Changes In Cell Surface Carbohydrates Research Articles

On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis.

This review focuses on the recent advances in investigations of the role of cell surface carbohydrates in tumor metastasis. It also summarizes the results of extensive studies of endogenous lectins, their structure, carbohydrate specificity and biological functions with the major emphasis on the significance of lectin-cell surface carbohydrate interactions in a metastatic process. Numerous data demonstrate that malignant transformation is associated with various and complex alterations in the glycosylation process. Some of these changes might provide a selective advantage for tumor cells during their progression to more invasive and metastatic phenotype. Cell glycosylation depends on the expression and function of various glycosyltransferases and glycosidases. Recently, transfection of genes encoding various glysosyltransferases gene in sense and antisense orientation helped to bring direct evidence that changes in cell surface carbohydrates are important for the metastatic behavior of tumor cells. Cell surface carbohydrates affect tumor cell interactions with normal cells or with the extracellular matrix during metastatic spread and growth. These interactions can be mediated via tumor cell carbohydrates and their binding proteins known as endogenous lectins. The family of the discovered endogenous lectins is rapidly expanding. The number of C-type lectins has reached 50 and at least 10 galectins have been identified. The biological significance of the endogenous lectins and their possible role in tumor growth and metastasis formation has started to unravel. Some lectins recognize the 'foreign' patterns of cell surface carbohydrates expressed by microorganisms and tumor cells, and play a role in innate and adaptive immunity. It was shown that lectins affect tumor cell survival, adhesion to the endothelium or extracellular matrix, as well as tumor vascularization and other processes that are crucial for metastatic spread and growth.

Blood-group-related carbohydrates are expressed in organotypic cultures of human skin and oral mucosa.

Cellular maturation and migration are usually associated with changes in cell-surface carbohydrates, but the relationship between these changes and cell behaviour is at present largely unknown. To investigate whether an organotypic culture system can be used as an in vitro model to study the function of cell-surface carbohydrates, we established organotypic cultures of skin and buccal mucosa. In these cultures, keratinocytes are grown at the air-liquid interface on a supporting matrix consisting of homologous fibroblasts embedded in a collagen type I gel. We examined the expression of blood-group-related carbohydrate structures, including Lewis x, sialylated Lewis x, Lewis y, Lewis a, and Lewis b, on the surface of epithelial cells in the cultures. We compared the results with the expression of more well-established markers, including cytokeratins, integrins, bullous pemphigoid antigen and laminin, in the same cultures. The organotypic skin and oral mucosa cultures showed a histological differentiation pattern analogous to that of normal skin and buccal mucosa, and a tissue-specific expression of carbohydrate structures and cytokeratins. However, both types of organotypic cultures also expressed markers which are normally seen during wound healing, including Lewis y, cytokeratin 16, and cytokeratin 19. We conclude that the organotypic cultures of oral mucosa and skin are suitable models for future studies of the function of cell-surface carbohydrates, although the expression of wound healing markers has to be taken into consideration.

Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA.

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes.

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2Kb or H-2Kd gene-transfected melanoma cells was associated with reduced adherence to endothelial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demonstrated reduced ability to invade Matrigel that paralleled up-regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2Dd/Ld genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2Kb transfected cells was associated with alterations in cell surface carbohydrates with appearance of alpha-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes.

Cell surface carbohydrates as prognostic markers in human carcinomas.

Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucin-like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour-associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since certain structures which are tumour-related in one organ may be normal constituents of other tissues. Tumour-associated carbohydrate changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some carbohydrate structures is associated with prognosis. Tn, sialyl-Tn, and T are cell membrane-bound mucin-like carbohydrate structures that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin-like structures. Their expression is strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression is at present unknown. The blood group-related carbohydrate structures Le(x), sialyl-Le(x), ABH, and Le(y) are examples of terminal carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl-Le(x) and E-selectin and studies indicate that Le(y) is related to cell motility. These findings are now the basis for tumour therapeutic experiments.

Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis.

A mouse embryonic stem (ES) cell line E14 and early mouse embryos were stained with a panel of 15 monoclonal antibodies recognizing sialylated or potentially sialylated carbohydrate determinants. Sialyl Le-x and sialyl Le-a were detected on the pre-implantation embryo from the 8-cell stage, and sialyl Le-a weakly on undifferentiated ES cells. Changes in cell surface carbohydrates occurred after induction of ES cell differentiation with retinoic acid (RA) and dibutyryl cAMP. Qualitative analysis of the neutral glycolipids of untreated and RA-treated ES cells using high-performance thin-layer chromatography (HPTLC) revealed few differences between the two types of culture. The major gangliosides in both cultures were indicative of an active 'a' ganglioside synthesis pathway. GD3, a precursor of the 'b' synthesis pathway, previously reported to be characteristic of embryonal carcinoma (EC) cells, was absent. RA-induced differentiation caused a shift in the spectrum to more complex gangliosides. Application of fast atom bombardment mass spectrometry (FAB-MS) to permethylated derivatives of individual bands permitted partial characterization of an unusual sialylated glycolipid and a rare ganglioside with the suggested structure of GalNAc-GD1a.

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

Blood group antigen staining pattern during experimental carcinogenesis in rat palate

During 4-nitroquinoline-1-oxide-induced carcinogenesis in the rat palate, animals were sacrificed at various intervals and stained for blood group antigens B and H (Type 2 chain) by an immunofluorescent method. In rats without signs of epithelial dysplasia, the staining pattern was identical with that in the normal control rats. In rats with definite or questionable (borderline cases) dysplasias, marked changes in blood group antigen staining pattern were seen. Thus, changes in cell-surface carbohydrates during malignant development in the rat palate seem to follow closely the histomorphological changes. As there is good evidence that carcinomas would eventually develop in all rats if they were not sacrificed, it seems that the blood group antigen staining pattern does not predict malignant development in the absence of histological suspicion.

β1-6 Branching of Asn-Linked Oligosaccharides Is Directly Associated with Metastasis

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.

Neural induction: embryonic determination elicits full expression of specific neuronal traits

ABSTRACT In Pleurodeles waltl, the early neuronal differentiation of precursor cells from late gastrula stage has been studied by culture in vitro from either isolated neural plate (NP) or isolated neural fold (NF). The aim of this study was to delineate the information acquired by ectodermal target cells during neural induction. By culturing these cells in vitro either with or without the underlying chordamesoderm, we showed that in the absence of chordamesodermal influence such NP or NF cells exhibited a high degree of biochemical and morphological differentiation as revealed by the synthesis and the storage of neurotransmitters, the activity of specific enzymes, as well as by the expression of neuronal markers: specific changes in cell surface carbohydrates, tetanus toxin binding sites and neuro filament polypeptides. Remarkable changes in the cell adhesive properties were the first events observed in the different central (NP) and peripheral (NF) types. In cocultures the chordamesodermal cells exert a beneficial influence on this differentiation, specially increasing acetylcholine synthesis. There are some differences between central (NP) or peripheral (NF) neuroblast response to this further notochord or mesodermal influence.

A dominant mutation to ricin resistance in Chinese hamster ovary cells induces UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III activity.

A biochemical basis for the LEC10 mutant phenotype of Chinese hamster ovary cells has been identified. Independent LEC10 mutants, originally selected for resistance to the toxicity of ricin, have been shown to exhibit reduced binding of 125I-ricin at the cell surface. Although this is indicative of structural changes in cell-surface carbohydrates, labeling of plasma membranes with galactose oxidase/[3H]borohydride revealed no significant differences between mutant and parental cells. Alterations in the carbohydrates synthesized by LEC10 cells were, however, resolved by lectin-affinity chromatography of glycopeptides from the G glycoprotein of vesicular stomatitis virus (VSV) grown in LEC10. LEC10/VSV glycopeptides contain a fraction which is not bound to concanavalin A-Sepharose but is strongly retarded on E-PHA (erythroagglutinin from Proteus vulgaris)-agarose. In contrast, CHO/VSV glycopeptides or those from a LEC 10 revertant (R.LEC 10/VSV) do not contain carbohydrates with these properties. High-field 1H NMR spectroscopy of the novel LEC10/VSV carbohydrates showed that they are complex, biantennary structures containing N-acetylglucosamine in beta(1,4)-linkage to the beta-linked core mannose residue. The presence of these structures correlates with the expression of the enzyme responsible for the addition of this "bisecting" GlcNAc residue, UDP-GlcNAc:glycopeptide beta-4-N-acetylglucosaminyltransferase III (GlcNAc-TIII). Parental Chinese hamster ovary cells and the LEC10 revertant possess no detectable GlcNAc-TIII activity. The combined evidence suggests that the LEC10 mutation induces the expression of the GlcNAc-TIII enzyme in Chinese hamster ovary cells.

Cell communication reduced by changes in cell surface carbohydrates

The possibility has been investigated that alterations in the plasma membrane interfere with the formation of communicating contacts between cells in vitro. To this end, various rodent cell lines and selected variant strains were simultaneously studied for the composition of membrane-bound carbohydrates and the capacity of forming intercellular communicative junctions. In addition, a variety of somatic cell hybrids of communication-incompetent mouse L cells and several normal, untransformed or neoplastic cells were investigated for the same parameters. Cell-to-cell communication was studied by measuring the cytoplasmic exchange of labelled uridine nucleotides. Maximal intercellular communication was found between cells displaying a normal size-distribution of membrane glycopeptides. In contrast, structural alterations in these molecules, reflected either by altered size-distribution or by a failure to incorporate terminal fucose, were associated with reduced or completely impaired capacity of establishing metabolic cooperation. In somatic cell hybrids complementation by normal genomes suggested the involvement of recessive genetic lesions affecting intercellular communication through disturbances in the genetic control of surface carbohydrate synthesis. The observed correlation between changes in plasma membrane molecules associated with tumour cells and impaired formation of functional cell-cell contacts may reflect a malfunction of tumour cells in their interactions with neighbouring cells or with signal molecules.

Lectin binding to neural crest cells. Changes of the cell surface during differentiation in vitro.

To examine possible changes in cell surface carbohydrates, fluorescent lectins were applied at various times during differentiation of neural crest cells in vitro. The pattern and intensity of binding of several lectins changed as the crest cells developed into melanocytes and adrenergic cells. Considerable amounts of concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to all unpigmented cells throughout the culture period. Melanocytes, however, bound much less of these lectins. Soy bean agglutinin (SBA), unlike Con A and WGA, only bound later in development to unpigmented cells at about the time when catecholamines were detected histochemically. Binding of SBA could be induced in younger cultures by pretreating the cells with neuraminidase. Melanocytes, however, did not bind detectable amounts of SBA even if treated with neuraminidase. The SBA-binding sites were often concentrated on cytoplasmic extensions and on contact points between neighboring cells, even when receptor mobility was restricted by prefixation of the cells or adsorption of lectin at 0 degrees C. All three lectins bound to cell processes resembling nerve fibers in particularly high amounts.