Developmentally regulated expression of peanut agglutinin (PNA)-specific glycans on murine thymocytes

Intrathymic maturation of T lymphocytes is characterized byvariable expression of O-linked Galß1,3GalNAc glycansreactive with peanut agglutinin (PNA) lectin. Recent studieson human thymocytes show that conversion from PNA⁺ to PNA^–phenotype is correlated with increased expression of     

Interaction of peanut agglutinin,a lectin specific for nonreducing terminal d-galactosyl residues,with embryonal carcinoma cells

Peanut agglutinin (PNA), a lectin specific for terminal d-galactosyl residues, was found to react with embryonal carcinoma cells, but not with their differentiated derivatives. Receptors for PNA were detectable at the surface of all cells of the quasinullipotent F9 line and on only 50% of the multipotent PCC3/A/1 line. The fraction of the PCC3/A/1 population which expresses the F9 antigen was found to be included in the subpopulation carrying the PNA receptors. PNA⁺ and PNA^? subpopulations of PCC3/A/1 were separated by a PNA-mediated reversible agglutination of PNA⁺ cells with rabbit erythrocytes. These subpopulations were essentially F9⁺ and F9^?, respectively.     

Identification of peanut agglutinin receptors on mouse testicular germ cells

Peanut agglutinin receptors expressed specifically in mouse testicular germ cells have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lectin blotting techniques. Two major components were estimated to have molecular weights of 86,000 and isoelectric points of 6.1 +/- 0.3 and 6.2 +/- 0.3. Minor components with molecular weights of 71,000-74,000 and isoelectric points of 6.1 +/- 0.3 were also detected. Specific expression of these receptors on testicular germ cells was confirmed using the testes of mutant mice, Sld/Sld, devoid of germ cells.     

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.     

Immobilized wheat germ agglutinin separates small oligosaccharides derived from poly-N-acetyllactosaminoglycans of embryonal carcinoma cells

Five pure oligosaccharides derived from poly-N-acetyllactosaminoglycans of teratocarcinoma cells were chromatographed on immobilized wheat germ agglutinin (WGA). Three of them, Gal beta 1-4GlcNAc, GlcNAc beta 1-3Gal, and GlcNAc beta 1-3Gal beta 1-4GlcNAc, revealed only weak binding, but GlcNAc beta 1-6Gal beta 1-4GlcNAc was bound moderately and GlcNAc beta 1-6Gal was bound quite strongly. The differences in the binding affinities were large enough to allow chromatographic separation of the five oligosaccharides into three distinct fractions in a small column of WGA-agarose.     

Cell surface carbohydrates of embryonal carcinoma cells: polysaccharidic side chains of F9 antigens and of receptors to two lectins, FBP and PNA

Three cell surface components of mouse embryonal carcinoma (EC) cells, F9 antigens and the receptors to the lectins FBP and PNA, have been isolated from radiolabeled EC cells by indirect immunoprecipitation. All three were efficiently labeled with fucose, galactose and glucosamine, but scarcely at all with mannose. The high molecular weight glycopeptides characteristic of early embryonic cells were released as the major glycopeptides upon pronase digestion of the three markers. The binding sites to the two lectins are present in the high molecular weight glycopeptides. Furthermore, a close correlation exists between the disappearance of the high molecular weight glycopeptides from differentiating EC cells and the disappearance of the three markers from the surface of these cells. The large glycopeptides from the three markers have the following properties in common. First, they are not mucin-type glycopeptides with short oligosaccharides, glycolipids and acidic mucopolysaccharides, nor are they products of incomplete pronase digestion of conventional complex-type glycopeptides. Second, they do not contain appreciable amounts of Fucα1→2Gal or Fucα1→6GlcNAc linkages. Third, a significant fraction of the glycopeptides have the GlcNAcβ→Gal sequence in their core structure. We propose that the cell surface markers of EC cells have a class of large carbohydrate chains not found in typical surface markers of adult cells such as H-2, la and LETS.     

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.     

Isolation and characterization of peanut agglutinin-resistant embryonal carcinoma cell-surface variants

The isolation and characterization of variant embryonal carcinoma (EC) cells possessing altered cell-surface structures is described. The lectin peanut agglutinin (PNA), which binds to EC cells but not their differentiated derivatives, was used to select the variants. Clones resistant to the cytotoxic effect of PNA were isolated at a frequency of 4 × 10^–5 following mutagenesis. The resistant phenotype was stable in the absence of selection in all eight clones tested. The increased frequency of resistant clones following mutagenesis and the stability of the phenotype suggests a mutational origin. Somatic cell hybrids constructed between wild-type cells and two different PNA-resistant cell lines were sensitive to PNA; this suggests that the resistant phenotype is recessive. Binding assays demonstrated that resistant cells exhibited a twofold to fourfold reduction in the total amount of PNA bound. Together with the recessive behavior of the phenotype, this suggests that resistant cells are deficient for PNA receptors. The PNA-resistant cells also showed reduced binding of monoclonal antibody against stage-specific embryonic antigen 1 (SSEA–1) in indirect cytotoxicity tests. All eight PNA-resistant lines isolated were tumorigenic in syngeneic mice and gave rise to well-differentiated teratocarcinomas. The PNA-resistant cells behaved like their wild-type parents in a cell recognition assay; when incubated in suspension with endodermal cells, they sorted out to form simple embryoid bodies (a core of EC cells surrounded by an endodermal rind). Thus, EC cells can form tumors, differentiate, and recognize differentiated cells in a sorting assay despite a reduction in expression of the embryo-specific cell surface structures (s) that bind PNA and anti-SSEA-1 antibody.     

Appearance of functional insulin receptors during the differentiation of embryonal carcinoma cells

The insulin-receptor binding activity and insulin-stimulated growth response of PC13 clone 5 cells were investigated for both the embryo carcinoma (EC) and retinoic acid-induced differentiated derivatives of this cell line. Whereas the EC cell was found to have very few, if any, receptors and showed no demonstrable dependence on insulin for growth, the differentiated derivative cell expressed a large number of insulin receptors and, when challenged with the hormone, showed stimulation of both DNA synthesis and cell division. The same data were obtained for five independent PC13 clones. These results, coupled with previous observations, lend weight to the suggestion that the appearance of specific receptors for growth regulatory substances may be a manifestation of a general change in growth-regulatory mechanisms accompanying EC cell differentiation and loss of malignancy.     

Nucleolar persistence in embryonal carcinoma cells

A series of embryonal carcinoma (EC) lines were found to have nucleoli which persisted through metaphase and anaphase in 60–88% of cells. When these cells differentiated, either spontaneously or upon chemical induction with retinoic acid or dimethylacetamide, the level of nucleolar persistence dropped to under 20%. Mouse embryo fibroblasts and mouse bone marrow cells had few persistent nucleoli. Persistent nucleoli in EC cells contained DNA, labeled with tritiated uridine and stained with ammoniacal silver, all of which support the hypothesis that rRNA synthesis continues in persistent nucleoli.     

Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells

Monoclonal antibodies to mouse epidermal growth factor (EGF) receptor were prepared by the immunization of rats with receptor glycoprotein purified from mouse liver by affinity chromatography on immobilized EGF. Purified mouse EGF receptor retained EGF-inducible autophosphorylating activity and was antigenic in rats and rabbits. The monoclonal antibodies cross react very poorly with human EGF receptor, while polyclonal rabbit antibodies immunoprecipitate human, rat and mouse EGF receptor equally well. The rabbit antibody blocks EGF binding to mouse fibroblast cells and, at 20-fold higher concentrations, stimulates uptake of tritiated thymidine into DNA. This indicates that antibodies bind at or close to the EGF-binding site and can mimic the effects of the growth factor. None of the monoclonals bind at the EGF site of the receptor. Immunoprecipitation, immunoblotting, 125I-EGF cross linking, 125I-surface labelling, immunohistochemistry and autophosphorylation techniques were used to delineate the basis for the induction of EGF receptors when OC15 embryonal carcinoma (EC) cells differentiate into endodermal derivatives (END). EGF-stimulated autophosphorylation of a 170 X 10(3) Mr protein in solubilized OC15 EC cells is readily detectable, although intact EC cells do not bind or respond to EGF by all other tests. The results suggest that cryptic EGF receptors are present in EC stem cells, a finding with implications in development.     

A simplified methodology for purification of peanut (Arachis hypogaea) agglutinin

The agglutinin from peanut (Arachis hypogaea) was readily isolated by affinity chromatography on acid-treated Sepharose 6B. The recovered lectin (50 mg/100 g seeds) appeared as a single band of Mr 32,000 on gel electrophoresis and its specific haemagglutination titre on desialylated human A red blood cells was very high (2(15)).     

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.     

Murine spleen lymphocytes bearing receptors for peanut agglutinin: VII. Separation and functional characterization

Murine spleen lymphocytes having receptors for peanut agglutinin (PNA) were visualized by a specific rosetting technique. The number of lymphocytes forming PNA rosettes (PNA_R⁺) is dependent on the PNA concentration used. T lymphocytes are the primary PNA-rosetting lymphocytes for low PNA concentration (2.5 μg/ml), while both T and B lymphocytes are involved in high PNA concentration (10 μg/ml). Functional studies show that when spleen lymphocytes are separated at a low PNA concentration, the PNA_R⁺ do not respond to T mitogens and suppress antigen-specific immune responses. However, when spleen cells are separated at a high PNA concentration, the PNA_R⁺ do not exhibit differential functional properties as compared to non-PNA-rosetting lymphocytes. The implication of these findings is discussed.     

Characterization of embryonic liver suppressor cells by peanut agglutinin.

Liver cells of 19-day-old mouse embryos were separated by peanut agglutinin (PNA) into two fractions. The fraction agglutinated with the PNA was found to be enriched for cells capable of suppressing the MLC reaction and the response to the mitogens Con A, PHA, and LPS. The fraction not agglutinated by PNA was significantly less suppressive. The response to DxS was not suppressed by any of these fractions. On the other hand, the response to LPS and DxS, but not to Con A or PHA, was expressed by the nonagglutinated fraction. It is thus inferred that the suppressor cells in the embryonic liver are separable from the potentially reactive cells.     

Junction formation in aggregated embryonal carcinoma cells

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.