g(m3)-ganglioside and Melanoma

Gangliosides are glycosphingolipids that are present in the plasma membranes of vertebrates and are involved in multiple cellular processes. In the Center of Molecular Immunology an NGcGM3 ganglioside based vaccine has been developed and is conceptualized as a targeted therapy in cancer. NGcGM3/VSSP vaccine had been used as treatment of metastatic melanoma patients and had showed to be safe and immunogenic. The treatment improved antitumoral response or maintain the response obtained with previous onco-specific treatment as chemotherapy. The results indicate that the vaccine improved overall survival of metastatic melanoma patients after first line-chemotherapy. The clinical trial ongoing currently will allow corroborating these results.

Topics: Cancer Vaccines; Clinical Trials as Topic; G(M3) Ganglioside; Humans; Immunotherapy; Melanoma; Proteolipids; Treatment Outcome

Different approaches to generating human monoclonal antibodies (MAbs) against tumor-associated ganglioside antigens have been carried out in several laboratories. A specific goal addressed by our laboratory is to produce human MAbs to several ganglioside antigens of relevance as therapeutic targets, such as the GM2, GD2, GD3 and GM3 gangliosides in melanoma. In vitro immunization of human B lymphocytes from normal donors was performed using liposomes containing gangliosides as the immunizing antigen combined with either complete tetanus toxoid or a synthetic peptide corresponding to a T helper epitope to stimulate in vitro immunization. Specific human anti-ganglioside antibodies were obtained, indicating that the antibody response found in vitro was antigen-driven. To overcome the widely reported problems concerning stability of immunoglobulin production by the antibody-secreting cell lines, a method of positive selection using GM3-coated magnetic beads has been developed in order to rescue unstable clones. Development of new methods to reproducibly generate ganglioside-specific human MAbs will amplify the possibilities for diagnostic and therapeutic applications.

Topics: Antibodies, Monoclonal; Antigens; B-Lymphocytes; Epitopes; G(M3) Ganglioside; Gangliosides; Humans; Immunization; Liposomes; Melanoma; Neoplasms; Radioimmunodetection; Radioimmunotherapy; T-Lymphocytes; Tetanus Toxoid

Topics: Animals; Antigens, Surface; Cells, Cultured; Epitopes; G(M3) Ganglioside; Gangliosides; Immunization, Passive; Killer Cells, Lymphokine-Activated; Liposomes; Lymphocyte Activation; Melanoma; Mice

NeuGcGM3 ganglioside is especially attractive because it is expressed on melanoma cells but it is minimally or not expressed at all on most normal human tissues. A Phase Ib/IIa clinical trial was carried out in patients with advanced cutaneous and ocular malignant melanomas, to evaluate immunogenicity and toxicity of an intramuscularly administered cancer vaccine and composed by NeuGcGM3 in a proteoliposome of Neisseria meningitides with Montanide ISA 51 as adjuvant. Twenty two patients were included, twelve at dose level of 200 microg and 10 at 400 microg. The first five doses were administered every other week and then monthly until 9 doses. 12 patients received additional immunizations. Vaccination induced specific anti-NeuGcGM3 IgM, IgG and IgA antibodies responses. Titers of IgM were greater for the highest vaccine doses. Vaccination also elicited DTH response in 45.5% of patients in the lower doses and 77.8% in the higher doses. Toxicities were mostly grade 1 or 2, according CTC-NCI criteria. Interestingly, 3 patients developed vitiligo at the lower dose (none in the highest dose) although the nominal antigen NeuGcGM3 is not present in melanocytes. Survival analysis was not the goal of this Phase I trial; nevertheless, the fact that seven patients are alive for more than 2 years after inclusion is noteworthy. Safety and immunogenicity with NeuGcGM3 vaccine treatment in advanced melanoma patients were established. The prognostic value of autoimmunity and the possibilities of dissociating anti-tumor immunity from autoimmunity deserve further research.

Topics: Adult; Aged; Bacterial Outer Membrane Proteins; Cancer Vaccines; Eye Neoplasms; Female; G(M3) Ganglioside; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Liposomes; Male; Mannitol; Melanoma; Middle Aged; Neisseria meningitidis; Oleic Acids; Skin Diseases; Survival Analysis; Treatment Outcome; Vaccination; Vitiligo

A human monoclonal antibody (L612 HuMAb) that binds to ganglioside GM3 has been developed in our laboratory. L612 HuMAb is a 100% human IgM protein. L612 HuMAb binds to cell surface of melanoma and can kill the cells in the presence of complement. The primary objective of this study was to test the toxicity and pharmacokinetics associated with administration of L612 HuMAb to melanoma patients whose tumor cells expressed GM3.. Nine patients with measurable metastatic melanoma (American Joint Committee on Cancer stage IV) were entered in the study. Eight had failed previous treatments that included chemotherapy, radiation therapy, melanoma cell vaccine, and/or biological therapy. All patients received a 48-h continuous infusion of L612 HuMAb at a dose of 960 mg, 1,440 mg, or 1,920 mg. Five of these patients received a second infusion and one patient received a third infusion, all with the previous dose.. Toxicity was limited to transient and mild pruritus and skin rash. One patient complained of pain at the site of subcutaneous metastases. Serum antibody levels peaked 24 to 48 h after starting the infusion. Two patients, one receiving a single course of 960 mg (612 mg/m(2)) and the second receiving two courses of 1,440 mg (911 mg/m(2)) followed by surgical therapy, are without evidence of disease >5 years after antibody infusion.. The human IgM monoclonal antibody, L612 HuMAb, was well tolerated. Infusion of L612 HuMAb appears to produce significant antitumor activity in melanoma patients.

Topics: Adult; Aged; Antibodies, Monoclonal; Drug Evaluation; Female; G(M3) Ganglioside; Humans; Immunoglobulin M; Infusions, Intravenous; Male; Melanoma; Middle Aged; Pilot Projects; Remission Induction; Skin Neoplasms

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.

Topics: Adult; Aged; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; Binding, Competitive; Cancer Vaccines; Female; G(M3) Ganglioside; Humans; Immunohistochemistry; Kinetics; Male; Melanoma; Middle Aged; N-Acetylneuraminic Acid

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Topics: Antibodies, Monoclonal; Cell Count; Cell Membrane; G(M3) Ganglioside; Humans; Melanoma

Tumor hypoxia drastically changes cancer phenotypes, including angiogenesis, invasion, and cell death. Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on plasma membranes and are involved in many biological processes, such as the endoplasmic reticulum stress response and apoptosis. In this study, we investigated the regulation and function of glycosphingolipids, which associate with lipid raft on mammalian plasma membranes under hypoxic condition.. B16F10 melanoma cells were subjected to chemical hypoxia and low pO. Hypoxia treatment decreased the expression of ganglioside GM3 synthase (GM3S; ST3GAL5), which synthesizes the common substrate of ganglioside biosynthesis. RNA interference of hypoxia inducible factor 1 subunit alpha (HIF-1α) inhibited hypoxia-induced GM3S suppression. Additionally, GM3S deficiency increased cellular resistance to oxidative stress and radiation therapy via upregulation of ERK.. Altered synthesis of glycosphingolipids downstream of HIF-1α signaling increased the resistance of melanoma cells to oxidative stress. Furthermore, GM3 has important role on cellular adaptive response to hypoxia.. This study indicates that tumor hypoxia regulates therapy-resistance via modulation of ganglioside synthesis.

Topics: Animals; Cell Line, Tumor; Female; G(M3) Ganglioside; Humans; Melanoma; Melanoma, Cutaneous Malignant; Melanoma, Experimental; Mice, Inbred C57BL; Oxidative Stress; Sialyltransferases; Skin Neoplasms; Tumor Hypoxia

GM3-ganglioside is known to be involved in melanoma proliferation. In order to modulate metastatic-related events, we have functionalized multi-walled carbon nanotubes (MWCNTs) with multiple copies of a GM3-lactone mimetic. The MWCNTs proved to guarantee the appropriate spatial arrangement of the mimetic allowing a stronger inhibition of migration and invasiveness of human melanoma (A375) cells compared to other multivalent constructs reported before. In addition, the effect of the multivalent tubular conjugate on the inhibition of specific tyrosine kinases, which are associated with the ganglioside complexes within the membrane domains, was demonstrated. Finally, the short-term fate of the conjugate was assessed, for the first time, by means of the 1H NMR relaxometry technique by exploiting the signal arising from the CNTs.

Topics: Antineoplastic Agents; Biomimetic Materials; Cell Line, Tumor; G(M3) Ganglioside; Humans; Melanoma; Models, Molecular; Molecular Conformation; Nanotubes, Carbon; Neoplasm Metastasis

GM3, the simplest ganglioside, regulates cell proliferation, migration, and invasion by influencing cell signaling at the membrane level. Although the classic N-acetylated form of GM3 (NeuAcLacCer) is commonly expressed and has been well studied, deacetylated GM3 (NeuNH2LacCer, d-GM3) has been poorly investigated, despite its presence in metastatic tumors but not in noninvasive melanomas or benign nevi. We have recently found that d-GM3 stimulates cell migration and invasion by activating urokinase plasminogen activator receptor (uPAR) signaling to augment matrix metalloproteinase-2 (MMP-2) function. However, the mechanisms by which d-GM3/uPAR increase MMP-2 expression and activation are not clear. By modifying the expression of d-GM3 genetically and biochemically, we found that decreasing d-GM3 expression inhibits, whereas overexpressing d-GM3 stimulates, p38 mitogen-activated protein kinase (MAPK) activity to influence MMP-2 expression and activation. p38 MAPK (p38) activation requires the formation of a membrane complex that contains uPAR, caveolin-1, and integrin α5β1 in membrane lipid rafts. In addition, knocking down or inhibiting focal adhesion kinase (FAK), phosphoinositide 3-kinase (PI3K), or Src kinase significantly reduces d-GM3-induced p38 phosphorylation and activation. Taken together, these results suggest that d-GM3 enhances the metastatic phenotype by activating p38 signaling through uPAR/integrin signaling with FAK, PI3K, and Src kinase as intermediates. Elucidation of the mechanisms by which d-GM3, a newly discovered, potential biomarker of metastatic melanomas, promotes cell metastasis will help us to understand the function of d-GM3 in metastatic melanomas and may lead to novel GM3-based cancer therapies.

Topics: Acetylation; Caveolin 1; Cell Line, Tumor; Cell Membrane; Cell Movement; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Protein-Tyrosine Kinases; G(M3) Ganglioside; Humans; Integrin alpha5beta1; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Melanoma; Membrane Microdomains; Neoplasm Invasiveness; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Receptors, Urokinase Plasminogen Activator; RNA, Small Interfering; Sialyltransferases; src-Family Kinases

A hydrolytically stable mimetic of the tumour antigen GM(3) lactone is used to decorate multivalent scaffolds. Two of them positively interfere on melanoma cell adhesion, migration and resistance to apoptosis (anoikis). Notably, their ability to hamper melanoma-cells adhesion and reduce the metastatic potential is enhanced when the two scaffolds, presenting a different shape, are used in combination.

Topics: Apoptosis; Biomimetic Materials; Cell Adhesion; Cell Movement; G(M3) Ganglioside; Humans; Melanoma

The aim of this study was to determine the expression of N-Glycolyl GM3 (NeuGcGM3) ganglioside in oral mucosal melanomas.. To assess the presence of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) mRNA, an RT-PCR assay was performed in melanoma cell line (ME), an oral mucosal ME, and two fresh oral mucosal melanoma tissues. Expression of NeuGcGM3 ganglioside was evaluated by immunohistochemistry in 44 primary oral mucosal melanomas and 10 oral melanotic nevi. Also, the expression of NeuGcGM3 was examined in ME by immunocytochemistry.. We first checked the expression of CMAH in ME and two fresh oral mucosal melanoma tissues. Presence of NeuGcGM3 ganglioside was evident in 37 of 44 cases (84.1%), showing a diffuse cytoplasmic and membranous staining. Patients with primary occurrence showed high levels of NeuGcGM3 ganglioside compared to patients with secondary occurrence. On the other hand, negative immunoreaction was evidenced in oral melanotic nevi. ME also presented the expression of NeuGcGM3 by immunocytochemistry.. In this work, we for the first time evaluated the expression of 14F7 MAb immunorecognition in oral mucosal melanomas. Our results were in agreement with the assumption that NeuGcGM3 ganglioside may be considered as target for passive and active immunotherapy in oral mucosal melanomas expressing this molecule and indicate less risk of recurrence and a better prognosis. Moreover, ME provides a platform for more studies on the specific function of NeuGcGM3 in oral mucosal melanomas.

Topics: Antibodies, Monoclonal; Cell Line, Tumor; Cell Membrane; China; Cytoplasm; Female; G(M3) Ganglioside; Humans; Immunoglobulin G; Immunohistochemistry; Male; Melanoma; Middle Aged; Mixed Function Oxygenases; Mouth Mucosa; Mouth Neoplasms; Neoplasm Recurrence, Local; Nevus, Pigmented

Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patient's immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM(3) ganglioside 1 and especially the expression of its metabolite GM(3) lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM(3) lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in vivo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM(1) was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.

Topics: Animals; Antibodies; Antibody Specificity; Antigen-Antibody Reactions; Carbohydrate Conformation; Computer Simulation; G(M3) Ganglioside; Hemocyanins; Humans; Immunotherapy; Melanoma; Mice; Molecular Mimicry

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

Topics: Antigens, CD; Apoptosis; Caspase Inhibitors; Caspases; Ceramides; Cysteine Proteinase Inhibitors; Dendritic Cells; Enzyme Activation; G(M3) Ganglioside; Gangliosides; Humans; Lactosylceramides; Melanoma; Monocytes; Oligopeptides; Tumor Escape

We have recently discovered that de-N-acetyl GM3 [NeuNH(2)LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82-95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3-positive) and poorly invasive (d-GM3-negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases.

Topics: Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Cell Movement; Enzyme Activation; Fluorescent Antibody Technique; G(M3) Ganglioside; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Neoplasm Invasiveness; Receptors, Urokinase Plasminogen Activator; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Skin Neoplasms; Transfection

GM3 has been shown to suppress TNFalpha expression in blood monocytes. However, we found that GM3 and TNFalpha were expressed in parallel in mouse melanoma B16 cells that were transfected with UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase cDNA in a sense or antisense direction or CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase siRNA. TNFalpha expression was increased by addition of GM3 to the B16 transfectants and decreased after treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis. These results clearly indicate that GM3 positively regulates TNFalpha expression in B16 cells. Phosphoinositide 3-kinase inhibitors, wortmannin and LY294,002, suppressed TNFalpha expression and Akt phosphorylation. GM3 was shown to increase phosphorylation of Akt in B16 cells and the B16-derived transfectants. Treatment of B16 cells with siRNA targeted to Akt1/2 resulted in TNFalpha suppression, indicating that Akt plays an important role in regulation of TNFalpha expression. Suppression of Akt1/2 rendered cells insensitive to GM3, suggesting that the GM3 signal may be transduced via Akt.

Topics: Androstadienes; Animals; Chromones; G(M3) Ganglioside; Melanoma; Mice; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-akt; Signal Transduction; Skin Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Wortmannin

L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications.. Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo.. Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was approximately 80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft.. A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; G(M3) Ganglioside; Humans; Melanoma; Recombinant Proteins; Skin Neoplasms

Gangliosides are ubiquitous, membrane-associated, glycosphingolipids, the composition and production of which is altered in many tumour cells. They have been shown to inhibit the in vitro generation and differentiation of dendritic cells (DCs) from progenitors, but their effect on human tissue-residing DCs is yet to be investigated. In the present study, we analysed the effect of GM3 and GD3 gangliosides purified from human melanoma tumours on the phenotypic and functional maturation of human epidermal Langerhans cells (LCs), the first immune barrier against the tumour cells. We showed that both gangliosides impaired spontaneous LC maturation induced by a short in vitro culture, as assessed by significant down-regulation of co-stimulation (CD40, CD54, CD80, CD86) and maturation markers (CD83, CCR7), which correlated to an impaired ability of the cells to mount allogeneic T cell proliferation. Furthermore, the ganglioside-treated cells displayed less ability to migrate towards CCL19/macrophage inflammatory protein 3 beta, the chemokine that specifically binds CCR7 and mediates LC migration to lymph nodes. Lastly, we showed that both GM3 and GD3 gangliosides enhance LC spontaneous apoptosis. Globally, these in vitro results might explain, at least in part, the altered number and distribution of LCs in melanoma-bearing patients. They underscore a new mechanism for gangliosides to impede the host immune response by inducing LC dysfunction in the tumour microenvironment.

Topics: Antigen Presentation; Antigens, CD; Apoptosis; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Chemokines; Epidermal Cells; Epidermis; G(M3) Ganglioside; Gangliosides; Humans; Langerhans Cells; Melanoma; T-Lymphocytes; Tumor Escape

Gangliosides are ubiquitous membrane-associated glycosphingolipids, which are involved in cell growth and differentiation. Most tumor cells synthesize and shed large amounts of gangliosides into their microenvironment, and many studies have unraveled their immunosuppressive properties. In the present study we analyzed the effects of GM3 and GD3 gangliosides, purified from human melanoma tumors, on the differentiation of monocyte-derived dendritic cells (MoDC). At concentrations close to those detected in the sera from melanoma patients, both gangliosides dose-dependently inhibit the phenotypic and functional differentiation of MoDC, as assessed by a strong down-regulation of CD1a, CD54, CD80, and CD40 Ags and impaired allostimulatory function on day 6 of culture. Furthermore, GM3 and GD3 gangliosides decreased the viable cell yield and induced significant DC apoptosis. Finally, addition of GD3 to differentiating DC impaired their subsequent maturation induced by CD154. The resulting DC produced low amounts of IL-12 and large amounts of IL-10, a cytokine pattern that might hamper an efficient antitumor immune response. In conclusion, the results demonstrate that gangliosides impair the phenotypic and functional differentiation of MoDC and induce their apoptosis, which may be an additional mechanism of human melanoma escape.

Topics: Antigens, Neoplasm; Apoptosis; CD40 Ligand; Cell Differentiation; Cell Division; Cells, Cultured; Dendritic Cells; Dinoprostone; G(M3) Ganglioside; Gangliosides; Growth Inhibitors; Humans; Interleukin-12; Ligands; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Melanoma; Monocytes; Up-Regulation

GM3 is a ganglioside that has been biochemically identified as dominating the cell surface of several human tumours, but is also found on human normal cells at much lower density. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concepts of tumour-associated antigen, immunogen, and toxicity. We have designed a GM3-based cancer vaccine for the treatment of human breast and melanoma tumours. Prior to the Phase I clinical trial, we carried out a 12-month dose repeated toxicity study in five male Macaca fascicularis monkeys. Four male monkeys were treated with placebo in a similar way. During the study, no differences were observed between control and treated monkeys related to daily clinical observations (other than local damage) including rectal temperature, blood pressure, respiratory and cardiac rates, weight gain, biochemical and hematological parameters (with the exception of transitory pathological changes), and anti-DNA and anti-nuclear antibodies, although treated monkeys consistently developed both IgM- and IgG-specific anti-GM3 antibodies. Sixty per cent of treated monkeys developed moderate local reactions at the injection site, which disappeared without sequels. We concluded that this GM3 cancer vaccine overcame in monkeys the natural tolerance to GM3 ganglioside evidenced by a strong immune response, while the local reactions elicited-were transitory without apparent important systemic toxicity effects.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Neoplasm; Body Weight; Breast Neoplasms; Cancer Vaccines; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dogs; Drug Evaluation, Preclinical; G(M3) Ganglioside; Hematologic Tests; Immunoglobulin G; Immunoglobulin M; Kidney Function Tests; Liver Function Tests; Macaca fascicularis; Male; Melanoma; Proteolipids; Skin Neoplasms; Toxicity Tests

The incidence of melanoma is estimated to be growing at the second fastest rate among all cancers in the United States. The progression of the melanocyte to a malignant melanoma involves various sequential steps: development of benign naevocellular naevus, preneoplastic dysplastic naevus, primary melanoma, and metastatic melanoma. Despite these clearly defined stages, very little is known about the molecular events leading to melanoma progression. We established a human congenital naevus cell line (UISO-CMN-1). UISO-CMN-1 cells were confirmed to have melanocytic origin by S100 immunoreactivity and the presence of melanin granules and melanosomes. UISO-CMN-1 cells, even though they showed structural and numerical abnormalities in karyotype, were non-tumorigenic when transplanted into athymic mice. However, following frequent exposure to ultraviolet C radiation, UISO-CMN-1 cells acquired tumorigenic potential. Transformation of UISO-CMN-1 cells into tumorigenic cells was accompanied by induction of ganglioside-2 expression without any significant changes in cellular ganglioside-3. These transformed and non-transformed UISO-CMN-1 cell lines can serve as excellent research tools for studying the molecular changes associated with melanoma development and progression, and for identifying agents that might prevent development of malignant melanoma.

Topics: Animals; Cell Line; Cell Line, Transformed; Female; G(M2) Ganglioside; G(M3) Ganglioside; Humans; Immunohistochemistry; Infant; Karyotyping; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Nevus; Transformation, Genetic; Ultraviolet Rays

The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.

Topics: Acetylation; Animals; Antibodies, Monoclonal; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; G(M3) Ganglioside; Gangliosides; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Melanoma; Neuraminic Acids; Tumor Cells, Cultured

The sensitivity and specificity of two influenza C virus assays, solid-phase and overlay assays, were investigated using naturally occurring 9-O-acetylated GD(3), rat serum glycoproteins containing 60% of N-acetyl-9-O-acetylneuraminic acid, and synthetically O-acetylated sialylated compounds. The sensitivity of the solid-phase assay was higher for glycoproteins containing N-acetyl-9-O-acetylneuraminic acid than for gangliosides, and also differed for various 9-O-acetylated gangliosides. The overlay assay was less sensitive for all glycoconjugates tested. For virus recognition the presentation of the sialic acid within the molecule and the structure of the sialic acid are essential. Investigation of gangliosides from human melanomas and normal skin with the influenza C virus assay showed an increase of O-acetylation of sialic acids in most tumour samples and the occurrence of several O-acetylated gangliosides.

Topics: Acetylation; Animals; Carbohydrate Sequence; Cattle; Chromatography, Thin Layer; G(M3) Ganglioside; Gammainfluenzavirus; Gangliosides; Glycoconjugates; Glycoproteins; Humans; Melanoma; Molecular Sequence Data; Neoplasm Metastasis; Rats; Sensitivity and Specificity; Sialic Acids; Skin

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund's adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Biomarkers, Tumor; Breast Neoplasms; Cholesterol, VLDL; Female; G(M3) Ganglioside; Glycolipids; Humans; Immunoglobulin G; Melanoma; Mice; Mice, Inbred BALB C; Organ Specificity

The biologic functions of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins (type I and IV collagens, fibronectin, and laminin) were investigated by using the G(D3)-deficient mutant clone (SK-MEL-28-N1) and the parent cell line SK-MEL28. SK-MEL-28-N1 (N1) (high G(M3) expression: G(M3), 97.3%; G(D3), 0%) was selected by treating SK-MEL-28 (high G(D3) but low G(M3): G(M3), 6.5%, G(D3), 93.5%) with an anti-G(D3) monoclonal antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells. The N1 clone showed significantly higher ability to adhere to type I and IV collagens and laminin than the parent clone SK-MEL-28. In the N1 clone, the expression of alpha2beta1 and alpha3beta1 integrin receptors was increased, whereas in SK-MEL-28, their expression was very low or undetectable. The treatment with monoclonal antibodies directed specifically to G(D3) expressed on SK-MEL-28 inhibited the cell attachment to type IV collagen (33% inhibition of control), fibronectin (59%), and laminin (71%). These findings suggest that gangliosides G(M3) (by influencing integrin receptor levels) and G(D3) (by interacting directly with matrix proteins) might play some functional roles in attachment to extracellular matrix proteins and thereby enhance the metastatic potency of melanoma cells.

Topics: Animals; Antibodies, Monoclonal; Cell Adhesion; Cell Line; Chromatography, Thin Layer; Extracellular Matrix Proteins; G(M3) Ganglioside; Gangliosides; Humans; Integrins; Melanoma; Rabbits; Receptors, Collagen; Skin Neoplasms

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Neoplasm; Antibody Specificity; Child; Enzyme-Linked Immunosorbent Assay; Female; G(M3) Ganglioside; Humans; Japan; Male; Melanoma; Middle Aged

N-Acylaminoethyl lactosides as lactosylceramide analogs as well as n-alkyl lactosides were examined for their ability to prime glycosphingolipid (GSL) synthesis in mouse melanoma B16 cells. Using compounds radiolabeled in a galactose residue and having nondegradable thioglucosidic linkages in lactoside, direct glycosylation was shown to occur at the terminal galactose residue of lactosides subsequent to uptake by cells and dissemination into Golgi compartments. B16 cells took in lactosides temperature-dependently to the point of saturation. All lactosides were taken up and glycosylated by B16 cells. C8-lactosides could not settle on the plasma membrane, while C16-lactosides remained within the cells. Glycosylation in all cases was cellular GSL-specific, suggesting the involvement of glycosyltransferases in GSL synthesis during glycosylation of lactosides. The priming of GSL synthesis by lactosides inhibited the cell surface expression of endogenous GM3 in B16 cells. Lactosylceramide analogs are thus shown useful as primers for glycosylation and to modify GSL expression, and these features should facilitate clarification of the functions of GSLs which have yet to be elucidated.

Topics: Animals; Disaccharides; G(M3) Ganglioside; Glycosides; Glycosphingolipids; Glycosylation; Golgi Apparatus; Lactosylceramides; Melanoma; Mice; Tumor Cells, Cultured

The inability of current therapy to prevent metastases arising from uveal melanoma often results in patient mortality. With the goal of developing a treatment for metastasis, gangliosides were studied as potential tumor-associated antigens. Our report describes the production of a metastatic liver variant (MH) from a human uveal melanoma cell line (SP6.5). Cells were injected into nude mouse spleens and liver metastases collected 2 months later. After 21 days of in vitro subculture, the cells were re-injected into normal nude mice spleen; 10 cycles (MH10) were performed. Gangliosides were extracted, purified, chromatographed on HPTLC plates and sprayed with a resorcinol-HCl reagent, the sialic acid spots being quantified by densitometry. Gangliosides were analyzed in each metastatic liver variant and compared with the SP6.5 s.c. tumor. The results showed a significant increase in GM3 and a significant decrease in GD3 and GD2 in the last metastatic variants obtained (MH5, MH8, MH9 and MH1O) compared with the primary s.c. tumor, SP6.5. Such evolution in the ganglioside pattern was maintained throughout the progression of the different liver variants. Our results indicate that precursor ganglioside GM3 and gangliosides GD3 and GD2 could be associated with neoplastic evolution of malignancy of human uveal melanoma in nude mice.

Topics: Animals; G(M3) Ganglioside; Gangliosides; Humans; Liver Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured; Uveal Neoplasms

Differential cell- and immuno-biological properties of two murine melanoma B16 variants, B16-F1 and F10, were investigated. Studies focused on the expression of proto-oncogene c-fos, sensitivities to LAK cells and/or IL-2, and modulation of the expression of ganglioside components after treatment with IL-2. Proto-oncogene c-fos was found to be highly expressed in F10 lines by an in situ hybridization technique and also in F10 lung metastatic nests by immunofluorescent staining with anti-c-fos antibody. F1 melanomas were more sensitive to local injection of IL-2. F10 melanomas hardly responded to IL-2 treatment, but successive injections of a combination of LAK cells and IL-2 did cause prolongation of survival rates, even of F10 melanoma-burdened mice. A major component of gangliosides of both F1 and F10 melanomas was GM3. Production of GM3 in F10 melanomas treated with IL-2 for 4 days increased, and, if the treatment was continued for 7 days, minor components of gangliosides, such as GM2, GM1, and GD1a, appeared only in F1 melanomas, while the increase of production of GM3 disappeared in both melanomas. These experimental results may provide clues for additional mechanisms which allow these two murine melanoma variants to show different implantation and metastasis rates.

Topics: Animals; Female; Fluorescent Antibody Technique; G(M1) Ganglioside; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Gene Expression Regulation, Neoplastic; Genes, fos; In Situ Hybridization; Interleukin-2; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Survival Rate; Tumor Cells, Cultured

We report that short-chain ceramide (Cer), C2- and C6-Cer, were immediately glycosylated and finally converted to short-chain Cer GM3 in B16 melanoma cells. By addition of either C2- or C6-Cer to a cell culture of B16 melanoma in the presence of [14C]Gal, the radiolabeled precursor, was incorporated into each of two novel glycosphingolipids (GSLs) within 30 min along with synthesis of normal GSLs. These novel GSLs were identified as C2-, C6-Cer cerebrosides and C2-, C6-Cer GM3, respectively. In comparison with C2-Cer, C6-Cer was found to be much more efficiently converted to the GSLs, whereas no glycosylated sphingosine was detectable when it was added in place of short-chain Cer.

Topics: Ceramides; G(M3) Ganglioside; Glycosphingolipids; Kinetics; Melanoma; Tumor Cells, Cultured

The optimal conditions were examined for selective re-N-acetylation with 14C or 3H.acetic anhydride of de-N-acetylated aminosugar-containing glycosphingolipids. Re-N-acetylation, which is nearly quantitative within 10 minutes in methanol, occurs selectively up to a maximal 100% yield when using a molar ratio of 5 mol of acetic anhydride per mole of aminosugar present in the glycosphingolipid. Above this molar ratio, it was observed some O-acetylation of carbohydrates which could be removed by mild alkali treatment. The method allows the choice of 14C- or 3H-labeling of glycosphingolipids with a final specific radioactivity which depends solely on the one of acetic anhydride. The binding of specific antibodies to glycosphingolipids, which was abolished upon de-N-acetylation, was again detectable after re-N-acetylation with radioactive acetic anhydride, suggesting that the native structures were recovered. This procedure of radiolabeling offers safety, rapidity and broad applicability to alkali-stable aminosugar-containing glycosphingolipids.

Topics: Acetylation; Animals; Brain Chemistry; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Erythrocytes; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Isotope Labeling; Melanoma; Tritium

The glycosphingolipid patterns were analyzed on two clones derived from a human melanoma cell line and selected for their respectively high and low metastatic ability in immunosuppressed newborn rats. Conversely to the weakly metastatic cells which exhibited a pattern similar to that of the parental cell line, highly metastatic human melanoma cells appeared to be deficient in ganglioside biosynthesis. An accumulation of lactosylceramide was found in the latter cells, with low amounts of GM3 as the only ganglioside detected and a fourfold decreased activity of GM3 synthase (EC 2.4.99.9). After subcutaneous injection of metastatic cells in newborn rats, the cells proliferating in the tumor induced at the injection site re-expressed the four common gangliosides of melanoma: GM3, GM2, GD3 and GD2, whereas the cells growing in the lungs as metastatic nodules were deficient in ganglioside synthesis and showed an accumulation of lactosylceramide. Taken together, our results suggest that the human melanoma cells which are able to escape from the primary tumor and invade the lungs have an impaired ganglioside biosynthesis with a deficient GM3 synthase.

Topics: Animals; Animals, Newborn; Antigens, CD; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Lactosylceramides; Melanoma; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Sialyltransferases; Tumor Cells, Cultured

Neuraminic acid is the core structure of most known sialic acids. In natural systems, the amino group at the 5 position of neuraminic acid residues is usually assumed to be acylated. Previously, synthetic de-N-acetyl-gangliosides (with free amino groups at the 5 position of neuraminic acids) have been shown to modulate cellular proliferation and tyrosine phosphokinase reactions. While indirect evidence has suggested that traces of these molecules exist naturally in certain tumor cells, further exploration has been hampered by the lack of a system showing consistent expression at an easily detectable level. Using synthetic compounds as antigens, we have developed highly specific monoclonal antibodies against de-N-acetyl-GM3 and de-N-acetyl-GD3 that require both the free amino group and the exocyclic side chain of sialic acids for recognition. Cultured human melanoma cells showed low but variably detectable levels of reactivity with these antibodies. The ability of various biologically active molecules to stimulate this reactivity was explored. Of many compounds tested, only the tyrosine kinase inhibitor genistein induced reactivity in a dose-dependent manner. Antibody reactivity with ganglioside extracts from genistein-treated cells was abolished by chemical re-N-acetylation and/or truncation of sialic acid side chains by mild periodate oxidation. High performance thin layer chromatography immuno-overlay analysis confirmed the presence of the novel compound de-N-acetyl-GD3 in these extracts. Several other tyrosine kinase inhibitors tested did not give the same increase in de-N-acetyl-ganglioside expression. However, the microtubule inhibitor nocodazole caused a similar accumulation of these molecules, particularly in non-adherent cells expected to be arrested at metaphase. Thus, genistein may induce de-N-acetyl-ganglioside expression by virtue of its known ability to arrest cells in the G2M phase, rather than as a general consequence of tyrosine kinase inhibition. These studies also provide a system in which to analyze the enzymatic basis of de-N-acetyl-ganglioside expression and their potential roles as growth regulating molecules.

Topics: Antibodies, Monoclonal; Benzoquinones; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; G(M3) Ganglioside; Genistein; Humans; Isoflavones; Lactams, Macrocyclic; Melanoma; Nocodazole; Protein-Tyrosine Kinases; Quinones; Rifabutin; Spectrometry, Mass, Fast Atom Bombardment; Tumor Cells, Cultured

In vitro immunization of human B-lymphocytes was performed with liposomes containing the monosialoganglioside GM3, with or without either complete tetanus toxoid or a synthetic T helper epitope derived from tetanus toxin (determinant 830-843). The immunized B-cells were Epstein-Barr virus transformed and the human anti-ganglioside antibody response was evaluated using an indirect ELISA against different mono- and disialogangliosides. Clones producing antigen-specific human antibodies of the IgM isotype against the ganglioside GM3 used as the immunogen were selected and one clone, IM-11, was further characterized. In addition, a method of positive selection using GM3-coated magnetic beads has been developed which allowed us to rescue unstable clones. The binding of the human antibody IM-11 to a large panel of glycosphingolipids separated on thin-layer plates was studied. The human MAb IM-11 was found to bind strongly to NeuAcGM3, IV3 NeuAcnLc4 and sulfate containing glycosphingolipids and weakly to NeuGcGM3. Immunohistological staining of melanoma and breast cancer biopsy sections showed a selective reactivity of IM-11 with tumor cells which varied among different tumors.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigens, Neoplasm; B-Lymphocytes; Breast Neoplasms; Carbohydrate Sequence; G(M3) Ganglioside; Humans; Immunization; Melanoma; Molecular Sequence Data

Previously we developed a murine monoclonal anti-idiotype (anti-id) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM3, that is, it carries the internal image of GM3. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (VH or VL) of the anti-id is responsible for the antigenicity of GM3. The TVE-1 VH and VL expression vectors were simultaneously transfected with either the VH or VL expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of VH and VL chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the VH-VL pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immunotherapy.

Topics: Animals; Antibodies, Anti-Idiotypic; Antigens, Neoplasm; G(M3) Ganglioside; Humans; Melanoma; Mice; Recombinant Fusion Proteins

A cDNA encoding a GM3-specific alpha-2,8-sialyltransferase (GD3 synthase) was obtained from an expression cDNA library of human melanoma cell line WM266-4 by enrichment of Namalwa KJM-1 cells highly expressing GD3 using an anti-GD3 antibody and a fluorescence-activated cell sorter. Selection of B-cell line Namalwa cells expressing transfected cDNAs in the presence of anti-GD3 monoclonal antibody KM641 gave a cDNA (pAMo-GD3) encoding a protein with a type II transmembrane topology as found for mammalian glycosyltransferases. The following evidence confirms that the cDNA encodes an alpha-2,8-sialyltransferase, which specifically converts GM3 to GD3. (i) Transfection of pAMo-GD3 into Namalwa KJM-1 cells leads to the appearance of GD3 and a GD3 synthase activity. (ii) Northern blot analysis revealed a correlation between the expression of this gene and GD3 in several cell lines. (iii) The putative COOH-terminal active domain of this cloned enzyme fused with protein A has been purified with IgG-Sepharose beads and has been shown to possess GD3-synthesizing activity, excluding the possibility that the cloned cDNA encodes a transacting factor inducing a GD3 synthase. The deduced primary sequence also contains the "sialyl motif" conserved among all the sialyltransferases cloned to date. The polymerase chain reaction analysis reveals that this gene is located on chromosome 12.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Cell Line; Chromatography, Thin Layer; Cloning, Molecular; Conserved Sequence; DNA Primers; DNA, Complementary; Flow Cytometry; G(M3) Ganglioside; Gangliosides; Gene Expression; Gene Library; Humans; Melanoma; Molecular Sequence Data; Phylogeny; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sialyltransferases; Substrate Specificity; Transfection; Tumor Cells, Cultured

In this study we report the characterization of a human monoclonal antibody (HuMab), L612, that reacts with ganglioside GM3 and has therapeutic application for the treatment of human neoplasms, particularly melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed B-cell line L612 was established. L612 HuMab bound specifically to neoplastic cell lines in culture and in tissue biopsy specimens such as melanoma, colon, breast, and lung cancer. The antibody did not bind to normal cells or biopsy tissue. HuMab L612 showed the highest reactivity to melanoma cells, particularly to those with high concentrations of GM3. Immunostaining on high-performance thin-layer chromatography plates demonstrated that L612 HuMab bound to GM3 purified from melanoma cells. Removal of the sialic acid from GM3 abolished antibody binding. HuMab L612 also reacted to GM4 purified from egg yolk, indicating that it recognizes an NeuAc alpha 2-3 galactose antigen determinant. HuMab L612 heavy and light chains were sequenced and determined to belong to the mu heavy chain variable subgroup III and kappa chain variable subgroup IV families, respectively. The studies indicate that the L612 HuMab has significant therapeutic potential for a wide variety of human cancers.

Topics: Adenocarcinoma; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Antigens, Neoplasm; B-Lymphocytes; Base Sequence; Breast Neoplasms; Carbohydrate Sequence; Cell Line, Transformed; Cloning, Molecular; Colonic Neoplasms; DNA Primers; Female; G(M3) Ganglioside; Gangliosides; Herpesvirus 4, Human; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Lung Neoplasms; Melanoma; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; Recombinant Proteins; Skin Neoplasms

beta-D-Xylosides are often used to competitively inhibit proteoglycan synthesis by serving as primers for free glycosaminoglycan (GAG) chain assembly. Quite unexpectedly, we found that when human melanoma cells and Chinese hamster ovary cells are labeled with [3H] galactose in the presence of 4-methyl umbelliferyl beta-D-xyloside (Xyl beta 4MU), a large portion of the labeled acceptor does not consist of the expected GAG chains, but of the novel GM3 ganglioside-like structure: Sia-alpha 2,3-[3H]Gal beta 1, 4Xyl beta 4MU. Moreover, formation of this derivative is associated with an inhibition of glycosphingolipid synthesis by up to 78% without affecting synthesis of other [3H]Gal-labeled glycoconjugates. Inhibition occurs rapidly and equally for all glycolipid species and is partially abrogated by brefeldin A. Inhibition requires the addition of a single galactose residue to the xyloside within the lumen of the Golgi apparatus. This addition appears to be carried out by galactosyl transferase I that normally synthesizes the core region of GAG chains. Although alpha-xyloside does not inhibit proteoglycan synthesis, it is galactosylated, but not sialylated, and is nearly as effective as a beta-xyloside at inhibiting glycolipid biosynthesis. Similar results were obtained for human macrophage U937, and differentiated or undifferentiated PC12 cells. However, in neuroblastoma cell line MR23, no low molecular weight xyloside products were made and glycolipid synthesis was not inhibited. These results suggest that some of the previously documented effects of beta-xylosides might result, in part, from their inhibition of glycolipid synthesis. The mechanism of inhibition is not a direct competition for glycolipid synthesizing enzymes; rather, it is an unexplained result of formation of Gal beta 1,4Xyl-1 (alpha or beta)4MU.

Topics: Animals; Anions; Brefeldin A; CHO Cells; Cricetinae; Cyclopentanes; G(M3) Ganglioside; Galactose; Glucuronidase; Glycolipids; Glycosides; Humans; Hymecromone; Macrophages; Melanoma; Neuroblastoma; PC12 Cells; Tritium; Tumor Cells, Cultured

The expression of gangliosides in non-malignant tissues (epidermis and pigmented nevus) and neoplastic lesions (melanoma, squamous cell carcinoma [SCC] and basal cell carcinoma [BCS]) of the human skin was analyzed immunohistochemically and biochemically to characterize the features associated with malignancy. Immunohistochemical staining with an anti-II3NeuAc-LacCer (GM3) monoclonal antibody (M2590 mAb) and an anti-II3(NeuAc)2-LacCer (GD3) mAb (R24) showed the expression of the gangliosides GM3 and GD3 to vary among the different tissues. M2590 clearly stained epidermal keratinocytes and the tumor cells of BCC and SCC, and strongly stained melanocytes and melanoma cells. In contrast, R24 did not stain epidermal keratinocytes and only faintly stained SCC cells, while it clearly stained BCC cells, and intensely stained melanocytes and melanoma cells. GM3 showed a similar level of staining among the tissue specimens, while the level of GD3 staining was quite variable among the tumor specimens. Biochemical analysis by thin-layer chromatography (TLC) with resorcinol staining and TLC immunostaining with either M2590 or R24 showed both GM3 and GD3 to be commonly expressed by both the normal and malignant skin tissues, including SCC. There was no close correlation between the intensity of immunohistochemical staining and the biochemically detected amounts of these gangliosides. This may have been partly due to the so-called cryptic expression of cell membrane gangliosides. Our results thus suggest that analysis of the tumor-associated expression of gangliosides requires several methods, since the sensitivity of the methods used may have a considerable effect on the diagnostic value of gangliosides as skin cancer markers.

Topics: Biomarkers, Tumor; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Dendritic Cells; Epidermis; G(M3) Ganglioside; Gangliosides; Gene Expression; Humans; Immunoenzyme Techniques; Melanocytes; Melanoma; Nevus, Pigmented; Phenotype; Skin; Skin Neoplasms

This report evaluates the relevance of the ratio of the melanoma-associated ganglioside, GD3, and its precursor, GM3, to prognosis and management of Stage II disease. Tumor biopsy specimens from 42 melanoma patients were examined for the ratio and found that GD3 and GM3 constitute 80% of the total lipid bound sialic acids (LBSA) of melanoma. Although the ratio of GM3:GD3 in melanocytes, the progenitors of melanoma, is 19:1 (based on %LBSA), it ranged from 15:1 to 1:5 in tumor biopsy specimens. Patients are categorized into three groups based on their GM3:GD3 ratio (%LBSA) of tumor tissues as Group I (ratio ranged from 15:1 to 1.5:1) (10 of 42), Group II (1.4:1 to 1:1.4) (13 of 42), and Group III (1:1.5 to 1:5) (19 of 42). When the overall survival of patients from the onset of Stage II disease was evaluated among different groups, patients belonging to Group I survived significantly longer than patients of Group II (P = 0.02) and Group III (P = 0.01). Interestingly, Group III expressed immunogenic gangliosides (GD2, GM2, and O-AcGD3) better than the other groups and may benefit more from therapies targeted against these gangliosides antigens. The results of this study indicate that GM3:GD3 ratio is an unusual but well-defined biochemical criteria that may be useful for prognosis and therapeutic management of the disease. Therefore, we propose a routine analysis of the GM3:GD3 ratio of tumors excised after surgery. Screening the ratio is feasible since melanoma expresses a simple profile of gangliosides unlike other forms of cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Female; G(M3) Ganglioside; Gangliosides; Humans; Lymphatic Metastasis; Male; Melanoma; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

Murine anti-idiotype monoclonal antibodies were generated against a human IgM monoclonal antibody (L612) that recognizes ganglioside GM3 on human melanoma. Hybridomas secreting antibodies that bound specifically to L612 were selected by enzyme-linked immunosorbent assay using L612 and three negative control human IgMs, including monoclonal anti-GM2 and anti-GD2 antibodies, as well as purified serum IgM, as antigen sources. GM3-binding inhibition and cell-binding inhibition assays were used to identify seven anti-idiotype monoclonal antibodies that recognized determinants located within the antigen-combining sites of L612. To determine whether these anti-idiotype monoclonal antibodies possessed the internal image of the original antigen, we immunized syngeneic BALB/c mice with one of the anti-idiotype monoclonal antibodies, 4C10, coupled with keyhole limpet hemocyanin. Sera from the immunized mice reacted strongly with an antigen-positive M12 melanoma cell line and with purified GM3. Because L612 detects and kills melanoma tumor cells in vitro and in vivo in the presence of complement without affecting normal tissues, anti-idiotype monoclonal antibodies carrying the internal image of GM3 may be an effective tool for active specific immunotherapy in patients with melanoma.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Antigens, Neoplasm; Culture Media; Enzyme-Linked Immunosorbent Assay; Epitopes; G(M3) Ganglioside; Humans; Hybridomas; Immune Adherence Reaction; Immunization; Immunoglobulin G; Immunoglobulin M; Immunotherapy; Melanoma; Mice; Mice, Inbred Strains; T-Lymphocytes, Cytotoxic

We investigated the ability of GM3-lactone liposomes to induce anti-melanoma T cell responses in mice. GM3-lactone liposomes, like murine B16 melanoma cells, induced anti-melanoma cytotoxic T cells (CTL) and also suppressor T cells (Ts). A small dose of GM3-lactone (0.0003 micrograms/ml) was enough to generate CTL in the in vitro primary response, whereas relatively large amounts of the antigen (0.03-0.3 microgram/ml) were required for anti-melanoma Ts induction. As the epitope for anti-melanoma Ts is NeuAc but not NeuGc residue on GM3, and anti-melanoma CTL are effectively induced by either GM3(NeuAc) or GM3(NeuGc)-lactone liposomes, GM3(NeuGc)-lactone or GM3(NeuGc) liposomes have potent activity as an artificial melanoma antigen to induce anti-melanoma CTL in vitro.

Topics: Animals; Antigens, Neoplasm; Carbohydrate Sequence; G(M3) Ganglioside; Gangliosides; Liposomes; Melanoma; Mice; Mice, Inbred Strains; Molecular Sequence Data; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

After the observation that human mAb 32-27M reacts only with melanoma and astrocytoma cells cultured in the presence of fetal bovine serum, a novel pathway for the uptake of exogenous gangliosides, their further biosynthesis, and expression at the cell surface as novel Ag has been elucidated. The addition of fetal bovine serum to melanoma and astrocytoma cells growing in synthetic medium (insulin-transferrin-selenium) resulted in reactivity with Ab32-27M. As antibody 32-27M detects N-glycolylneuraminic acid (NeuGc)-containing gangliosides, the effect of adding a number of different gangliosides to melanoma and astrocytoma cells cultured in the synthetic medium was studied. Only the addition of NeuGc-GM3 resulted in the development of Ab32-27M reactivity. The identity of the antigenic structures developed after addition of fetal bovine serum or NeuGc-GM3 was determined by analysis of the gangliosides from both samples. The major component detected in melanoma cell lines was shown to be N-acetylneuraminic acid-NeuGc-GD3. Another, slower moving component, present in some melanomas and in astrocytomas may be N-acetylneuraminic acid-NeuGc-GD2. The cell type specificity for these processes can be most readily explained by postulating that all cells can take up exogenous gangliosides but only melanoma and astrocytoma cells have sufficiently high levels of GM3 alpha 2----8-sialyltransferase for the conversion of added NeuGc-GM3 to disialogangliosides to be effective. These results demonstrate a novel pathway for exogenous glycolipid processing that can lead to novel Ag expression but may also play a role in normal glycolipid metabolism and function.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Neoplasm; Antigens, Surface; Astrocytoma; Cattle; Cell Line; Cell-Free System; Epitopes; Fetal Blood; G(M3) Ganglioside; Gangliosides; Humans; Melanoma; Neuraminic Acids; Tumor Cells, Cultured

We have shown that a syngenic monoclonal antibody, M2590, established after immunization of C57BL/6 mice with B16 melanoma cells, recognized GM3 (NeuAc) ganglioside. Although GM3 is widely distributed among various normal cells and tissues, the antibody did not react with them. However, it reacted exclusively with melanoma cells from mouse, hamster and human. Preliminary experiments suggested that proteins and lipids as well as GM3 density on B16 cells are involved in the reactivity of GM3 with the antibody. Then, we investigated the biological function of the melanoma antigen, which was secreted from B16 cells into the culture medium. This soluble antigen was shown to suppress the positive immune responses by inhibiting CTL activity in the effector phase and by induction of specific suppressor T cells (Ts) that block CTL generation in the induction phase. Liposomes containing GM3 (NeuAc) but not GM3 (NeuGc) can effectively induce the melanoma specific Ts as did the soluble antigen. The results indicated the tumor cells can escape from host-immune system by stimulating the repertoire of Ts for self-antigen, GM3. To understand the biological role of GM3, we have established mutant clones of no-expressor of GM3 recognized by M2590. The clones were found to have lower attachment to laminin and type IV collagen and poor ability of lung metastasis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; G(M3) Ganglioside; Lymphocyte Activation; Melanoma; Mice; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Tumor Cells, Cultured

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside GM3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of GM3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of GM3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.

Topics: Animals; Antibodies, Monoclonal; Carbohydrate Sequence; Ceramides; Chromatography, Thin Layer; Dogs; Enzyme-Linked Immunosorbent Assay; Erythrocytes; G(M3) Ganglioside; Glycolipids; Humans; Mass Spectrometry; Melanoma; Molecular Sequence Data; Spectrometry, Mass, Fast Atom Bombardment

The present study investigates the chemical structure of a ganglioside, detected by monoclonal antibody (MAb) MacG1, which reacts with intracytoplasmic granules of tumor-infiltrating macrophages. The results obtained by enzymatic hydrolysis and fast-atom bombardment-mass spectrometry reveal that MAb MacG1 reacts with a subcomponent of the ganglioside GM3 found in melanoma and bovine brain. MAb MacG1 might be a powerful tool to distinguish among GM3 species and could help to define their possibly different biological functions.

Topics: Animals; Antibodies, Monoclonal; Antigens; Brain; Cattle; G(M3) Ganglioside; Gangliosides; Humans; Melanoma; Molecular Structure; Tissue Distribution

Gangliosides appear to be important target molecules for immunological effector mechanisms on neuro-ectodermal tumors. Therefore in vitro studies were performed to examine whether ganglioside GD3, which is highly expressed on the cell surface of cultured human melanoma cells, is being shed into the culture medium. Measurable quantities of gangliosides GM3 and in particular GD3 were shed by the melanoma cells we have tested as detected on thin-layer chromatograms (TLC) stained with orcinol. Ganglioside GD3 was also evidenced by immunostaining with anti-GD3 MAb and by ELISA. The concentration of GD3 in the supernatant of human melanoma cells depended on the ganglioside pattern of the cell line. Cells containing high levels of GD3 shed large amounts, cells with low levels shed no detectable GD3. Ganglioside GD3 was detectable in sera, but no major quantitative differences were observed in sera of patients with GD3-positive tumors and normal controls. This points to a local accumulation of ganglioside GD3 at the tumor site.

Topics: G(M3) Ganglioside; Gangliosides; Humans; Melanoma; Tumor Cells, Cultured

A novel ganglioside, de-N-acetyl-GM3 (neuraminyllactosylceramide, II3NeuNH2LacCer), was found in the monosialoganglioside fraction of A431 cells and B16 melanoma cells by high-performance liquid chromatography, thin-layer chromatography, and immunoblotting with its specific monoclonal antibody DH5. This novel type of membrane ganglioside strongly enhanced the kinase activity associated with the epidermal growth factor (EGF) receptor, and it showed 32, 35, and 12% growth stimulation as compared with control cultures of A431, Swiss 3T3, and B16 melanoma cells, respectively. Exogenously added de-N-acetyl-GM3 did not alter the affinity of EGF binding to its receptor. These properties of de-N-acetyl-GM3 are in striking contrast to those of GM3 and its lyso derivative (lyso-GM3) which were previously shown to inhibit EGF receptor kinase activity and to inhibit growth in the same cells. These data indicate that de-N-acetylation at the sialic acid moiety of GM3 ganglioside is an important mechanism for modulation of EGF-dependent cell growth. The mechanism is antagonistic to that of GM3-dependent modulation of receptor function.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; ErbB Receptors; G(M3) Ganglioside; Gangliosides; Immunosorbent Techniques; Melanoma; Mice; Promoter Regions, Genetic; Protein-Tyrosine Kinases

In previous studies, an IgM monoclonal antibody (M2590), established after immunization of C57BL/6 mice with syngeneic B16 melanoma cells, was found to react with melanoma cells, but not with various normal cells and tissues (Taniguchi, M., and Wakabayashi, S., Jpn. J. Cancer Res., 75:418-426, 1984). The structure defined by this antibody was identified as GM3 (Hirabayashi, Y., et al., J. Biol. Chem., 260:13328-13333, 1985) organized in membranes at high density, although the real immunogen was suggested to be GM3 lactone (Nores, G. A., et al., J. Immunol., 139:3171-3176, 1987). Since GM3 lactone was found to be highly immunogenic, we subsequently immunized C57BL/6 mice with GM3 lactone coated on Salmonella minnesotae and established hybridoma DH2, secreting an IgG3 antibody showing preferential reactivity with GM3 lactone over GM3 under certain conditions. The reactivity of the DH2 antibody was competitively inhibited by M2590, and it showed a preferential reactivity with melanoma cells and displayed various immunochemical and immunobiological properties similar to those of M2590. However, DH2 antibody inhibited melanoma cell growth in vivo, induced antibody-dependent cytotoxicity in vitro, and showed a preferential accumulation in melanoma growth in vivo. These properties are characteristic of the IgG3 subclass, in striking contrast to IgM antibody M2590, which does not inhibit cell growth in vivo or in vitro and does not induce antibody-dependent cytotoxicity. Thus, immunization with lactone forms of tumor-associated ganglioside antigens might be useful in the production of antibodies and prevention of tumor cell growth in vivo (antitumor vaccines).

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Cell Division; Cells, Cultured; Dogs; G(M3) Ganglioside; Gangliosides; Immunoglobulin G; Melanoma; Mice; Mice, Inbred C57BL; Rats

Expression of the gangliosides GM3, GD3 and GD2 was studied in tissue sections from 19 naevi, 29 primary and 83 metastatic melanoma using the ABC immunoperoxidase technique. GM3 was not detected in normal skin whereas GD2 was detected on the basal and stratum spinosum of the epidermis and on peripheral nerves in the dermis. GD3 was expressed on melanocytes but not on most other components of normal skin. However, GD3 was strongly expressed on epidermis adjacent to naevi and primary melanoma whereas GD2, in contrast to that in normal skin, was not expressed on the epidermis adjacent to 26/29 primary melanoma. All naevi were positive for GM3 and GD3 except that GM3 was not detected on junctional components of naevi. GD2 was not expressed on naevi except in areas showing neuroid differentiation. Studies on melanoma revealed that approximately 60% of primary and 75% of metastatic melanoma expressed GM3 to a varying extent. With 2 exceptions, all primary and metastatic melanomas expressed GD3 although there was variable expression within most of the individual tumours. GD2 was detected in only approximately 25% of primary and 50% of metastatic melanomas. Both GD2 and GD3 were detected on lymphocytes surrounding melanoma. The higher expression of GD2 on metastases compared to primary melanomas was consistent with the view that GD2 expression was associated with increased metastatic potential. However, the low proportion of metastases expressing GD2 and the absence of any correlation with thickness of the primary tumour suggested that GD2 expression was not a reliable marker of metastatic potential. No differences could be detected in ganglioside expression on metastases in skin or lymph nodes. These results appear to have implications for the use of MAbs against gangliosides in therapy of melanoma and in the study of melanocytic differentiation.

Topics: Antigens; G(M3) Ganglioside; Gangliosides; Humans; In Vitro Techniques; Melanoma; Nevus; Skin; Skin Neoplasms

The specificity of antibody to NeuGc alpha 2-3Gal beta 1-4Glc-cer (GM3(NeuGc] was carefully reexamined by the method of enzyme-immunostaining on a thin layer plate. The affinity-purified antibody was found to react with NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuGc] and NeuGc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuAc], but not with NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuGc)) or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuAc]. From this result together with the previous results, it (GD3(NeuAc-NeuAc], From this result together with the previous results, it could be concluded that the antibody recognizes the outer portion of molecular species of sialic acids in the gangliosides. By using this antibody, the expression of Hanganutziu-Deicher (HD) gangliosides could be demonstrated in human malignant melanoma. The molecular species were different among individuals examined. Among HD-antigenic gangliosides, GM3(NeuGc) was commonly found in melanoma tissues. One of the patients examined expressed GD3(NeuGc-NeuGc) and GD 3(NeuGc-NeuAc), which may be characteristic gangliosides in human melanomas, since these gangliosides could not be detected in human colon cancer or human fetal tissues.

Topics: Antigens, Heterophile; Carbohydrate Sequence; Chromatography, Thin Layer; Densitometry; G(M3) Ganglioside; Gangliosides; Humans; Melanoma

A convenient, rapid, and reproducible assay was developed to evaluate the immunoreactivity of radiolabeled monoclonal antibodies against three different human melanoma-associated antigens, p97, a proteoglycan and a GD3 ganglioside. A cloned melanoma cell line (M 2669 CL 13) was selected as the target and, when fixed with paraformaldehyde, showed binding as good as or better than that obtained with live cells for the three antigens. Fixed cells retained good binding properties stored at 4 degrees C for over 6 mo. This assay has general applicability to other antigen-antibody systems for testing chemically modified monoclonal antibodies or fragments during the development of a radiopharmaceutical or as a routine quality control measure for clinical agents.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Cell Count; Cell Line; Evaluation Studies as Topic; G(M3) Ganglioside; Humans; Immunoassay; Immunoglobulin Fab Fragments; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Proteoglycans

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigens; Cell Line; Enzyme-Linked Immunosorbent Assay; Epitopes; G(M3) Ganglioside; Gangliosides; Immunoenzyme Techniques; Mass Spectrometry; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Radioimmunoassay; Species Specificity

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Epitopes; G(M3) Ganglioside; Gangliosides; Killer Cells, Natural; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred C57BL; Neoplasm Proteins

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.

Topics: Cell Line; G(M3) Ganglioside; Gangliosides; Humans; Male; Melanocytes; Melanoma; Tetradecanoylphorbol Acetate