globotriaosylceramide and Burkitt-Lymphoma

Silurus asotus (catfish) egg lectin (SAL) has potent affinity to Gal alpha-linked carbohydrate chains of not only glycoproteins but also glycosphingolipids such as globotriaosylceramide (Gb3). SAL selectively bound to Gb3 localized in glycosphingolipid-enriched microdomain (GEM) of Gb3-expressing (Gb3(+)) Burkitt's lymphoma cells. Since treatment of Gb3(+) cells with SAL caused an increase in externalization of phosphatidylserine via activation of P-glycoprotein, and apoptotic volume decrease via activation of G-protein activated K(+) channel-1, SAL may function as an inducer of early apoptotic signal; however, neither caspase-8 and -3 activation nor DNA fragmentation was observed. We therefore investigated whether cell proliferation and viability were altered in SAL-treated Raji cells. SAL caused reduction of Raji cell proliferation without cytotoxicity. Although SAL did not induce apoptotic cell death to Gb3-expressing cells, it functionally behaved as a regulator of cell proliferation. SAL activated the suppression system of cell proliferation, such as down-regulation of c-myc and cdk4, and up-regulation of p21 and p27, inducing G1 arrest of the cell cycle, and consequently inhibited cell proliferation of Raji cells. Therefore, we conclude that SAL leads the cells to early apoptotic status but not late apoptotic (necrotic) status via binding to Gb3 existing in GEM, and that this binding is a prerequisite condition to induce cell cycle stop signal.

Topics: Animals; Burkitt Lymphoma; Cell Proliferation; Depression, Chemical; Fish Proteins; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lectins; Phosphatidylserines; Potassium; Protein Binding; Signal Transduction; Trihexosylceramides

Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway.

Topics: Analysis of Variance; Animals; Blotting, Western; Burkitt Lymphoma; Catfishes; Cell Line, Tumor; Cytokines; DNA Primers; Electrophoresis, Polyacrylamide Gel; Fish Proteins; Flow Cytometry; Humans; Lectins; Melibiose; Microscopy, Fluorescence; Osmeriformes; Phycoerythrin; Protein Binding; Real-Time Polymerase Chain Reaction; Signal Transduction; Trihexosylceramides

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galβ1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Topics: Amino Acid Sequence; Animals; Apoptosis; Burkitt Lymphoma; Cell Line, Tumor; Humans; K562 Cells; Lectins; Molecular Sequence Data; Mytilus; Peptide Mapping; Polysaccharides; Sequence Alignment; Trihexosylceramides

Verotoxin (VT-1) is a cytotoxin, produced by Shigella dysenteriae type 1 or by Shiga toxin-producing Escherichia coli, which binds specifically to globotriaosylceramide (Gb3). This glycosphingolipid is a B cell differentiation antigen (Gb3/CD77) strongly expressed on Burkitt's lymphoma cells. We have previously shown that, in these cells, VT-1 induces apoptosis via a caspase- and mitochondria-dependent pathway. In this report, we provide new insights into this signal transduction pathway. First, we demonstrate that VT-1-induced apoptosis requires degradation of the caspase-8 inhibitory molecule c-FLIPL and that this degradation occurs through the ubiquitin-proteasome pathway. Furthermore, we show that mitochondrial activation is mainly due to i) cleavage and activation of the pro-apoptotic Bcl-2 family member Bid by caspase-8 and ii) Bax relocalization to mitochondrial membranes which lead to cytochrome c release. However, tBid is not involved in Bax relocalization, and relocalization is most likely controlled by the extent of Bax phosphorylation: in non-treated BL cells, p38 MAPK participates in the retention of Bax in the cytoplasm in an inactive form whereas in VT-1 treated cells, protein phosphatase 2A is activated and induces Bax relocalization to mitochondria.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Burkitt Lymphoma; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line; Cytochromes c; Humans; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Protein Phosphatase 2; Shiga Toxin 1; Shigella dysenteriae; Signal Transduction; Trihexosylceramides; Ubiquitin

Silurus asotus (catfish) egg lectin (SAL) has a strong affinity to Gal alpha-linked carbohydrate chains of not only glycoproteins but also glycosphingolipids such as globotriaosylceramide (Gb3). SAL uniformly bound to surfaces of Gb3-expressing (Gb3+) Burkitt's lymphoma cells, while Gb3 molecules were interspersed on the surfaces of Gb3+ cells. After a short period of treating Raji and Daudi cells with SAL, each cell size was 10 and 25% smaller than that of untreated cells, respectively. Treatment of Gb3+ cells with SAL caused an increase in binding of annexin V, however, neither caspase activation nor DNA fragmentation was observed after treatment with SAL for 22 h. Since SAL did not induce cell death in Gb3+ cells, SAL may function as an inducer of early apoptotic signal. We have revealed that SAL did not bind to D-threo-1-phenyl-2-decanoylamino-3-morphorino-1-propanol (D-PDMP)-treated Raji cells, and no cell shrinkage was observed in Gb3-deficient Raji cells treated with SAL, indicating that Gb3 localized in the glycosphingolipid-enriched microdomain (GEM) was involved in SAL-induced cell shrinkage through activation of voltage-gated potassium channel Kv1.3, and that the glycoprotein ligands on Gb3-deficient Raji cells treated with SAL were not included in this phenomenon. These results suggest that SAL leads the cells to early apoptotic status via binding to Gb3 existing in GEM, and that this binding is a prerequisite condition to induce early stage of apoptosis.

Topics: Animals; Annexin A5; Apoptosis; Burkitt Lymphoma; Catfishes; Cations, Monovalent; Cell Line, Tumor; Cell Membrane; Fish Proteins; Humans; Kv1.3 Potassium Channel; Lectins; Phosphatidylserines; Potassium; Protein Binding; Trihexosylceramides

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.

Topics: Adhesins, Bacterial; Animals; Antibodies, Monoclonal; Bacterial Adhesion; Binding Sites; Burkitt Lymphoma; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Crystallography, X-Ray; Disaccharides; Globosides; Humans; Hydrogen Bonding; Lectins; Microarray Analysis; Models, Molecular; Molecular Sequence Data; Molecular Structure; Polysaccharides; Protein Binding; Protein Structure, Quaternary; Protein Structure, Secondary; Pseudomonas aeruginosa; Substrate Specificity; Trihexosylceramides; Water

Silurus asotus lectin (SAL) is a member of the rhamnose-binding lectin (RBL) family, and recognizes globotriaosylceramide (Gb3) on the cell surface of Burkitt's lymphoma cell lines, such as Raji and Daudi cells. The variation of gene expression in the treatment of both cells with SAL was analyzed using the differential display (DD) method with combination of 16 kinds of arbitary primers and 3 kinds of anchor primers. Treatment of Raji cells with SAL down-regulated mitochondria-associated granulocyte-macropharge colony-stimulating factor (GM-CSF) signaling molecule (Magmas) gene, and up-regulated N-myc downstream regulated gene (NDRG) 3. On the other hand, treatment of Daudi cells with SAL down-regulated Rad50 gene. Since Magmas gene expression was repressed in SAL-treated Raji cells, but did not change in SAL-treated D-threo-1-phenyl-2-decanoylamino-3-morphorino-1-propanol (PDMP)-pretreated Raji cells, it was clear that the expression of Magmas, NDRG3 or Rad50 was regulated by SAL binding to Gb3.

Topics: Acid Anhydride Hydrolases; Animals; Base Sequence; Burkitt Lymphoma; Catfishes; Cell Line, Tumor; Chromatography, Ion Exchange; Databases, Genetic; DNA Primers; DNA Repair Enzymes; DNA-Binding Proteins; Gene Expression; Genes, myc; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Intracellular Signaling Peptides and Proteins; Lectins; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Molecular Sequence Data; Mutagens; Nerve Tissue Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trihexosylceramides

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.

Topics: Antibodies; Antigens, CD20; Apoptosis; Biomarkers; Burkitt Lymphoma; Cell Line, Tumor; Cholera Toxin; Humans; Lipid Bilayers; Membrane Microdomains; Models, Biological; Proto-Oncogene Proteins c-bcl-2; Shiga Toxins; Trihexosylceramides

Globotriasosylceramide (Gb3), a neutral glycosphingolipid, is the B-cell differentiation antigen CD77 and acts as the receptor for most Shiga toxins, including verotoxin-1 (VT-1). We have shown that both anti-Gb3/CD77 mAb and VT-1 induce apoptosis in Burkitt's lymphoma cells. We compared the apoptotic pathways induced by these two molecules by selecting cell lines sensitive to only one of these inducers or to both. In all these cell lines (including the apoptosis-resistant line), VT-1 was transported to the endoplasmic reticulum and inhibited protein synthesis similarly, suggesting that VT-1-induced apoptosis is dissociated from these processes. VT-1 triggered a caspase- and mitochondria-dependent pathway (rapid activation of caspases 8 and 3 associated with a loss of mitochondrial membrane potential (Deltapsim) and the release of cytochrome c from mitochondria). In contrast, the anti-Gb3/CD77 mAb-induced pathway was caspase-independent and only involved partial depolarization of mitochondria. Antioxidant compounds had only marginal effects on VT-1-induced apoptosis but strongly protected cells from anti-Gb3/CD77 mAb-induced apoptosis. VT-1- and anti-Gb3/CD77 mAb-treated cells displayed very different features on electron microscopy. These results clearly indicate that the binding of different ligands to Gb3/CD77 triggers completely different apoptotic pathways.

Topics: Antioxidants; Apoptosis; Biological Transport; Blotting, Western; Burkitt Lymphoma; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Death; Cell Line, Tumor; Cytosol; Endoplasmic Reticulum; Glycosylation; Humans; Ligands; Membrane Potentials; Microscopy, Electron; Shiga Toxins; Signal Transduction; Time Factors; Transfection; Trihexosylceramides

The Shiga toxins (Stx1 and Stx2), produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli, consist of one A subunit and five B subunits. The Stx1 and Stx2 B subunits form a pentameric structure that binds to globotriaosylceramide (Gb3-Cer) receptors on eukaryotic cells and promotes endocytosis. The A subunit then inhibits protein biosynthesis, which triggers apoptosis in the affected cell. In addition to its Gb3-Cer binding activity, the data in the following report demonstrate that the Stx2 B pentamer induces apoptosis in Ramos Burkitt's lymphoma B cells independently of A subunit activity. Apoptosis was not observed in A subunit-free preparations of the Stx1 B pentamer which competitively inhibited Stx2 B pentamer-mediated apoptosis. The pancaspase inhibitor, Z-VAD-fmk, prevented apoptosis in Ramos cells exposed to the Stx2 B subunit, Stx1 or Stx2. Brefeldin A, an inhibitor of the Golgi transport system, also prevented Stx2 B subunit-mediated apoptosis. These observations suggest that the Stx2 B subunit must be internalized, via Gb3-Cer receptors, to induce Ramos cell apoptosis. Moreover, unlike the two holotoxins, Stx2 B subunit-mediated apoptosis does not involve inhibition of protein biosynthesis. This study provides further insight into the pathogenic potential of this family of potent bacterial exotoxins.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Brefeldin A; Burkitt Lymphoma; Cloning, Molecular; Cysteine Proteinase Inhibitors; Protein Binding; Protein Biosynthesis; Protein Subunits; Proto-Oncogene Proteins c-bcl-2; Receptors, Cell Surface; Recombinant Proteins; Shiga Toxin 1; Shiga Toxin 2; Trihexosylceramides

The unique manifestation of the inherited immunodeficiency, X-linked lymphoproliferative disease (XLP), is the impaired control of EBV infection. The gene, which carries mutations or is deleted in the patients, has been identified (Xq25). The encoded protein (SAP, 128 aa) contains a single SH2 domain and binds to signaling lymphocytic activation molecule (SLAM) and to other related surface molecules that are expressed on activated T, B and NK cells. SAP modifies signal transduction through its association with these molecules. Initially it was assumed that SAP acts passively by interfering and blocking active interactions involving other SH2 carrying molecules. We demonstrated that SAP protein is expressed in activated T and NK, but not in activated B cells. This finding is in line with the fact that in vitro performance of effector cells derived from XLP patients is impaired. However, it is still not known why the severe symptoms (fatal mononucleosis or malignant lymphoproliferation in the survivors of the primary infection) are elicited by EBV. We studied SAP expression in several Burkitt lymphoma (BL) derived lines. In contrast to normal B cells, certain lines expressed SAP. These were all type I cells in the Burkitt line nomenclature: they expressed only one of the EBV encoded proteins (EBNA-1) and their phenotype corresponded to resting B cells. Lymphoblastoid cell lines and type III BLs, whose phenotype resembled activated B cells and expressed all nine EBV encoded proteins, were devoid of SAP. The relationship between cell activation and SAP expression is reciprocal in T and B cells i.e. BL lines, activated T and NK cells express SAP, while BL blasts do not express SAP. This opposite relationship may be exploited for studies about the function of SAP.

Topics: Antigens, CD; B-Lymphocytes; Burkitt Lymphoma; Carrier Proteins; Cell Transformation, Viral; Cells, Cultured; Genetic Linkage; Glycoproteins; Herpesvirus 4, Human; Humans; Immunoglobulins; Infectious Mononucleosis; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Killer Cells, Natural; Lymphocyte Activation; Lymphoproliferative Disorders; Receptors, Cell Surface; Signaling Lymphocytic Activation Molecule Associated Protein; Signaling Lymphocytic Activation Molecule Family Member 1; T-Lymphocytes; Trihexosylceramides; Tumor Cells, Cultured; X Chromosome

The Burkitt lymphoma-derived Daudi cell line is often used as an in vitro model for germinal center B-cell function. Globotriaosyl ceramide (CD77), a marker for germinal center B-cells, is present on Daudi cells but is deficient in the Daudi-derived mutant VT500 cell line. Previous results showed a correlation in these cells between CD77 expression and expression of the B-cell protein CD19 and indicated that CD19/CD77 interaction is a mechanism for B-cell adhesion. Roles for CD77 in IFN-alpha-induced growth inhibition and anti-viral activity also have been described previously. Through flow cytometric analysis and adhesion assays, we investigated whether expression of CD77 was required for cell adhesion pathways induced by IFN or antibodies against additional B-cell surface molecules: CD20, CD22, CD38, CD40, CD81 and HLA-D proteins. In contrast to the pronounced homotypic adhesion induced by treatment with interferon-alpha in Daudi cells, no increase in adhesion was observed in IFN-treated VT500 cells. Of the B-cell proteins tested, only CD22-mediated adhesion and surface expression was stronger in Daudi than in VT500 cells. These results indicate that CD77 may be required for IFN and CD22-associated adhesion pathways, but CD77 is not a universal component of adhesion pathways in these cells.

Topics: Antibodies, Monoclonal; Burkitt Lymphoma; Cell Adhesion; Flow Cytometry; Humans; Interferons; Membrane Proteins; Trihexosylceramides; Tumor Cells, Cultured

Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed.

Topics: Antibodies, Monoclonal; B-Lymphocytes; bcl-X Protein; Burkitt Lymphoma; CD40 Ligand; Cell Death; Cell Line; DNA; Escherichia coli; Humans; Ionomycin; Protein Subunits; Proto-Oncogene Proteins c-bcl-2; Receptors, Cell Surface; Shiga Toxin 1; Signal Transduction; Trihexosylceramides

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitt's lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Topics: Apoptosis; B-Lymphocytes; Burkitt Lymphoma; Enzyme Activation; Humans; Shiga Toxin 1; Signal Transduction; src-Family Kinases; Trihexosylceramides; Tumor Cells, Cultured

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in alpha-interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon-alpha receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon-alpha were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.

Topics: Antigens, CD19; Binding Sites; Burkitt Lymphoma; Cell Division; Humans; Interferon alpha-2; Interferon-alpha; Mutation; Recombinant Proteins; Shiga Toxins; Signal Transduction; Trihexosylceramides; Tumor Cells, Cultured

A region of the N-terminal extracellular domain of the B-cell restricted cell differentiation antigen, CD19, has high amino acid sequence similarity to the receptor binding subunit B of verotoxin 1 (VT), an Escherichia coli elaborated cytotoxin, which specifically binds to the cell surface glycolipid, globotriaosylceramide, also known as the germinal center (GC) B-cell differentiation antigen, CD77. We have previously provided evidence of the association of CD19 and CD77 on the cell surface and in CD19-mediated homotypic adhesion of the Daudi Burkitt Lymphoma cell line, one normal counterpart of which is a subset of GC B cells. Evidence for the role of CD77 in CD19-induced apoptosis is now presented. Initial cell surface distribution, antibody-induced redistribution, internalization, and intracellular routing of CD19 were studied by confocal microscopy, IF, and postembedding IEM in CD77+ve and CD77-ve cells to investigate the possible role of CD77 in CD19 internalization and signaling. Daudi Burkitt's lymphoma cells were used as CD77+ve cells and as CD77-ve cells, Daudi mutant VT500 cells, and Daudi cells treated with PPMP, an inhibitor of CD77 synthesis, were used. Antibody ligated CD19 surface redistribution, internalization, and subcellular distribution of internalized CD19 was found to be different in CD77+ve and CD77-ve cells. A delay in internalization of antibody-CD19 complex was observed in CD77-ve cells. Internalized CD19 was targeted to the nuclear envelope in CD77+ve cells in a manner similar to that reported for VT, but not in CD77-ve cells. Internalization of CD77 by ligation with verotoxin prevented the internalization of ligated CD19. Induction of apoptosis following crosslinking of cell surface CD19 was greater in CD77+ve cells than in CD77-ve cells. The nuclear targeting of internalized CD19 and induction of apoptosis following CD19 crosslinking only in CD77+ve cells indicates a role for CD77-dependent CD19 retrograde transport from the B cell surface via the ER to the nuclear envelope in CD19-mediated signal transduction for apoptosis.

Topics: Antigen-Antibody Reactions; Antigens, CD19; Biological Transport; Burkitt Lymphoma; Fluorescent Antibody Technique, Indirect; Germinal Center; Humans; Microscopy, Electron; Nuclear Envelope; Receptors, Cell Surface; Trihexosylceramides; Tumor Cells, Cultured

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to be multifunctional factors. Contrasting with their enzyme-inhibitory activity, TIMPs also promote cell growth. Previously, we have reported an enhanced expression of TIMP-1 by normal reactive B cells and high-grade lymphomas. In the present study, a series of Burkitt's lymphoma (BL) cell lines were analyzed for their expression of TIMP-1. TIMP-1 expression correlates with upregulation of activation and survival markers. TIMP-1-negative cells express the phenotype associated with group I BL lines and Epstein-Barr virus (EBV)-negative, nonendemic BLs (CD10+, CD38+, sIg+, and CD77+). However, TIMP-1+ BL lines showed group II/III BL phenotype, downregulation of the above markers, and upregulation and secretion of the activation marker CD23. Also, TIMP-1+ cells have high levels of CD40 expression. To determine whether TIMP-1 is directly involved in the BL phenotype, an EBV-negative BL line JD38 was infected with timp-1-expressing retrovirus and analyzed. In the absence of EBV, upregulation of TIMP-1 is sufficient to induce the same phenotype seen in TIMP-1+, EBV+ BL lines (CD10-, CD38-, sIg-, CD77-, CD23+, CD40 bright). This study not only suggests a role for TIMP-1 in BLs, but also supports its value as a prognostic factor. This is a US government work. There are no restrictions on its use.

Topics: Apoptosis; B-Lymphocyte Subsets; Burkitt Lymphoma; Cell Differentiation; Embryonal Carcinoma Stem Cells; Germinal Center; Herpesviridae Infections; Herpesvirus 4, Human; Humans; Immunoglobulin M; Neoplasm Proteins; Neoplastic Stem Cells; Neprilysin; Phenotype; Receptors, IgE; Recombinant Fusion Proteins; Tissue Inhibitor of Metalloproteinase-1; Transfection; Trihexosylceramides; Tumor Cells, Cultured; Tumor Virus Infections

A new monoclonal antibody (TU-1) directed against the Gal alpha 1-4Gal beta 1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer(Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Gal beta 1-4Glc beta 1-1'Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer), galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), sulfatide (HSO3-3Gal beta 1-1'Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Gal alpha 1-4Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Biomarkers; Burkitt Lymphoma; Carbohydrate Sequence; Cell Differentiation; Cell Line; Child; Enzyme-Linked Immunosorbent Assay; Glycolipids; HL-60 Cells; Humans; Immunohistochemistry; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Palatine Tonsil; T-Lymphocytes; Thymus Gland; Trihexosylceramides; Tumor Cells, Cultured

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77-induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.

Topics: Apoptosis; Burkitt Lymphoma; Calcium; Calcium Channels; Ceramides; Chelating Agents; Colforsin; Cyclic AMP; Egtazic Acid; Humans; Ion Transport; Ionophores; Signal Transduction; Trihexosylceramides; Tumor Cells, Cultured

Shiga-like toxin-1 (SLT-1) is a bacterial toxin that kills cells by inhibiting protein synthesis. SLT-1 is composed of one cytotoxic A-subunit and five B-subunits that bind to CD77, a cell-surface glycolipid. In the human hematopoietic system, CD77 expression is restricted to a subset of activated B cells and derived cancers. Here we report that SLT-1 treatment of murine bone marrow ex vivo effectively cures severe combined immunodeficient mice of a human B-cell lymphoma xenograft while sparing normal hematopoietic precursor cells. Flow cytometry results using fluorescein isothiocyanate-labeled SLT-1 B-subunit show the high prevalence of expression of SLT-1 receptors (CD77) in human non-Hodgkin's lymphomas, especially follicular lymphomas. These results suggest the use of SLT-1 for the purging of human bone marrow before autologous bone marrow transplant in the case of CD77+ B-cell lymphomas as just one of many possible uses.

Topics: Animals; Antineoplastic Agents; Bacterial Toxins; Bone Marrow; Bone Marrow Purging; Bone Marrow Transplantation; Burkitt Lymphoma; Female; Humans; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; Radiation Chimera; Shiga Toxin 1; Specific Pathogen-Free Organisms; Transplantation, Heterologous; Trihexosylceramides; Tumor Stem Cell Assay

Earlier studies have shown that Burkitt's lymphoma (BL) cell lines can be divided into 2 major groups: group I, which retain the original BL biopsy phenotype with expression of CD10 and CD77 antigens and lack of B-cell activation markers, and group III, which, after several in vitro passages, progress toward an "LCL-Like" phenotype with loss of CD10 and C77 expression and up-regulation of B-cell activation antigens. In previous studies we have shown that several glycolipid molecules constitute stage-specific antigens for B cells and that sequential shifts in the 3 major glycolipid series are observed during B-cell differentiation, these changes being mostly due to sequential activations of the corresponding glycosyltransferases. In the present work, 10 BL cell lines with group I or group III phenotype have been examined for cell surface expression of 5 glycolipid antigens (LacCer, GM3, Gb3/CD77, Gb4 and GM2), total glycolipid content and enzymatic activities of 4 glycosyltransferases (GM3, Gb3, Gb4 and GM2 synthetases). We now report that group I and group III BL cells differ in their glycolipid metabolism and express either mostly globoseries or ganglioseries compounds. Indeed, Gb3 is the major glycolipid of group I cells, whereas GM3 and GM2 are the 2 major components of group III cells, and these phenotypic differences are mainly due to differential activities of the corresponding glycosyltransferases: group I cells have high Gb3 synthetase activities and low or no GM3 and GM2 synthetase activities, whereas group III cells have high GM3 and GM2 synthetase activities and low Gb3 synthetase activities. Finally, we also show that, unlike LCL, group III BL cells do not synthesize Gb4.

Topics: Burkitt Lymphoma; G(M2) Ganglioside; G(M3) Ganglioside; Galactosyltransferases; Glycosphingolipids; Glycosyltransferases; Humans; N-Acetylgalactosaminyltransferases; Phenotype; Polypeptide N-acetylgalactosaminyltransferase; Sialyltransferases; Trihexosylceramides; Tumor Cells, Cultured

We report the fractionation of freshly isolated subsets of tonsillar lymphocytes according to cell density and double sorting for the differential expression of CD10 and CD77, and their analysis for the presence of Epstein Barr virus genome by nested PCR. All the subsets of tonsillar lymphocytes gave unequivocal evidence for the presence of EBV DNA, when obtained from EBV seropositive individuals. Confirmation of all cases examined demonstrates that B lymphocytes from the germinal centers of tonsils are also involved in carrying the EBV infection in vivo.

Topics: Adolescent; B-Lymphocyte Subsets; Base Sequence; Burkitt Lymphoma; Cell Count; Cell Fractionation; Cell Separation; Child; Child, Preschool; DNA, Viral; Genome, Viral; Herpesvirus 4, Human; Humans; Lymphocyte Subsets; Lymphocytes; Molecular Sequence Data; Neprilysin; Palatine Tonsil; Polymerase Chain Reaction; Trihexosylceramides

The growth transformation of human B cells by Epstein-Barr virus (EBV) is controlled by the coordinate expression of 10 latent viral genes. This transforming capacity is believed to be fundamental to the involvement of the virus in human malignancies of B cell origin. EBV-negative Burkitt's lymphoma (dG75) clones stably expressing one of these EBV-coded antigens, EBNA-4, have been established using an episomal-based plasmid. EBNA-4 expression was found to upregulate the cytoskeletal protein vimentin as well as surface expression of the activation antigen CD40. In addition, the presence of EBNA-4 resulted in downregulation of the Burkitt's lymphoma-associated antigen (BLA/CD77). These studies show for the first time that EBNA-4 modulates the expression of several cellular genes implicated in cell-growth transformation.

Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Antigens, Viral; B-Lymphocytes; Burkitt Lymphoma; Callithrix; Cell Transformation, Viral; DNA-Binding Proteins; Down-Regulation; Epstein-Barr Virus Nuclear Antigens; Humans; Transfection; Trihexosylceramides; Tumor Cells, Cultured; Up-Regulation; Vimentin

Gb3/CD77 is a glycolipid antigen, specifically expressed on two different B-cell populations, Burkitt's lymphoma and a subset of tonsillar B-lymphocytes located in germinal centers, which could be the normal counterpart of Burkitt cells. Both Gb3/CD77(+) populations have recently been shown to enter programmed cell death (apoptosis) readily. Here we show that verotoxin, also called Shiga-like toxin, which is known to bind to the carbohydrate moiety of Gb3/CD77, induces cell death in Gb3/CD77(+) Burkitt's lymphoma cells, not only by inhibiting protein synthesis as classically described but also through an additional mechanism, namely apoptosis. Furthermore a recombinant B-subunit of verotoxin, which carries only the binding property of the holotoxin, also induces apoptosis in Gb3/CD77(+) cells. Gb3/CD77 could thus represent the first example of a glycolipid antigen able to transduce a signal leading to apoptosis.

Topics: Antigens, CD; Apoptosis; Bacterial Toxins; Burkitt Lymphoma; Cell Line; Cell Survival; DNA Damage; DNA, Neoplasm; Humans; Molecular Weight; Recombinant Proteins; Restriction Mapping; Shiga Toxin 1; Trihexosylceramides; Tumor Cells, Cultured

Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.

Topics: Animals; Bacterial Toxins; Burkitt Lymphoma; Cell Line; Cell Survival; Cytotoxins; Galactosyltransferases; Glycolipids; Humans; Phenotype; Shiga Toxin 1; Trihexosylceramides; Vero Cells

Fresh and transformed human B lineage cells were found to be sensitive to the cytotoxic action of Shiga-like toxin (SLT), a bacterial cytotoxin. The toxin was specifically bound by the glycolipids globotriosylceramide and galabiosylceramide expressed on the surface of sensitive cells. Mutant Daudi cells selected for resistance to SLT cytotoxicity (SLTR20) were deficient in SLT-binding glycolipids and failed to bind SLT to their surface, suggesting a role for these glycolipids in the mediation of SLT cytotoxicity. Of a number of normal and transformed lymphoid and myeloid cells screened for SLT sensitivity, only B lymphoid cells were susceptible to SLT action. Moreover, B lymphoid cells were the only cells expressing the SLT binding glycolipids. In vitro B cell activation studies with Epstein-Barr virus and pokeweed mitogen both indicated that the vast majority of SLT-sensitive B cells belong to the IgG and IgA committed subset, whereas most IgM and IgM/D producing cells were resistant to SLT toxicity. The selective elimination of IgG and IgA committed cells may explain the production of only IgM class anti-SLT antibodies in Shigella-infected humans leading to the failure of long-term immunity to dysenteric disease.

Topics: Antibodies, Bacterial; B-Lymphocytes; Bacterial Toxins; Burkitt Lymphoma; Cells, Cultured; Colitis; Cytotoxins; Escherichia coli; Escherichia coli Infections; Gangliosides; Glycolipids; Hemolytic-Uremic Syndrome; Humans; Lymphocyte Activation; Receptors, Cell Surface; Shiga Toxin 1; Trihexosylceramides; Tumor Cells, Cultured

To investigate a role for globotriaosylceramide (Gb3) as a tumor-associated antigen, variant cells resistant to treatment with complement and monoclonal antibody 38-13, which recognizes Gb3, were selected from a Burkitt's lymphoma cell line, Ramos. Variant cells displayed a clear decrease of antibody-binding capacity whereas the amount of Gb3 at their plasma membrane was not significantly different from that of Ramos parental cells. This demonstrated a reduced accessibility of Gb3 at the surface of variant cells. In parallel, no changes in other surface antigens were recorded as compared to those in Ramos cells. No changes of proliferative properties in suspension culture or of c-myc expression were observed although variant cells showed a decreased colony-forming capacity in agar. Variant cells showed a significant reduction in tumorigenic potential when injected s.c. into nude mice. The decreased tumorigenicity appeared related to the low antibody-binding capacity because both tumorigenicity and Gb3 antigenicity were recovered in parallel in revertant cells growing in suspension culture. In vivo, after two transplantations of variant cells into mice, cells isolated from the few induced tumors still retained the low antibody-binding capacity.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Burkitt Lymphoma; Cell Division; Flow Cytometry; Galactose Oxidase; Gene Expression Regulation, Neoplastic; Globosides; Glycosphingolipids; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Trihexosylceramides; Tumor Cells, Cultured

The results described here provided an example of a human IgM monoclonal antibody against a tumor-associated glycolipid and of the unusual properties of its corresponding immunotoxin (IT). The monoclonal antibody referred to as 38-13 has been previously described and reacted with the globotriaosylceramide [Gb3:Gal(alpha 1----4)-Gal(beta 1----4)-Glc(beta 1----1)ceramide] specifically expressed on surface membrane of Burkitt's lymphoma (BL) cells. An immunotoxin (38-13 IT) combined with the pokeweed antiviral protein (PAP) toxin via S-S bridges showed paradoxically a lower cytotoxic effect in BL Ramos cells than in non-BL cells such as leukemic mouse L1210 cells, while these cells appeared not to be involved by flow cytometric analysis and complement-dependent cytotoxicity. Consequently, the inhibitory effect of selective galactose analogs on the binding and the cytotoxicity of 38-13 antibody conjugated or not to PAP toxin was compared on BL and non-BL cells. Only the galactose blocked in alpha configuration provided a fine inhibition of 38-13 binding on BL Ramos cells and both alpha and beta-galactose allowed us to establish a clear distinction between the pathway entry of 38-13 IT in BL and non-BL cells; in close correlation with the 38-13 binding specificity the 38-13 IT cytotoxic effect in Ramos BL cells could also be prevented by alpha-Gal only, suggesting that this toxic action is probably mediated through the IT binding to Gb3 antigenic sites. In contrast, on apparently irrelevant L1210 cells, 38-13 IT showed a cytotoxic effect which was inhibited preferentially by lactose (Gal in beta configuration). It was discussed that IT binding alone to either antigenic sites which are inhibited by the hapten alpha-Gal, or nonspecific sites which can compete with the hapten beta-Gal is unable to induce efficient killing of cells. But cooperation of both bindings might give an attractive explanation of IT cytotoxic effect. It was concluded that the unexpected activity of 38-13 IT in non-BL cells probably could be mediated through an active macromolecular transport process which could implicate a beta-galactoside-binding protein (lectin).

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Binding, Competitive; Burkitt Lymphoma; Complement System Proteins; Cytotoxicity, Immunologic; Epitopes; Galactose; Globosides; Glycosphingolipids; Humans; Immunoglobulin M; Immunotoxins; Leukemia L1210; N-Glycosyl Hydrolases; Plant Proteins; Ribosome Inactivating Proteins, Type 1; Ricin; Structure-Activity Relationship; Trihexosylceramides

The cellular specificity of the Escherichia coli-derived verotoxin is of particular interest because of its extreme toxicity and high selectivity toward certain primate cells. The human Burkitt lymphoma cell line (Daudi) is highly susceptible to the cytotoxicity of verotoxin and contains large amounts of the verotoxin-binding glycolipids on its surface. A mutant selected from Daudi cells for verotoxin resistance was found to be deficient in the verotoxin-binding glycolipids, globotriosylceramide and galabiosylceramide, and failed to bind verotoxin to its surface; interestingly, these mutant cells were found to be cross-resistant to inhibition of growth by alpha-interferon. Mutant cells also lack the high affinity component of alpha-interferon binding. These observations suggest that, in addition to providing the functional cell-surface receptor for verotoxin, these glycolipids may also play a role in the modulation of the affinity of alpha-interferon for its membrane protein receptors.

Topics: alpha-Galactosidase; Bacterial Toxins; Burkitt Lymphoma; DNA Replication; Gangliosides; Globosides; Glycolipids; Glycosphingolipids; Humans; Interferon Type I; Kinetics; Receptors, Immunologic; Receptors, Interferon; Shiga Toxin 1; Trihexosylceramides; Tumor Cells, Cultured

The deliberate transfer of globotriaosylceramide (Gb3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.

Topics: Animals; Burkitt Lymphoma; Cell Line; Cloning, Molecular; Gene Expression Regulation; Globosides; Glycosphingolipids; Hexosyltransferases; Leukemia L5178; Mice; Transfection; Trihexosylceramides

The kinetics of ATP release from Burkitt lymphoma target cells following complement activation by rat monoclonal IgM antibody have been used to develop a standard assay for antibody-complement mediated cytotoxicity. This assay permitted the study of the reactivity of the monoclonal IgM with Burkitt and non-Burkitt cell lines and to perform IgM-complement mediated cytotoxicity inhibition experiments with oligosaccharides. This led to a precise definition of the antigenic determinant recognized by the monoclonal IgM on globotriaosylceramide.

Topics: Adenosine Triphosphate; Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigens, Neoplasm; Burkitt Lymphoma; Carbohydrates; Cell Line; Complement Activation; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Depression, Chemical; Epitopes; Fabry Disease; Globosides; Glycosphingolipids; Humans; Immunoglobulin M; Kinetics; Rats; Trihexosylceramides

We have previously described a monoclonal antibody, referred to as 38.13, reacting with most Burkitt's lymphoma (BL) derived lines, either containing the Epstein-Barr virus (EBV) or not, but not reacting with EBV-positive lymphoblastoid cell lines (LCL). (PNAS 78: 6485, 1981; Int. J. Cancer 29: 653, 1982). The antibody reacted with 41 BL lines out of 57 tested so far. The target antigen. BLA, was shown to be a neutral glycolipid, identified as globotriaosylceramide (Gal alpha 1----4 Gal beta 1----4 Glc beta 1----1 Ceramide). This substance is known as Pk blood group antigen, a normal intermediate in the P substance synthesis. Pk antigen is detectable only on the erythrocytes of very rare individuals with the Pk phenotype. We showed that 38.13 antibody stained erythrocytes and fibroblasts from 2 Pk subjects, whereas it was unreactive with erythrocytes and fibroblasts of all the other individuals tested. The accumulation of globotriaosylceramide on BL cells could reflect some functional disturbance of the enzymes in the P substance synthesis pathway.

Topics: Antibodies, Monoclonal; Blood Group Antigens; Burkitt Lymphoma; Cell Line; Epitopes; Globosides; Humans; P Blood-Group System; Trihexosylceramides

In our previous study, a Burkitt lymphoma-associated antigen defined by a monoclonal antibody, designated 38.13, was characterized as globotriaosylceramide (Gb3, Gal alpha 1----4 Gal beta 1----4 Glc beta 1----1 Cer) (Nudelman, E., Kannagi, R., Hakomori, S., Parsons, M., Lipinski, M., Wiels, J., Fellous, M., and Tursz, T. (1983) Science (Wash. D.C.) 220, 509-511). Consequently, we have studied the enzymatic basis and organization of Gb3 expression in Burkitt as compared with non-Burkitt lymphoblastoid cell lines. Burkitt lymphoma cell lines (Ramos, Daudi, Put) were characterized by a high chemical quantity of Gb3, high enzyme activity for synthesis of Gb3 (UDP-Gal:LacCer alpha-galactosyltransferase), and a high degree of surface exposure of Gb3, as determined by galactose oxidase/NaB[3H]4 and by cytofluorometry with the monoclonal antibody to Gb3 (38.13). Non-Burkitt lymphoblastoid cell lines (Priess, Remb1, and ARH77) were characterized by the absence of Gb3 at the cell surface detected by cytofluorometry or cell-surface labeling. The cell lines Priess and Remb1 did not contain Gb3 and showed a low alpha-galactosyltransferase activity for Gb3 synthesis. However, the cell line ARH77, though it did not express Gb3 at the cell surface, was found to contain a large chemical quantity of Gb3 and a high level of alpha-galactosyltransferase activity for Gb3 synthesis. However, Gb3 of ARH77 cells was exposed by sialidase treatment, but not by protease treatment, although Gb3 itself was not sialylated. The crypticity of Gb3 in ARH77 cells could be associated with an adjacent sialosyl residue of a second glycoconjugate at the cell surface, in the same way as Gg3 in mouse lymphoma L5178 (Urdal, D. L., and Hakomori, S. (1983) J. Biol. Chem. 258, 6869-6874). Thus, the expression in Burkitt and non-Burkitt lymphoma is dependent on (i) Gb3 synthesis due to alpha-galactosyltransferase activity and (ii) membrane organization of Gb3, which may be controlled through interaction with the sialosyl residue of a second glycoconjugate.

Topics: Antigens, Neoplasm; Burkitt Lymphoma; Cell Line; Chromatography, Thin Layer; Flow Cytometry; Galactosyltransferases; Globosides; Glycolipids; Glycosphingolipids; Humans; Leukemia, Lymphoid; Lymphocytes; Trihexosylceramides

The antigen defined by a rat monoclonal antibody directed to a Burkitt lymphoma cell line was identified as globotriaosylceramide [Gal alpha (1 leads to 4)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide]. The antibody demonstrated a strict steric specificity since it did not react with globoisotriaosylceramide [Gal alpha (1 leads to 3)-Gal beta (1 leads to 4)-Glc beta (1 leads to 1)-ceramide], the positional isomer of the antigen associated with the Burkitt lymphoma. Chemical analysis of various Burkitt lymphoma cell lines revealed that the Burkitt lymphoma cells contained more than 100 times as much of the glycolipid antigen as was found in other human lymphoma and leukemia cell lines.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Burkitt Lymphoma; Cell Line; Cell Transformation, Neoplastic; Erythrocytes; Globosides; Glycosphingolipids; Humans; Rabbits; Rats; Trihexosylceramides