Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells.

We reported previously that non-invasive bladder cancer expresses high level of GM3 ganglioside, whereas invasive tumors have low levels. Since glycosphingolipid synthesis in Golgi is modified greatly by a macrocyclic lactone isolated from fungi, brefeldin A (BFA), we studied effects of BFA on expression of glycosphingolipids and on invasiveness of bladder cancer cell lines. Only GM3 synthesis in invasive tumors was greatly enhanced upon treatment with BFA; synthesis of other glycosphingolipids with lacto-series type 2 or globo-series structure in both invasive and non-invasive tumors was not changed. Invasiveness of bladder cancer cells was greatly decreased in association with the great increase of GM3 synthesis induced by BFA treatment. Level of sialyl-Lex expressed in invasive cell line YTS1, which provides the adhesive property of the cells to E-selectin, was unchanged upon BFA treatment. All the bladder cancer cell lines, regardless of invasiveness, highly express tetraspanin CD9. GM3 has been implicated as a co-factor of CD9 in control of tumor cell motility. Down-regulation of CD9 is associated with metastatic properties of tumor cells and survival of patients with colonic cancer. Therefore, enhanced synthesis of GM3 induced by BFA, causing decrease of invasiveness in bladder cancer, is ascribable to the capability of GM3 to interconnect integrin with CD9, in analogy to colonic cancer and perhaps many other types of cancer.

Association of MUC-1 and SPGL-1 with low-density microdomain in T-lymphocytes: a preliminary note.

Two mucin-type glycoproteins, MUC-1 and P-selectin glycoprotein ligand-1 (PSGL-1), and glycosphingolipids (GSLs), expressed in human T-cell line HUT78, were highly enriched in low-density buoyant fraction (termed "GEM"), together with CD45, Yes, Fyn, and lck(56). Enrichment of MUC-1, PSGL-1 and GSLs, together with these signal transducer molecules in low-density membrane fraction was observable when fraction was prepared from cells either in nonionic detergent Brij 58 or in hypertonic alkaline conditions (500 mM Na(2)CO(3)). On pretreatment of cells with cholesterol-binding reagent methyl beta-cyclodextrin, levels of MUC-1 and PSGL-1 together with the above signal transducers in GEM was greatly reduced, and they were translocated into high-density membrane fraction. Similar association of lck(56), Yes, Fyn, and cSrc together with MUC-1 was also found in GEM fraction of mouse T-cell lymphoma EL4 cells expressing MUC-1 through transfection of its gene. These findings indicate the presence of another glycosyl cluster ("glycocluster"), in addition to the previously well-established GSL cluster organized with signal transducer molecules in GEM fraction, and its possible functional role in T-cells.

A one pot synthesis of mono- and di-lactosyl sphingosines.

The importance of analogues of lactosyl ceramides as basic structures of many natural glycosphingolipids provided a rationale for developing an efficient synthetic route to these compounds. We report herein a novel approach to synthesize several members of this family. Glycosylation of N-diphenylmethylene-spingosine, which exists in an imine-oxazolidine tautomeric mixture, with acetobromolactose under a modified Koenigs-Knorr condition yielded lactosyl beta-(1 --> 1) sphingosine, lactosyl beta-(1 --> 3) sphingosine and dilactosyl sphingosine in good yields. A similar glycosylation could be applicable to the synthesis of other glycosphingolipids.

Monoclonal antibody (5F3) defining renal cell carcinoma-associated antigen disialosyl globopentaosylceramide (V3NeuAcIV6NeuAcGb5), and distribution pattern of the antigen in tumor and normal tissues.

Renal cell carcinoma (RCC) has been characterized by high expression of three types of disialogangliosides: two based on lacto-series type 1 structure (disialosyl Lc(4), GalNAc disialosyl Lc(4)), the other based on globo-series structure (disialosyl globopentaosylceramide; disialosyl Gb5). The present study established a mAb, 5F3, directed to disialosyl Gb5. 5F3 was established after immunization with RCC cell line ACHN. The major disialoganglioside antigen isolated from ACHN cells, showing specific reactivity with 5F3, was characterized unequivocally as disialosyl Gb5 (V(3)NeuAcIV(6)NeuAcGb5) by identification of the core structure as globopentaosylceramide (Gb5) after enzymatic and acid hydrolysis, and by 2-dimensional (1)H-NMR spectroscopy. 5F3 does not react with monosialosyl Gb5 (V(3)NeuAcGb5), Gb5, or any lacto-series structures. 5F3 strongly stained 19 of 41 cases of primary RCC tissue. It reacted with proximal tubules (but not distal tubules) of kidney, microglial cells of cerebrum and cerebellum, goblet cells of stomach and intestine, smooth muscle of various organs. It did not react with parenchymatous cells of various organs, except for kidney epithelia and prostate stroma. Immunostaining of RCC tissue by mAb 5F3, in combination with staining by other antibodies directed to globo-series and lacto-series structures, has prognostic significance in defining metastatic potential of RCC.

Human alpha (1,3)-fucosyltransferase IV (FUTIV) gene expression is regulated by elk-1 in the U937 cell line.

The alpha1,3-fucosyltransferase IV (FucTIV) encoded by its gene (FUTIV) is responsible for synthesis of Le(x) (Galbeta4[Fucalpha3]GlcNAcbeta3Galbeta1,R), which causes compaction in the morula stage of the preimplantation mouse embryo, as well as alpha1,3-fucosylation at multiple internal GlcNAc of unbranched poly-N-acetyllactosamine, termed "myeloglycan," the physiological epitope of E-selectin. Since myeloglycan-type structure is also expressed in various types of human cancer and may mediate E-selectin-dependent metastasis, expression of FUTIV is oncodevelopmentally regulated. The mechanisms controlling FUTIV expression remain to be clarified. In this report, we further characterize FUTIV gene structure and define a non-TATA box-dependent transcriptional start region just upstream from the translational start. FUTIV promoter/reporter fusion constructs defined a "full-length" promoter and highly active fragments in the macrophage-derived U937 and myeloid HL60 cell lines. One highly active fragment contains a consensus binding site for the Ets-1 transcription factor (Withers, D. A., and Hakomori, S. (1997) Glycoconj. J. 14, 764). Gel shift analysis shows specific binding to this site in nuclear extracts from U937 cells. Mutation of the Ets consensus site significantly reduces FUTIV promoter activity in both cell lines. Gel supershift and dominant negative cotransfection experiments identified the Ets family member Elk-1 as one component binding and regulating the FUTIV promoter in U937 cells. The significance of FUTIV regulation by Elk-1 is discussed.

Soluble fibronectin interaction with cell surface and extracellular matrix is mediated by carbohydrate-to-carbohydrate interaction.

Cell adhesion and spreading on solid phase fibronectin (FN), coated on plate or presented in extracellular matrix, are mediated by integrin receptors alpha5beta1, alpha4beta1, etc., although binding of "soluble-form FN" to cell surface varies extensively depending on glycosylation status of FN per se. Deposition or incorporation at the cell surface or pericellular matrix of soluble-form FN from body fluids or synthesized de novo takes place through a yet-unknown (perhaps integrin-independent) mechanism. Here we present evidence that the mechanism involves carbohydrate-to-carbohydrate interaction. Binding or incorporation of soluble-form placental or hepatoma FN to cell surface or pericellular matrix is highly dependent on the specific glycosylation status of FN per se and combination with glycosylation status of the cell surface, and is greatly promoted by a certain type of coexisting (shedded) glycosphingolipid. A few lines of study indicate that the process is mediated by interaction of FN carbohydrate with cell surface carbohydrate. The great enhancement of the binding process by glycosphingolipid is based on dual interaction of glycosphingolipid carbohydrate with FN carbohydrate and with cell surface carbohydrate. Here we present an example of promotion of binding of soluble-form FN from placenta or from hepatoma cells, having a specific carbohydrate epitope termed "disialyl-I," to K562 or VA13 cell surface in the presence of glycosphingolipid Gg3, which interacts specifically with disialyl-I.

Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain.

Glycosphingolipids (GSLs) and sphingomyelin in animal cells are clustered and organized as membrane microdomains closely associated with various signal transducer molecules such as cSrc, Src family kinases, small G-proteins (e.g., RhoA, Ras), and focal adhesion kinase. GSL clustering in such microdomains causes adhesion to complementary GSLs on the surface of counterpart cells or presented on plastic surfaces, through carbohydrate-to-carbohydrate interaction. GSL-dependent cell adhesion in microdomain causes activation of the signal transducers, leading to cell phenotypic changes. A retrospective of the development of this concept, and current status of our studies, are presented.

The globoseries glycosphingolipid sialosyl galactosyl globoside is found in urinary tract tissues and is a preferred binding receptor In vitro for uropathogenic Escherichia coli expressing pap-encoded adhesins.

Women with a history of recurrent Escherichia coli urinary tract infections (UTIs) are significantly more likely to be nonsecretors of blood group antigens than are women without such a history, and vaginal epithelial cells (VEC) from women who are nonsecretors show enhanced adherence of uropathogenic E. coli isolates compared with cells from secretors. We previously extracted glycosphingolipids (GSLs) from native VEC and determined that nonsecretors (but not secretors) selectively express two extended globoseries GSLs, sialosyl galactosyl globoside (SGG) and disialosyl galactosyl globoside (DSGG), which specifically bound uropathogenic E. coli R45 expressing a P adhesin. In this study, we demonstrated, by purifying the compounds from this source, that SGG and DSGG are expressed in human kidney tissue. We also demonstrated that SGG and DSGG isolated from human kidneys bind uropathogenic E. coli isolates expressing each of the three classes of pap-encoded adhesins, including cloned isolates expressing PapG from J96, PrsG from J96, and PapG from IA2, and the wild-type isolates IA2 and R45. We metabolically 35S labeled these five E. coli isolates and measured their relative binding affinities to serial dilutions of SGG and DSGG as well as to globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4), two other globoseries GSLs present in urogenital tissues. Each of the five E. coli isolates bound to SGG with the highest apparent avidity compared with their binding to DSGG, Gb3, and Gb4, and each isolate had a unique pattern of GSL binding affinity. These studies further suggest that SGG likely plays an important role in the pathogenesis of UTI and that its presence may account for the increased binding of E. coli to uroepithelial cells from nonsecretors and for the increased susceptibility of nonsecretors to recurrent UTI.

A new procedure for establishing functional monoclonal antibodies capable of inhibiting E- or P-selectin-dependent cell adhesion.

Employing a new procedure, we established many monoclonal antibodies (mAbs) which inhibit E- or P-selectin-dependent cell adhesion. One of these mAbs is capable of staining selectin in paraffin-embedded histological sections. The procedure is based on immunization of BALB/c mice with irradiated mouse myeloma NS-1 cells (syngeneic HAT-sensitive fusion partner cells) transfected with cDNA encoding human E- or P-selectin. Resulting NS-1 transfectant cells permanently express human E- or P-selectin as immunogen. The mAbs are useful for detecting selectins by flow cytometric and immunohistological methods, and for inhibiting selectin-dependent adhesion in experimental models. In contrast, the majority of anti-selectin mAbs previously established do not have these capabilities.

A specific cell surface glycoconjugate controlling cell motility: evidence by functional monoclonal antibodies that inhibit cell motility and tumor cell metastasis.

The biochemical basis of cell motility has been viewed as a complex process involving cell surface membrane proteins, integrin receptors, growth factors and their receptors, and cytoskeletal components [Rosen & Goldberg (1989) In Vitro 25, 1079]. The possible involvement of glycoconjugates at the cell surface in controlling cell motility has not been systematically investigated. We addressed this question using functional monoclonal antibodies (MAbs), which inhibit cell motility and the metastatic potential of tumor cells, as probes. Two such MAbs, derived from two independent processes of immunization and selection, were found to directed to a common specific carbohydrate structure, Fuc alpha 1----2Gal beta 1----R. MAb MIA-15-5 was established after immunization of mice with small cell lung carcinoma line PC7 and selected on the basis of inhibition of U937 and HEL cell migration. MAb MIA-22-20 was established after immunization with lung adenocarcinoma line MAC-10 and selected on the basis of inhibition of MAC-10 cell migration. These two MAbs were both IgM and were consistently reactive with the Fuc alpha 1----2Gal beta 1----R structure, regardless of the identity of the R group. Various other anti-H MAbs, specific to carrier isotype, did not affect cell motility. MAb MIA-15-5 reacted with 30-40% of high-metastatic variant BL6 of mouse melanoma B16 line but with only less than 5% of low-metastatic variant F1. Metastatic deposition to lung after injection of BL6 cells was inhibited if MAb MIA-15-5 was injected within 3 h but was not inhibited by injection of other anti-H antibodies under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization.

Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N- and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both.

Murine embryonal carcinoma cell-surface sialyl LeX is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan.

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl LeX was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (LeX or X hapten). Sialyl LeX was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl LeX and i antigens but not LeX, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.

Transformation by the oncogene v-fms: the effects of castanospermine on transformation-related parameters.

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.

A monoclonal antibody defining a binary N-acetyllactosaminyl structure in lactoisooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6): a useful probe for determining differential glycosylation patterns between normal and transformed human fibroblasts.

Monoclonal antibodies A5 and C6 have been reported previously to recognize developmentally regulated determinants involving N-acetyllactosamine [Fenderson B. A., O'Brien D. A., Millette C. F. and Eddy E. M. (1984) Devl Biol. 103, 117-128]. In the present study, the specificity of these antibodies was determined by solid-phase radioimmunoassay and by thin-layer chromatography immunostaining using purified glycolipid standards. Antibody A5 recognized N-acetyllactosamine (type 2 chain; Gal beta 1----4GlcNAc beta 1----3R), irrespective of branching status. In contrast antibody C6 recognized the binary N-acetyllactosamine structure carried on lactoisooctaosylceramide. Antibody C6 did not react with sialosyl or alpha-galactosyl derivatives of the isooctaosyl structure, including human G10, G8 and bovine G9. Thus, unlike other anti-I antibodies, C6 provides a specific probe for both branching status and absence of terminal chain modification. Monoclonal antibodies A5, C6 and anti-I(Ma) were used to investigate glycosylation changes associated with oncogenic transformation. In contrast to results with lectins, these antibodies preferentially labeled the major glycoproteins of SV40-transformed human embryonic lung fibroblasts, including GP80, GP180, GP200 and GP250. The results suggest that increased expression of unsubstituted polylactosamine core structure at the cell surface follows SV40-transformation.

Carbohydrate determinants associated with carcinoembryonic antigen (CEA).

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.

Tissue distribution of the Pk antigen as determined by a monoclonal antibody.

Using an anti-Pk monoclonal antibody (mAb) designated CPK-1, the expression of the Pk antigen was assessed on normal human tissue from non-Pk individuals. Although the Pk antigen was detected on fibroblasts and blood vessels as previously reported, it was also found on smooth muscle cells of the digestive tract and the urogenital system. Pk was also found on glandular cells of the stomach, oesophagus and prostate. Additionally, CPK-1 reacted weakly with oesophagus squamous cells, and a small number of glomeruli and tubules in the kidney. The mechanism of expression of the Pk determinant in non-Pk individuals is discussed.

Monoclonal antibodies PMN 6, PMN 29, and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-N-fucopentaose III.

Three monoclonal antibodies, PMN 6, PMN 29, and PM-81, bind myeloid cells. Antibodies PMN 6 and PMN 29 bind specifically to granulocytes but differ in their ability to bind some other cell lines [E. D. Ball, R. F. Graziano, L. Shen, and M. W. Fanger (1982) Proc. Natl. Acad. Sci. USA 79, 5374-5378]. Antibody PM-81, in addition to granulocytes, also binds to eosinophils, monocytes, and most acute myelocytic leukemia cells [E. D. Ball, R. F. Graziano, and M. W. Fanger (1983) J. Immunol. 130, 2937-2941]. Despite these differences, the binding of all three antibodies to cells was inhibited by the oligosaccharide, lacto-N-fucopentaose III [Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc]. Solid-phase radioimmunoassays using purified glycolipids containing sugar sequences found in lacto-N-fucopentaose III demonstrated different binding characteristics for each antibody. PM-81 bound lower concentrations of glycolipids than PMN 29, while PMN 6 required the highest concentration of glycolipids for binding. Autoradiography of thin-layer chromatograms of glycolipid antigens supported these results. The binding of these monoclonal antibodies to cells probably depends on the density of antigens on the cell surface, each antibody requiring a different density. Thus, cells containing antigen below a certain threshold concentration may not bind low-affinity antibodies.

A granulocyte reactive monoclonal antibody, 1G10, identifies the Gal beta 1-4 (Fuc alpha 1-3)GlcNAc (X determinant) expressed in HL-60 cells on both glycolipid and glycoprotein molecules.

1G10, a monoclonal IgM antibody that identifies a differentiation antigen on human granulocytes and a subpopulation of monocytes, was found to react specifically with glycosphingolipids bearing the Gal beta 1-4(Fuc alpha 1-3)GlcNAc hapten (X determinant). This carbohydrate determinant was found on both glycolipid and glycoprotein molecules isolated from HL-60 cells (a promyelocytic leukemia cell line). Thus, this highly conserved carbohydrate-defined determinant previously described on mouse embryonic and mouse and human carcinoma cells is also expressed as a tissue-specific differentiation antigen on normal human granulocytes.