Inhibition of adhesion of F9 embryonal carcinoma cells to substratum by a novel monoclonal antibody, TEC-05, reactive with a developmentally regulated carbohydrate epitope

Embryonal carcinoma cells carry on their surfaces carbohydrate antigens that are also expressed in early embryonic cells. We report here the expression and properties of a new developmentally regulated carbohydrate epitope, which is defined by a monoclonal antibody TEC-05. This antibody was generated by immunization of a rat with mouse embryonal carcinoma cells P19S1801A1. By immunofluorescence, the TEC-5 epitope was first detected on 8-cell-stage mouse embryos and was present on all subsequent stages of preimplantation development. Absorption analysis revealed that TEC-5 epitope was expressed only on a limited number of adult mouse tissues. In the direct radioantibody binding assay, TEC-05 reacted strongly with OTF9-63 cells and with some of the mouse embryonal carcinoma cell lines tested. Its reaction with differentiated cell lines was weak or undetectable. In the course of differentiation of OTF9-63 cells induced by retinoic acid, the epitope disappeared with the onset of morphological differentiation. The binding of the antibody to OTF9-63 cells was inhibited to 50% by 10-50 microM N-acetyllactosamine and lactose. Immunolabelling of extracts from OTF9-63 cells separated by sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis revealed that TEC-5 epitope was carried by high-molecular-weight glycoconjugates (molecular weight greater than 100,000). Molecules, isolated from [3H]-fucose-labelled OTF9-63 cells by indirect immunoprecipitation with TEC-05 antibody, were degraded by extensive pronase digestion or mild alkaline treatment to large carbohydrate chains that were excluded from a Sephadex G-50 column. Direct evidence that TEC-05 antibody bound to embryoglycan was obtained using a modified Farr's assay. The antibody was found to inhibit adhesion of F9 and OTF9-63 cells to substratum. The inhibitory effect, which could be abrogated by lactose, seemed to be specific, because another IgM monoclonal antibody which also binds to embryoglycan had no effect. Combined data indicated that TEC-05 antibody recognizes a carbohydrate epitope which is involved in cell-substratum adhesion of F9 cells and which provides a new marker for structure-function studies of stage-specific embryonic antigens.     

A monoclonal antibody detaches embryonic skeletal muscle from extracellular matrices

We have described a monoclonal antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin- coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies with differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 10(5) sites per myoblast with an apparent Kd in the range of 2-5 x 10(-9) molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of approximately 120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an obvious candidate.     

Embryonic stem cells deficient in I beta1,6-N-acetylglucosaminyltransferase exhibit reduced expression of embryoglycan and the loss of a Lewis X antigen, 4C9

Embryoglycan is a class of branched high-molecular-weight poly-N-acetyllactosamines characteristically expressed in early embryonic cells and has been shown to be involved in the intercellular adhesion of early embryonic cells in vitro. Branching of poly-N-acetyllactosamine chains is performed by beta1,6-N-acetylglucosaminylation of the galactosyl residue. We previously knocked out the gene encoding I beta1, 6-N-acetylglucosaminyltransferase (IGnT), and the resultant deficient mice were born without any abnormality, although the mice exhibited various deficits in later life. In the present investigation, we produced embryonic stem (ES) cells from IGnT-deficient embryos. The mutant ES cells exhibited a reduced capability in embryoglycan synthesis. Thus, IGnT is a major enzyme involved in the branching of poly-N-acetyllactosamine chains in embryoglycan. Since ES cells are equivalent to multipotential cells of the embryonic ectoderm in early postimplantation embryos, this result indicates that an abundance of embryoglycan in these cells is not essential for normal embryogenesis. The IGnT-deficient ES cells continued to express SSEA-1, but lacked the expression of 4C9 antigen, although the epitope of 4C9 antigen was confirmed to be Lewis X by a transfection experiment. The result establishes the distinct nature of 4C9 antigenicity, which requires either Lewis X epitope on I-branch or clustering of Lewis X epitope, best accomplished by poly-N-acetyllactosamine branching. Alpha6-integrin was newly identified as a carrier of embryoglycan. The IGnT-deficient ES cells adhered to dishes coated with laminin, which is a ligand for alpha6-integrin, significantly less than wild-type ES cells, raising the possibility that embryoglycan in ES cells enhances alpha6-integrin-dependent adhesion in vitro.     

Inhibition of embryonic neural retina cell-substratum adhesion with a monoclonal antibody

The C1H3 monoclonal antibody recognizes two distinct developmentally regulated cell surface antigens, with molecular masses of 170,000 and 140,000 daltons, in embryonic chick neural retina (Cole, G. J., and Glaser, L. (1984) Proc. Natl. Acad. Sci. U. S. A., in press). In vitro, the 170,000-dalton polypeptide is released by retinal cells into the surrounding culture medium and is present in material sedimentable at 100,000 X g. This pelletable material contains particles designated as adherons (Schubert, D., LaCorbiere, M., Klier, F. G., and Birdwell, G. (1983) J. Cell Biol. 96, 990-998) which promote cell-substratum adhesion of chick neural retina cells. In the present study, evidence is provided that the C1H3 monoclonal antibody inhibits cell adhesion to adheron-coated dishes when bound either to cells or to the adherons. The failure of other monoclonal antibodies, that bind to retinal cells with equal abundance, to disrupt adhesion demonstrates that the effect is specific. These data suggest that the neural-specific 170,000-dalton C1H3 polypeptide is the neural cell-adhesion molecule which is responsible for the ability of adherons to bind to cells.     

Isolation and partial characterization of endothelial cell extracellular complexes

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.     

Effects of Succinyl Concanavalin A and Tunicamycin on F9 Embryonal Carcinoma Cells: Inhibition of Cell Spreading

Succinyl concanavalin A at a concentration of 50 μg/ml inhibited spreading of F9 embryonal carcinoma cells, while at this concentration it inhibited cell growth only partially. Thus, in the presence of succinyl concanavalin A, F9 cells grew as rounded cell aggregates that sometimes became detached from the substratum. Tunicamycin at a concentration of 1 μg/ml had a similar effect on F9 cells. These results suggest that a mannosyl glycoprotein(s) is involved in cell-substratum interaction of the cells. Furthermore, tunicamycin partially inhibited the biosynthesis of embryoglycan, the large glycoprotein-bound carbohydrates of early embryonic cells.     

Drosophila PS integrins recognize vertebrate vitronectin and function as cell-substratum adhesion receptors in vitro.

Using the Drosophila cell line MLDmBG-1, a monoclonal antibody aBG-1 that can inhibit not only cell clumping but also cell spreading was generated. This antibody immunoprecipitates a complex of molecules consisting of a major 120 x 10(3) Mr and other components. To characterize the 120 x 10(3) Mr component, we purified it, generated antibodies to it, and cloned its cDNA. Sequencing of this cDNA suggests that the 120 x 10(3) Mr molecule is identical to PS beta, a beta chain of Drosophila integrins. The other components immunoprecipitated included two alpha chains of Drosophila integrins, PS1 alpha and PS2 alpha, as revealed using specific antibodies to these molecules. These suggest that aBG-1 recognizes the PS beta associated with PS1 alpha or PS2 alpha. However, immunostaining of embryos and larvae with aBG-1 showed that the staining pattern is similar to that for PS2 alpha but not for PS beta, suggesting that the antibody preferentially recognizes the PS beta associated with particular alpha chains in situ. We then attempted to characterize the ligands for these integrin complexes, using culture dishes coated with various vertebrate matrix proteins. These cells spread very well on dishes coated with vitronectin and, to a lesser extent, on those with fibronectin. This spreading was partially inhibited by aBG-1, but not by other control antibodies or RGD peptides. The cell attachment to these substrata was not affected by the antibody. The cells also can attach to dishes coated with laminin but without spreading, and this attachment was not inhibited by aBG-1. Furthermore, they do not attach to dishes coated with collagen type I, type IV, and fibrinogen. These results indicate that Drosophila PS integrins can recognize vertebrate vitronectin, and also fibronectin with a weaker affinity, at sites other than RGD sequences, and thus can function in cell-substratum adhesion.     

Flow cytometric analysis of sialyl Lewis A antigen on human cancer cells by using F(ab')2μ fragments prepared from a mouse IgM monoclonal antibody

F(ab')2 fragments, herein designated as F(ab')2μ fragments, were prepared from a mouse IgM monoclonal antibody specific to sialyl Lewis A antigen. The fragments were applied to flow cytometry to analyze the antigen on human cancer cells. The binding of the fragments to the antigen-positive cells was stronger than that of the original IgM. The non-specific binding of the IgM antibody to the antigen-negative cells was much decreased by using the F(ab')2μ fragments. These results indicate that the F(ab')2μ fragments are more suitable than the original IgM monoclonal antibody in flow cytometric analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

20-alpha-Hydroxysteroid dehydrogenase from pseudopregnant rat ovary: obtention and characterization of a monoclonal antibody against the enzyme activity.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD) was purified from pseudopregnant rat ovaries and used as antigen for the development of a monoclonal antibody by the hybridoma technique. Spleen cells of BALB/c mice immunized with purified 20-alpha-HSD were fused with SP2/0 mouse myeloma cells. Among the colonies of hybrid cells, one (designated mAb-HSD 11) was found to be secreting antibodies (IgM) able to inhibit 20-alpha-HSD activity. The antibody-secreting hybridome was amplified by ascitic fluid production and the monoclonal antibody purified by Bakerbond ABx procedure. Purified mAb-HSD 11 was able to inhibit 20-alpha-HSD activity in a dose-dependent manner. Studies of Michaelis constants of 20-alpha-HSD indicate that this monoclonal antibody increases the Km for 20-alpha-dihydroprogesterone and decreases the Vmax.     

Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan.

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.     

Central nervous system antigen P84 can serve as a substrate for neurite outgrowth

Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.     

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.     

Antibody 624H12, which detects lung cancer at early stages, recognizes a sugar sequence in the glycosphingolipid difucosylneolactonorhexaosylceramide (V3FucIII3FunLc6Cer)

Immunocytochemical staining of cells in sputum by rat monoclonal antibody 624H12 detects lung cancer 2 years prior to its detection by conventional diagnostic techniques. The antigen recognized by antibody 624H12 is a sugar sequence in the glycosphingolipid difucosylneolactonorhexaosylceramide (V3FucIII3FucnLc6Cer) whose structure is (formula see; text) Both fucosyl residues are required for high affinity binding by the antibody. The antigen was expressed in 35 of 45 specimens of cancer tissue from patients with early stage non small cell lung cancer. There was no correlation between antigen expression and patient survival.     

A Novel Monoclonal Antibody disrupting Cell Type Specific Substratum Adhesion of Frog (Xenopus laevis) Epithelial Cells and Endothelial Cells

We isolated a mouse monoclonal antibody (FAD-II) that disrupts cell-substratum adhesion of amphibian ( Xenopus laevis ) epithelial cells and endothelial cells. The effect of the antibody was cell-type specific, and the antibody had no effect on fibroblastic cells while fibronectin peptide blocked cell-substratum adhesion of all the cell types examined. In developing frog embryos, the epitopes recognized by the antibody were detected in pronephrotic ducts and in other tissue cells of embryos (from stage 33/34 afterwards). In adult tissues, the antibody mainly recognized antigens in extracelluar matrices. The antigens recognized by the antibody seems to be novel glycoepitopes in frog cells.     

1H-NMR spectroscopy of the glycoprotein-bound large carbohydrates from embryonal carcinoma cells

400 MHz NMR spectrum was recorded for the glycoprotein -bound large carbohydrates (embryoglycan) isolated from F9 embryonal carcinoma cells. Two intense signals at 4.13 ppm and 4.69 ppm were assigned to be H-4 of galactosyl residues substituted at C-3 and H-1 of G1cNAc beta 1----3, respectively. The result is consistent with the proposal that the fundamental building unit of the large glycan is G1cNAc beta 1----3Ga1 beta. Furthermore, the spectral data confirmed a conclusion obtained by glycosidase digestion that fucosyl residues are linked mostly to N-acetylglucosamine rather than galactose.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.     

细胞表面糖链末端唾液酸和岩藻糖和某些细胞生物学行为的关系

用神经氨酸酶和α-L-岩藻糖苷酶分别切除人肝癌细胞株7721细胞表面糖链中的末端唾液酸（SA）和岩藻糖（Fuc)残基来研究表面聚糖结构和某些细胞生物学行为之间的关系。选择细胞对纤连蛋白（Fn）,层黏蛋白（Ln)和人脐静脉内皮细胞（HUVEC）的黏附能力,细胞趋化性迁移以及趋化性侵袭作为细胞行为的指标。结果表明：表面人脐静脉内皮细胞（HUVEC）的黏附能力,细胞趋化性迁移以及趋化性侵袭作为细胞行为的指标。结果表明：表面糖链末端SA对细胞黏附至Fn并不必需,对细胞黏附至Ln和细胞的趋化性侵袭却至为重要,而对细胞黏附至HUVEC以及趋化性迁移则为关键性残基。与SA相比,Fuc可能参与细胞Fn,Ln和HUVEC的黏附,但对趋化性迁移以及趋化性侵袭并不重要。细胞对HUVEC的黏附以及趋化性迁移和侵袭可被唾液酸化Lewis X(SLe^x)单抗抑制,但不被未唾液酸化的Lewis X(Le^x)单抗抑制,这一结果支持SA在上述三种细胞过程中Fuc残基重要。     

Functional characterization of chondroitin sulfate proteoglycans of brain: interactions with neurons and neural cell adhesion molecules

Ng-CAM and N-CAM are cell adhesion molecules (CAMs), and each CAM can bind homophilically as demonstrated by the ability of CAM-coated beads (Covaspheres) to self-aggregate. We have found that the extent of aggregation of Covaspheres coated with either Ng-CAM or N-CAM was strongly inhibited by the intact 1D1 and 3F8 chondroitin sulfate proteoglycans of rat brain, and by the core glycoproteins resulting from chondroitinase treatment of the proteoglycans. Much higher concentrations of rat chondrosarcoma chondroitin sulfate proteoglycan (aggrecan) core proteins had no significant effect in these assays. The 1D1 and 3F8 proteoglycans also inhibited binding of neurons to Ng-CAM when mixtures of these proteins were adsorbed to polystyrene dishes. Direct binding of neurons to the proteoglycan core glycoproteins from brain but not from chondrosarcoma was demonstrated using an assay in which cell-substrate contact was initiated by centrifugation, and neuronal binding to the 1D1 proteoglycans was specifically inhibited by the 1D1 monoclonal antibody. Different forms of the 1D1 proteoglycan have been identified in developing and adult brain. The early postnatal form (neurocan) was found to bind neurons more effectively than the adult proteoglycan, which represents the C-terminal half of the larger neurocan core protein. Our results therefore indicate that certain brain proteoglycans can bind to neurons, and that Ng-CAM and N-CAM may be heterophilic ligands for neurocan and the 3F8 proteoglycan. The ability of these brain proteoglycans to inhibit adhesion of cells to CAMs may be one mechanism to modulate cell adhesion and migration in the nervous system.