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.     

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.     

Establishment of a pluripotent embryonal carcinoma cell line not expressing SSEA-1 and ECMA-7 phenotypes

A murine embryonal carcinoma (EC) cell line heterozygous for t0 recessive lethal mutation has been established from an embryo-derived transplantable teratocarcinoma TC1Ph of the genotype (129-T/t0 X C3H/Di)t0/+. The EC cell line, designated EC1Ph, and two cloned sublines, EC1Ph/a and EC1Ph/b, maintain the diploid karyotype (40, XY) and give rise to teratocarcinomas with differentiated derivatives of EC cells after inoculation into syngeneic recipients. The cloned sublines express low or zero amounts of SSEA-1 and ECMA-7 stage-specific antigens. At some passages, the EC1Ph line and the cloned subline EC1Ph/b express a significant quantity of class I H-2 antigens. This unusual EC phenotype resembles that of human teratocarcinoma 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.     

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.     

Retinoic acid-induced changes in differentiation-defective embryonal carcinoma RAC65 cells.

RAC65 is a mutant clone of mouse embryonal carcinoma cells, P19, which does not undergo terminal differentiation upon treatment with retinoic acid (RA). RAC65 cells express a truncated RA receptor alpha (RAR alpha) which, however, does not fully explain their defect. Here we show that RAC65 cells exhibit an additional defect in RAR alpha mRNA which may reflect a defect in RNA splicing. The parental and mutant cells also differ in their capacities to bind [3H]RA into nuclear fractions and in expression of cellular RA binding protein (CRABP) mRNA after treatment with RA. The combined data suggest that the defect in RAC65 RAR alpha results in reduced expression of the CRABP gene after RA treatment and, therefore, increased flow of RA into the nucleus.     

Serological characterization of a pluripotent mouse embryonal stem cell line, two transformed derivatives, and an endoderm-like cell line

The pluripotent mouse embryonal stem cell line BLC 1 and two transformants derived from it by DNA transformation (T1 and T2/K26) as well as the blastocyst-derived cell line BLC 3 were tested for the presence of cell surface antigens recognized by the monoclonal antibodies ECMA-7, anti-SSEA-1 and M 1/22.25, and intermediate filament proteins labeled by the monoclonal antibodies TROMA 1 and TROMA 2 using a three-step indirect immunofluorescence technique. According to present concepts and in agreement with previous data (Wobus et al., 1984a), the results obtained indicate that BLC 1, T1 and T2/K26 are undifferentiated embryonal stem cells, and BLC 3 is an endoderm-like cell line.     

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.     

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.     

Teratocarcinoma X friend erythroleukemia cell hybrids resemble their pluripotent embryonal carcinoma parent.

Five independent clones of somatic cell hybrids have been produced by fusing FBU Friend erythroblastic leukemia cells with cells of the pluripotent teratocarcinoma-derived embryonal carcinoma line PCC4azal. All five lines closely resemble their PCC4azal parent. They look like embryonal carcinoma cells by phase contrast and electron microscopy, have high levels of alkaline phosphatase but low levels of acetylcholinesterase, and, like PCC4azal, express both LDH-A and LDH-B. Tumors produced from hybrid lines often contain large amounts of differentiated tissue, including representatives of all three of the classical germ layers. These results suggest that the genome of a pluripotent mammalian cell, far from being unconditionally susceptible to whatever signals differentiated cells employ to maintain their stable phenotype, may itself be able to “reset” the genome of the differentiated cell.     

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.     

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RARα

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.     

Mode of cell surface marker changes during retinoic acid-induced differentiation in pluripotent embryonal carcinoma cells

Pluripotent teratocarcinoma cell line, 311, was cultured in the presence of retinoic acid (RA) and studied for the processes of early marker changes associated with cell differentiation. The cell populations that have lost peanut agglutinin (PNA), Lotus tetragonolobus agglutinin (LTA) or wheat germ agglutinin (WGA) receptor increased in proportion to the period since the start of RA treatment. The kinetics of the appearance of these receptor-negative cell populations suggests that the differentiating cells lose lectin receptors in the order of PNA, LTA and WGA. However, the changes in F9 antigen(s) and LTA receptor occurred at an equal frequency in PNA+ and PNA- cells, indicating that, although the loss of lectin receptors takes place in a distinct order, the change in each receptor itself proceeds independently of the state of other lectin receptors.     

Commitment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.     

States of developmental commitment of a mouse embryonal carcinoma cell line differentiating along a neural pathway

The embryonal carcinoma cell line PCC7-S-AzaR1 (clone 1009) has been shown to differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP into cells of predominantly neural properties (Paulin, D., H. Jakob, F. Jacob, K. Weber, and M. Osborn. 1982. Differentiation. 22:90-99). By analyzing the marker expression of derivatives in further detail, we characterized the two major cell phenotypes as neuron- and fibroblast-like and the two minor ones as astroglia- and endothelial-like. The stability of developmental commitment of clone 1009 was tested by recloning. The isolated subclones exhibited different patterns of chemically induced derivatives, with some of them (denoted N-clones) producing only a single (neuronal) cell type. As shown by long-term cultures in the absence of retinoic acid, the properties of isolated subclones remained essentially stable. In contrast to the clones producing neuron-like and other derivatives upon induced differentiation, the (exclusively neuronal) derivatives of N-clones detached and died within a few days in culture. If maintained in the presence of other neural cell types, however, their survival was dramatically extended indicating a requirement for specific interactions with other cells of the same tissue. The patterns of derivatives obtained from N-clones depended on the chemical nature of the substrate on which they were grown. Thus, when seeded on laminin-coated surfaces before induced differentiation, N-clones developed not only to neuron-like derivatives but rather to the same four derivatives observed with the original cell pool. These and further results suggest a common cell lineage of the identified phenotypes. The isolated subclones of uninduced cells probably represent different states of commitment within the same developmental pathway. Their stability offers the opportunity to analyze the nature of cellular commitment on the cellular, molecular, and genetic levels. This makes the family of clones derived from PCC7-S-AzaR1 (clone 1009) cells an advantageous in vitro model of mammalian brain early ontogenesis.     

The isolation of a canavanine-resistant mouse embryonal carcinoma cell line which has reduced arginine transport

A mouse embryonal carcinoma cell line resistant to the toxic arginine analogue -canavanine has been isolated. Kinetic studies of the transport of arginine in the canavanine-sensitive parental cell indicate that there are two arginine uptake systems which operate at different substrate concentrations. The canavanine-resistant variant shows a reduction in the rate at which it can transport arginine at all substrate concentrations. This is not, however, due to the complete loss of either uptake system. The observation that the rate of arginine transport at high substrate concentrations is reduced in the variant can be explained, at least in part, by an increase in chromosome number and cell volume. This is not true of the reduction in the low substrate concentration uptake system. The observation that the reductions in the two uptake systems can be dissociated in this way provides support for the conclusion, based on the kinetic data from the parental cell, that there are two independent arginine transport systems in this mouse embryonal carcinoma cell line.     

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.