The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

SSEA-1, a stage-specific embryonic antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells

Molecules carrying SSEA-1 were isolated from [3H]galactose-labeled embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. The antigenic molecules were degraded by extensive pronase digestion or mild alkaline treatment, and the majority of the products thus formed were so large as to be excluded from a column of Sephadex G-50. Therefore, the major carbohydrate constituent of the antigenic molecule was embryoglycan, the glycoprotein-bound large glycan in early embryonic cells. Furthermore, the binding of isolated embryoglycan with anti-SSEA-1 was directly shown by a modified Farr's assay. From these results we concluded that SSEA-1 determinant was carried by the large glycan.     

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.     

High-mannose glycopeptides from embryonal carcinoma cells

Endo-beta-N-acetylglucosaminidase H released four major oligosaccharides from high-mannose glycopeptides prepared from embryonal carcinoma cells. The oligosacchaides were indistinguishable from (Man)9GlcNAc, (Man)8GlcNAc, (Man)7GlcNAc, and (Man)6GlcNAc isolated from fibroblasts. This result suggests that the biosynthetic pathway of asparagine-linked oligosaccharides in early embryonic cells is controlled as in adult cells, at least to the initial stage of processing of the nascent oligosaccharide transferred from lipid-linked intermediate.     

Derivation of a nondifferentiating clone from multipotential PSA1 embryonal carcinoma cells

The ability of PSA1 embryonal carcinoma cells to differentiate when grown as clones on a monolayer of feeder cells was assessed using morphological criteria. The first appearance of a differentiated phenotype within a clone occurred at different times for individual clones after 10 days of culture, this being apparently unrelated to clone size or cell density. Those clones which showed no morphological evidence of differentiation after several weeks (about 5% of the clones observed) were selected and recloned with the aim of finding variant lines which were stably deficient in their differentiating ability. Undifferentiated clones - identified and selected after about 3 weeks of growth - were of three different types after recloning: those similar to control cultures of PSA1, those having delayed and reduced differentiation frequency, and those having variable frequencies of differentiation in replicate reclonings. The isolation of a variant with a more complete differentiation deficiency was accomplished by selecting ten nondifferentiating clones growing isolated in individual culture wells after 5 weeks of culture. One of these, T2H9, proved to be a stable, differentiation-deficient variant subline with less than 3% of its clones showing any morphological evidence of differentiation in five repeated reclonings. It was also determined that the frequency of undifferentiated clones in embryonal carcinoma cultures increased from 0.3% to 54% after 11 months of in vitro aging, i.e., approximately 200 cell doublings. The isolation of clonal embryonal carcinoma cell derivatives which are stable, heritable differentiation variants provides resources for somatic-cell genetic analysis of stem-cell pluripotency.     

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.     

Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

Purification and properties of N-acetylglucosaminide alpha 1----3-fucosyltransferase from embryonal carcinoma cells

A membrane-bound alpha-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on GDP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65 000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to alpha-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of beta-galactoside alpha 1----2-fucosyltransferase and N-acetylglucosaminide alpha 1----4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above.     

Purification and properties of N-acetylglucosaminide beta 1----4 galactosyltransferase from embryonal carcinoma cells

N-Acetylglucosaminide beta 1----4 galactosyltransferase was chromatographically purified about 1,700-fold from F9 embryonal carcinoma cells after solubilization with Triton X-100, using N-acetylglucosamine as the acceptor. As the last step of the purification, affinity chromatography was performed either on N-acetylglucosamine-Sepharose or on alpha-lactalbumin-Sepharose: in both cases, two protein bands with molecular weights of around 68,000 and 59,000 were detected by SDS-polyacrylamide gel electrophoresis of the purified preparations. The enzymological properties including behavior toward alpha-lactalbumin were very similar to those of the enzyme from other sources. The specificity of the enzyme was confirmed by determining the structure of the product; it was mostly Gal beta 1----4GlcNAc. beta-Galactosidase-treated embryoglycan (poly-N-acetyllactosamine) and asialo-agalactofetuin could serve as acceptors with the purified enzyme. Thus, the embryonic enzyme, apparently involved in the synthesis of poly-N-acetyllactosamines, has properties similar in several respects to those of the beta-galactosyltransferases so far studied.     

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.     

Isolation of metabolic cooperation-defective variants from mouse embryonal carcinoma cells

We describe the isolation from the HGPRT^? embryonal carcinoma cell line PC13TG8 of a variant, R5/3, defective in metabolic cooperation. This was achieved in two stages via an intermediate, R2/1, using a selective system in which the HGPRT^? embryonal carcinoma cells were co-cultured with HGPRT⁺ cells in 6-thioguanine. R5/3 cells show increased survival compared with PC13TG8 when retested under selection conditions, and a reduction in grain count index when tested by autoradiography as recipients of [³H]hypoxanthine-labelled nucleotides from wild-type donors and as donors of [³H]thymidine- and [³H]adenine-labelled nucleotides to suitably marked recipients. However, a low residual fraction of heavily labelled recipients is found in all autoradiographic experiments with R5/3 cells. This is not due to heterogeneity of either donor or recipient populations. We also describe the development of a colony-formation assay for metabolic cooperation based on the “kiss of life” phenomenon, in which R5/3 shows very poor survival compared with PC13TG8. R2/1 shows behaviour intermediate between PC13TG8 and R5/3 in all the tests described above. We conclude that two steps can be identified in the change of phenotype by which R5/3 is derived from PC13TG8, and that both steps modify the ability of the cells to form permeable junctions.     

Estimation of liposome integrity by 1H-NMR spectroscopy

A fast and simple method of 1H-NMR spectroscopic control of liposome membrane integrity is suggested. The method is based on the redistribution of intensities between two singlet 1H-NMR signals--from intraliposomal marker compound (nitrilotriacetic acid sodium salt, 1H-NMR signal at 4 p.p.m.) and from its complex with Eu3+ added to the external medium (NMR signal of the complex at - 1 p.p.m.). The method permits registration of the kinetics of liposome destruction under the action of detergent or serum. It is shown that the presence of cholesterol in the membrane makes it more stable.     

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.     

Differentiation of F9 embryonal carcinoma cells

We found that monolayer cultures of F9 cells induced to differentiate with trans-retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step.     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.     

Metabolism of retinoids by embryonal carcinoma cells

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.