Flow Cytoenzymology of the Early Differentiation of Mouse Embryonal Carcinoma Cells

Dual parameter flow cytoenzymology was used to detect biochemical differentiation of embryonal carcinoma cells, the undifferentiated, multipotent stem cells of teratocarcinomas. With the use of fluorogenic substrates, two enzyme systems, alkaline phosphatase (EC 3.1.3.1.) and carboxyl esterase (EC 3.1.1.), were studied. Embryonal carcinoma cells passaged in vitro for several years retained high alkaline phosphatase activities similar to those of embryonal carcinoma cells in embryoid bodies grown in vivo. Similar to the embryonal carcinoma cells in vivo, the in vitro embryonal carcinoma cells were capable of giving rise to progeny with greatly decreased levels of alkaline phosphatase. The embryonal carcinoma cell alkaline phosphatase was inhibited by l-p-bromotetramisole, suggesting a relationship between this enzyme and somatic, nonintestinal alkaline phosphatase isoenzymes. Determinations of esterase activities in viable teratocarcinoma cells showed that prior to any evidence of morphologic differentiation, the embryonal carcinoma cells are quite heterogeneous with regard to esterase activities.     

Saccharides on teratocarcinoma cell plasma membranes their investigation with radioactively labelled lectins

We have studied the interaction of five lectins differing in their sugar specificity, with the surface of clonal cell lines derived from transplantable murine teratocarcinoma. The results show that the differentiation from primitive embryonal carcinoma cells into parietal yolk sac cells is accompanied by changes in cell surface saccharides. These changes consist of a marked decrease in the total number of binding sites for the l-fucose-specific lectin of Lotus tetragonolobus and a large increase in the total number of binding sites for wax bean agglutinin. It is suggested that these differences can be used as markers in the study of this early embryonic differentiation. No agglutination of primitive embryonal carcinoma cells or of parietal yolk sac cells by low concentrations (10 μg/ml) of concanavalin A, soybean agglutinin or the fucose binding proteins was observed.     

Inhibition of ornithine decarboxylase induces embryonal carcinoma cell differentiation

Murine embryonal carcinoma cells can be induced to differentiate in vitro by various physical and chemical means. We report here that inhibition of ornithine decarboxylase activity with a specific enzyme-activated inhibitor, alpha-difluoromethylornithine, can induce differentiation in embryonal carcinoma cells. The differentiated phenotype can be distinguished from undifferentiated embryonal carcinoma cells by altered cellular morphology, biochemical and cell surface antigenic properties. These results suggest that alterations in the levels of cellular polyamines may play a role in embryonal carcinoma cell differentiation.     

Transforming growth factor-β and its receptor are differentially regulated in human embryonal carcinoma cells

The human embryonal carcinoma cell lines Tera-2 clone 13 and NTera-2 clone D1 can be induced by retinoic acid to differentiate in vitro into neuroectodermal derivatives. The undifferentiated cells are rapidly proliferating and tumorigenic, whereas retinoic-acid-treated cells possess a decreased growth rate, lose their transformed phenotype and show a finite lifespan. Differentiation is accompanied by a marked increase in the levels of mRNA for TGF-beta 1 and TGF-beta 2 and the production of TGF-beta activity. Just like murine embryonal carcinoma cells the growth of Tera-2 clone 13 cells is not affected by the addition of either TGF-beta 1 or TGF-beta 2 to the culture medium. In contrast to published data on murine embryonal carcinoma cells, Tera-2 clone 13 and NTera-2 clone D1 cells bind TGF-beta 1 with high affinity, which is due to the presence of type-III TGF-beta receptors. Furthermore, and again in contrast to murine embryonal carcinoma cells, treatment of the human embryonal carcinoma cells with retinoic acid causes a nearly complete loss of TGF-beta 1 binding sites. These results are discussed in the light of similarities and differences in the regulation of growth and differentiation of human and murine embryonal carcinoma cell lines.     

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.     

Induction of F9 embryonal carcinoma cell differentiation by inhibition of polyamine synthesis

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

A Reproducible Colonial Morphology Formed by Individual Pluripotent Embryonal Carcinoma Cells in Culture and Selection of Variants

Embryonal carcinoma cells are stem cells equivalent to those of the early embryo which can be grown in vitro and which under certain conditions can differentiate into many cell types. Events in this differentiation process are numerous and complex, thus a system for the analysis of clonal differentiation is essential. In this paper I report that individual pluripotent embryonal carcinoma cells can each give rise to colonies, in the absence of a feeder layer but in the presence of β-mercaptoethanol, that show a distinctive and reproducible gross morphology. Embryonal carcinoma cell lines can be derived from the stem cells in these colonies. Furthermore, variant cell lines can be derived from those colonies showing an altered gross morphology. These lines when cloned as above give rise to colonies showing a gross colonial morphology different to that of wild-type. These variant lines have been shown to be embryonal carcinoma cell lines. These findings indicate that genetic and cell lineage analysis of embryonal carcinoma cell differentiation might be possible.     

Stimulation of the Clonal Growth and Differentiation of Feeder Layer Dependent Mouse Embryonal Carcinoma Cells by β-Mercaptoethanol

Embryonal carcinoma cells are the undetermined stem cells of teratocarcinomas. Supplementation of culture medium with β-mercaptoethanol permits the feeder layer independent clonal growth and differentiation of normally feeder layer dependent embryonal carcinoma cell lines. Differentiated cells within the clones appeared less than 6 days after plating and were distinguished from embryonal carcinoma cells by their morphology, lack of histochemically detectable alkaline phosphatase activity, and secretion of plasminogen activator. Over 70% of the colonies secreted plasminogen activator after 6 days.
In comparison, a different embryonal carcinoma cell line which has lost the potential for substantial differentiation, either in vitro or in vivo forms very few clones (< 1%) which secrete plasminogen activator. Embryonal carcinoma cells derived from the rare clones which secrete plasminogen activator have the same frequency of production of plasminogen activator secreting colonies as the parental cell line.     

A Putative G-Protein-Coupled Receptor, H218, Is Down-regulated during the Retinoic Acid-Induced Differentiation of F9 Embryonal Carcinoma Cells

We have previously cloned a novel guanine nucleotide-binding protein (G-protein)-coupled receptor, H218, that has sequence similarity to a lysophosphatidic acid receptor, edg2. We present here Northern analysis indicating that the H218 mRNA is expressed in undifferentiated F9 embryonal carcinoma cells. The H218 message is down-regulated and its stability is decreased during retinoic acid- and dibutyryl cAMP-induced differentiation. Treatment by various receptor-selective retinoids indicated that retinoic acid receptor β or γ signaling, but not retinoid X receptor activation, is required for the down-regulation of H218 mRNA. Activation of the H218 receptor may contribute to the phenotype of undifferentiated F9 embryonal carcinoma cells.     

Effects of differentiation of embryonal carcinoma cells (P19) on mitochondrial DNA content in vitro

Summary The embryonal carcinoma cell line P19 is derived from mouse teratocarcinomas. These pluripotent cells can be induced to differentiate into a variety of cell types by exposure to various drugs. We used retinoic acid to induce embryonal carcinoma cells to differentiate into neuronlike cells. In this study, we show that changes occur in mitochondria during differentiation of embryonal carcinoma cells to neuronlike cells. We found that various morphologic parameters such as mitochondrial fractional area and mitochondrial size decrease as embryonal carcinoma cells differentiate into neuronlike cells. Similar changes were also observed in mitochondrial DNA content. Stereologic analysis of cell preparations provided a measure of mitochondrial fractional area per cell and mtDNA content was assessed by radiolabeled mtDNA probe. This study establishes that mitochondria are regulated as cells differentiate. This study was financially supported by the Medical Research Council of Canada.     

Alterations in the developmental potential of embryonal carcinoma cells in mixed aggregates of nullipotent and pluripotent cells.

Pluripotent embryonal carcinoma cells can be triggered to differentiate in vitro by allowing them to form multicellular aggregates. Nullipotent embryonal carcinoma cells form aggregates, but further development is blocked. Pluripotent and nullipotent embryonal carcinoma cell lines were co-cultured to form mixed aggregates in order to determine whether a developmental signal produced by the pluripotent cell could induce the nullipotent cells to differentiate. Unlike pure pluripotent cell aggregates, aggregates from cultures initiated with a 1:1 mixture of pluripotent (PSA-1) and nullipotent (F9) cells formed endoderm but failed to differentiate further. The nullipotent cells did not produce a detectable soluble inhibitor of differentiation. A hypoxanthine phosphoribosyltransferase-deficient subclone of the nullipotent cell line was used so that the fate of both nullipotent and pluripotent cells could be followed in autoradiographs of histological sections of aggregates labeled with ³H-hypoxanthine. Seven day old aggregates of pure pluripotent cell cultures contained endoderm, ectoderm and embryonal carcinoma cells. On the other hand, in 7 day old mixed cell aggregates, almost all the pluripotent cells became endoderm located on the outer surface of the aggregate. The nullipotent cells in the mixed aggregates assumed an internal position and remained embryonal carcinoma cells. Following the efficiency of plating of pluripotential cells in pure and mixed aggregates as a function of time showed that viable pluripotent embryonal carcinoma cells were lost at a 10 fold greater rate in mixed cell aggregates than in pure pluripotent cell aggregates. We conclude that nullipotent embryonal carcinoma cells in mixed aggregates with pluripotent cells exert a limitation on the ability of these pluripotent cells to differentiate.     

Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development

The patterns of protein synthesis in teratocarcinoma stem cells (embryonal carcinoma cells) and in mouse embryos at various stages of preimplantation development were studied using SDS-polyacrylamide slab gel electrophoresis with autoradiography. Significant differences were observed in comparisons of embryonal carcinoma cells with isolated inner cell masses (ICMs) or with embryonic cells at earlier stages of development. However, no such differences in the overall pattern of protein synthesis were found when the embryonal carcinoma cells were compared with the embryonic ectoderm (that portion of the ICM which remains after endoderm differentiation). Both synthesize at least one prominent 55,000-dalton protein that is not detected in embryonic cells at earlier stages of development. This protein can thus be used as a biochemical marker of ectoderm formation during embryonic development. The pattern of protein synthesis common to embryonal carcinoma cells and embryonic ectoderm is not shared by other cultured cell types.     

Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.     

Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells

Embryonal carcinoma cells are pluripotent stem cells derived from teratocarcinomas and are considered to be the malignant counterparts of human embryonic stem cells. As there are few reliable experimental systems available to study the molecular mechanisms governing normal embryogenesis, well-characterized human embryonal carcinoma stem cell lines may provide a robust and simple model to study certain aspects of pluripotency and cellular differentiation. Here, we have analysed NTERA-2 cL.D1 cells at molecular and cellular levels during expansion and differentiation, via formation of cell aggregates similar to embryoid bodies in embryonic stem cells. Thus, human embryonal carcinoma cells may provide a valuable insight into cell fate determination, into the embryonic ectoderm, mesoderm and endoderm and their downstream derivatives.     

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.