Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Analysis of a nontumorigenic embryonal carcinoma cell line

Embryonal carcinoma (EC) cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected EC cells with a retrovirus in an effort to obtain by insertional mutagenesis cell lines defective in either differentiative or oncogenic potentials. One such cell line, identified originally by its unique morphological phenotype, is abnormal with respect to both parameters. These cells do not differentiate along typical EC cell lineages, possibly having lost their ability to elaborate endodermal derivatives. They do, however, retain certain cell surface markers characteristic of EC cells and lose these markers after exposure to retinoic acid. Most significantly, they also fail to form tumors in vivo in syngeneic mice, although they grow as well as the parental cells in vitro. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site, suggesting that a single gene may regulate both the tumorigenic and differentiative capacities of the cell.     

alpha-Difluoromethylornithine induces differentiation of a human embryonal carcinoma cell line in vitro

Human embryonal carcinoma cells could serve as a useful model system for analysis of early human development. A limited number of human embryonal carcinoma cell lines have been generated from in vivo tumors. We report here that alpha-difluoromethylornithine, a specific enzyme-activated inhibitor of ornithine decarboxylase activity, can induce differentiation in human embryonal carcinoma cells. The differentiated phenotype could be distinguished from undifferentiated cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the intracellular levels of polyamines may play a role in human embryonal carcinoma cell differentiation, and possibly human embryogenesis.     

A cellular 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line. Purification and characterization

A cellular binding protein for 3,3',5-triiodo-L-thyronine (T3) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from A431 human epidermoid carcinoma cells. The binding activity is T3 specific. Analysis of the equilibrium binding data indicated that the binding protein has one class of binding sites for T3 with a Kd of (17 +/- 3) nM and Bmax of (1.8 +/- 0.6) pmol/50 micrograms of protein. The pH optimum for binding is 6.8. The T3 binding protein elutes from Sephadex G-200 in an included peak which has a Stokes radius of 40 A and sediments on glycerol gradients at 3.7 S. By affinity labeling with [3,5-125I]thyroxine a protein with a molecular weight of 58,000 was specifically labeled. Its isoelectric point was determined to be 7.1, which is different from the reported pIs of other thyroid hormone binding proteins. p58 was successively purified to apparent homogeneity by chromatography on Sephadex G-200, QAE-Sephadex, SP-Sephadex, and hydroxylapatite. Approximately 50 micrograms of purified protein was obtained from 2.5 X 10(9) cells with a yield of 1.1%. The purified protein retains its binding activity. The specific binding activity is enriched by approximately 1000-fold. With the availability of a purified protein with T3 binding activity, it becomes possible to study its cellular function.     

Induced muscle differentiation in an embryonal carcinoma cell line.

Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.     

Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line.

Two glycoproteins, GP-1 and GP-2, have been isolated from an extracellular membrane synthesized in cell culture by an embryonal carcinoma-derived cell line. The amino acid and carbohydrate compositions have been determined. Both proteins are rich in half-cystine residues and contain approximately 12-15% carbohydrate. Antibodies have been obtained against one of the glycoproteins, GP-2, in rabbits. The antibody reacts with basement membranes from adult mouse and human kidney glomeruli and tubules, and all basement membranes tested from mouse embryonic tissues. The molecular properties of GP-2 are superficially similar to LETS protein; however, immunological and other criteria show that they are distinct proteins. The presence of LETS protein and GP-2 in basement membranes suggests that there are subtle interactions which are important in adhesion of epithelial cells to basement membranes.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Purification and partial characterization of high-molecular-weight forms of ectopic calcitonin from a human bronchial carcinoma cell line.

Studies on the high-molecular-weight immunoreactive calcitonin produced ectopically in culture by an epidermoid bronchial carcinoma cell line are reported. In cell-exposed medium, the principal component has a molecular weight of 40000 and molecules of mol.wts. 13000 and 10000 also occur. Only a trace amount of material co-eluting with 35000-mol.wt. human calcitonin is detectable. None of the calcitonins show cross-reactivity with anti-corticotropin serum. The 40000-mol.wt. immunoreactive calcitonin is readily proteolysed to the 13000- and 10000-mol.wt. components, but the 10000-mol.wt. component behaves as a comparatively stable 'core' molecule. By using immunoprecipitation and high-pressure liquid chromatography (h.p.l.c.), it is possible to prepare radiochemically homogeneous 10000-mol.wt. immunoreactive calcitonin from cells grown in the presence of individual 35S- or 3H-labelled amino acids. Peptide mapping of enzymic digests of this material by h.p.l.c. shows that it contains peptides in common with synthetic human calcitonin.     

A differentiation-defective concanavalin-A-resistant variant of a pluripotent embryonal carcinoma cell line

A concanavalin-A(Con A)-resistant variant of the pluripotent mouse embryonal carcinoma cell line, PSA1-NG2, was isolated. This variant, designated NG2-2.16, fails to exhibit the extensive spontaneous differentiation displayed by PSA1-NG2 in colonies in vitro and in tumours in vivo. The molecular nature of the defect in NG2-2.16 cells was not revealed by quantitative studies of the binding, uptake and metabolism of tritiated Con A, or by Western blotting of membrane and whole cell homogenates, thus indicating the defect to be the result of a more subtle molecular alteration. Statistical evidence suggests that the same mutation is responsible for both the Con A resistance and the lack of spontaneous differentiation. NG2-2.16 cells were induced to differentiate by exposure to retinoic acid, suggesting that the mutation affects the regulation of differentiation rather than the potential for differentiation.     

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of M_r 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of M_r 62 000 isolated from lung lavage of patients with alveolar proteinosis.     

Characterization of a glycoprotein isolated from a continuous tumor-cell line of human lung carcinoma

A glycoprotein with a molecular weight of 62 000 has been isolated from a tumor-cell line, A549, and purified to homogeneity by gel chromatography. The glycoprotein contained sialic acid, galactose, mannose, N-acetylglucosamine and a relatively high amount of glutamic acid and proline. The data indicated that the overall composition of this glycoprotein was different from that of the glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis. The glycoprotein did not react with the antiserum raised against glycoprotein of Mr 62 000 isolated from lung lavage of patients with alveolar proteinosis.     

Isolation of a revertant clone from a variant embryonal carcinoma cell line deficient in metabolic co-operation

A revertant clone with restored capacity for metabolic cooperation has been isolated from the cooperation-defective variant embryonal carcinoma cell line R5/3. The properties of the revertant clone provide evidence that deficiencies shown by R5/3 in intercellular transfer of nucleotides and sodium ions are the result of a common genetic lesion which can be dissociated from a secondary lesion causing increased thioguanine resistance and from an increase in ploidy.     

Induction of NILE/L1 glycoprotein during neuronal differentiation of the embryonal carcinoma cell line EC 1003

Abstract. A new clone of the mouse embryonal carcinoma cell line 1003 (EC 1003.16) can be maintained in an undifferentiated state in serum-containing medium. Shifting these cells to serum-free, hormonally defined medium causes them to differentiate morphologically and acquire a number of molecular properties characteristic of neurons. Whereas undifferentiated cells lack the NILE/L1 glycoprotein, expression of this neuronal cell adhesion niolecule is induced in the differentiating cells. Message for NILE/L1 becomes detectable after 5 days in serum-free medium, and cell-surface NILE/L1 can first be seen at this same time. Changes in two other cell adhesion molecules occur in parallel with the induction of NILE/L1. Fibronectin receptor is present on un- differentiated cells, but is down-regulated by the differentiating neurons. The neural cell adhesion molecule (N- CAM) undergoes a shift from the very adhesive adult form to the less adhesive, highly sialylated embryonic form. These changes would appear to emphasize the role of NILE/L1 in adhesive interactions involving differentiating neurons. Some changes in ganglioside expression also occur during EC 1003.16 differentiation. Undifferentiated cells express the D 1.1 ganglioside but lack gangliosides that are reactive with the monoclonal antibody A,B, Differentiating cells lose D 1.1 and become A,B,-positive. Since D 1.1 is characteristic of undifferentiated neuroepithelial cells and A,B, reactivity is a marker for several types of differentiated neurons, these changes in vitro appear to mimic events that occur in vivo.     

Clones defective in metabolic cooperation selected from a pluripotent feeder-dependent mouse embryonal carcinoma cell line

Starting from embryonal carcinoma (e.c.) cells capable of extensive differentiation in culture, the technique of thioguanine 'kiss of death' has been used to select four independent metabolic cooperation-defective variants. The communication ability of these variant cell lines has been quantified by autoradiographic measurement of the transfer of uridine nucleotides, and also by an assay of the extent of junction-mediated rescue from ouabain toxicity by resistant fibroblasts. The cell lines which are defective in ability to transfer nucleotides, as measured by the uridine nucleotide transfer assay, are also defective in their ability to differentiate into endoderm and to form the cavitated 'embryoid bodies' which are produced by the parental cell line when grown in suspension culture. However, it is not clear whether this is related to the defects in metabolic cooperation, since clones which had been subjected to the same selective conditions but which cooperate normally have also lost some of the capacity to undergo this differentiation. Endoderm differentiation was classified into two categories, one being visceral endoderm and the other, primary plus parietal endoderm, on the basis of morphology, immunocytochemical staining for alpha-fetoprotein, and basement membrane formation. With the exception of correlations arising from variations between experiments and differences between cell lines, there is no statistical association between these two categories of differentiation. The formation of cavities was observed only in embryoid bodies with endoderm differentiation: the present of either category was a sufficient condition for cavitation to occur.     

HLA class I homologous transcripts in the human embryonal carcinoma cell line Tera-2

We have used the human teratocarcinoma-derived embryonal carcinoma cell line Tera-2 cl. 13 to explore the putative expression of novel HLA class I(-like) genes. Serological analyses revealed that Tera-2 cells do not express polymorphic HLA class I (-A, -B, -C) specificities, but do express HLA class I-like antigens. These phenotypic properties parallel those of certain mouse embryonal carcinoma cells. To study the expression of HLA class I(-like) genes in the Tera-2 cells two different approaches were used. Screening of a Tera-2 cDNA library with a full-length HLA class I cDNA probe under conditions that would allow for the identification of relatively distinct HLA class I-like sequences yielded 27 positive clones, all of which were of the regular HLA-A, -B, -C type. Reverse northern hybridizations of the restriction enzyme-digested Tlab region comprising cosmids with Tera-2 cDNA as the probe resulted in the identification of several putative human genes whose equivalents map within the mouse Tla region. However, none of these genes appeared to be structurally related to HLA class I. A putative H3.3 histone gene was identified in the proximal Tla region of the C57BL/10 mouse. It is concluded that no structural homologues of mouse Qa/Tla genes are expressed in the human developmental cell line Tera-2.     

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

Elevated levels of glycoprotein gp200 in progeria fibroblasts

The glycosylation of proteins in fibroblasts from people with the premature ageing disease Hutchinson-Gilford Progeria Syndrome (progeria) was investigated.Protein was prepared from fibroblast cell lines established from skin biopsy taken from progeria patients and control donors. Glycoproteins were labelled by the covalent attachment of the steroid hapten digoxygenin to the sugar group. After separation of total protein by SDS-PAGE and electroblotting onto Immobilon-P, glycoproteins were detected by enzyme immunoassay.We have observed a glycoprotein of M_r 200 kDa which is consistently present in protein preparations from progeria fibroblasts and which is absent, or markedly reduced, in preparations from control fibroblasts. This suggests that it may be useful as a marker for progeria. Similar analysis of progeria lymphoblast and control lymphoblast cultures did not show this altered pattern of glycosylated proteins, indicating that it may be cell-type specific.Glycoproteins were also detected by labelling fibroblastsin vitro with D-[6-³H] glucosamine hydrochloride followed by SDS-PAGE of isolated protein and subsequent fluorography. Profiles of glycoproteins from progeria and control fibroblasts were consistent with those obtained from labelling of carbohydrate groups with digoxygenin. Protease digestion of cell protein verified that the band at M_r 200 kDa contains a protein core.Characteristic features of progeria primarily involve the connective tissue and include wrinkled and loose skin, loss of soft tissue, thin limbs and stiff joints. Death of progeria patients is usually a result of cardiovascular abnormalities. The most consistent manifestations thus involve the connective tissue.The glycoprotein of M_r 200 kDa which we have observed in progeria fibroblastsin vitro could reflect a perturbation in glycosylation which may underly the connective tissue defects seen in progeria.Abbreviations EMEM Eagle's Minimum Essential Medium with Earle's Salts - FCS Fetal Calf Serum - PBS Ca²⁺- and Mg²⁺-free Phosphate-Buffered Saline - PDL Population Doubling Level - SDS Sodium Dodecyl Sulphate - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis     

Differentiation potential of human embryonal carcinoma cell lines

We have established 17 embryonal carcinoma (EC) cell lines from human testicular germ cell tumors by using three different methods of in vitro cultivation. Cultures of only three of these cell lines, and of clones derived from two of them, differentiate extensively when the cells are seeded at low density. A comparison is presented of the results obtained with the three methods used to establish and maintain these cell lines, and some properties of the three pluripotential EC lines are summarized.     

Characterization of human embryonal carcinoma cell lines derived from testicular germ-cell tumors

Four human embryonal carcinoma (EC) cell lines (ITO, NEC 8, NEC 14, NEC 15) derived independently from testicular germ-cell tumors were established in vitro. In their xenografted tumor tissues, all of them exhibited histological characteristics consistent with EC. The cell-biological characterization of these human EC cell lines was investigated with reference to well-known murine EC cell lines. This included examination of their morphology, growth, tumorigenic potential, karyotype, cell-aggregate formation, HLA expression, large glycopeptides, AFP and HCG production, plasminogen-activator secretion, and LDH profiles. Three (ITO, NEC 14, NEC 15) of these human EC cell lines shared cell-biological characteristics consistent with typical EC, but one of them (NEC 8) differed from the others with respect to its rapid growth, high tumorigenic potential, formation of solid cell aggregates, and less differentiated, solid histological pattern. Thus, it is suggested that there are several developmentally different types of human EC cells. The relationship between the properties of these human EC cell lines and their differentiation potential is discussed.