma tumor patients. The same antibody had no reaction with CSF of other neurological patients, normal rat brain tissue extract, oncofetal antigens such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), plant lectins, and other body fluids like plural and ascites. Conclusions: This antilectin antibody may be useful in the evaluation of patients with primary brain tumor (glioma).