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At least 19 recordsLinked to original sources

PubMed1987

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas.

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.

Animals↗

PubMed1995

Identification of a transposon-related RNA down-regulated by retinoic acid in embryonal carcinoma and embryonic stem cells.

The differential display polymerase chain reaction was employed to identify changes in mRNA expression during retinoic acid-induced differentiation in embryonal carcinoma PCC4.aza1R cells. In this study, we report on one cDNA, EC1, that was identified by this method. EC-1 encodes a 0.6-kb mRNA that is present in PCC4.aza1R cells and down-regulated by retinoic acid. Sequence analysis revealed that EC-1 exhibits a 47% identity with the early transposon RNA ETn and does not contain a long open reading frame. EC-1 mRNA expression was reduced by 50% after 24 h of treatment with 10 nM retinoic acid and was undetectable after 48 h. Down-regulation of EC-1 mRNA was observed at retinoic acid concentrations as low as 0.1 nM. EC-1 was found to be expressed in several other embryonal carcinoma cell lines as well as in embryonic stem cells but was undetectable in differentiated cell types obtained after RA treatment. Northern blot analysis using RNA from multiple mouse tissues demonstrated that the expression of EC-1 is restricted to the testis. Treatment of PCC4.aza1R cells with an RAR-selective agonist also repressed the expression of EC-1 mRNA while treatment with an RXR-selective agonist reduced EC-1 expression slightly. The RAR alpha-specific antagonist Ro 41-5253 had little effect on the down-regulation of EC-1 by retinoic acid. Our observations indicate that the repression of EC-1 is associated with the induction of differentiation in embryonal carcinoma and embryonic stem cells and involves an RAR-activated signaling pathway.

Animals↗

PubMed1997

Differential regulation of the Wilms' tumor gene, WT1, during differentiation of embryonal carcinoma and embryonic stem cells.

The expression pattern of the Wilms' tumor suppressor gene, WT1, during embryonal development suggests a role for the WT1 proteins in the differentiation of specific tissues. This notion is supported by the observation that WT1 knock-out mice fall to develop kidneys and gonads. We describe here the changes in the expression and DNA binding activity of the WT1 gene product in P19 embryonal carcinoma cells and embryonic stem cells triggered to differentiate by either retinoic acid (RA) or DMSO. In exponentially growing P19 embryonal carcinoma (EC) cells, WT1 mRNA and proteins were undetectable. During RA-induced but not DMSO-induced differentiation of P19 EC cells, WT1 expression and DNA binding are strongly activated. Treatment of embryonic stem cells with RA resulted in a similar activation of WT1. Immunohistochemical analysis showed that WT1 is expressed in endodermal, glial, and epithelial cell types. In addition, DNA binding by EGR-1, a transcription factor structurally related to WT1, increased during differentiation of P19 EC and embryonic stem cells. To investigate the possible functional consequences of DNA binding by WT1, we examined the expression levels of two putative transcriptional targets of WT1, the insulin-like growth factor 1 receptor and epidermal growth factor receptor. We found that after an initial induction, decreasing expression of the insulin-like growth factor I receptor is correlated with increasing WT1 expression. Our results demonstrate that expression of WT1 is induced in specific cell types during RA-induced differentiation of P19 EC cells, reflecting the tissue-specific expression of WT1 in vivo. Therefore, we believe that P19 EC cells are a suitable system to study activation and function of WT1 during differentiation.

Blotting, Western↗

PubMed1991

Octamer-dependent regulation of the kFGF gene in embryonal carcinoma and embryonic stem cells.

Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors.

Animals↗

PubMed1995

Induction of a hyaluronan receptor, CD44, during embryonal carcinoma and embryonic stem cell differentiation.

This paper describes the expression profile of the CD44 glycoprotein during differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. We have recently shown that CD44 is expressed in discrete embryonic structures and, in view of this, we sought an in vitro differentiation model of development in which we could study more readily the structure and function of the CD44 molecule. The P19 EC and CGR8 ES cells were chosen as they have the capacity to develop down the cardiac muscle pathway and we have previously demonstrated that CD44 is expressed abundantly in the embryonic myocardium. The differentiation process in both cell types is accompanied by an induction of CD44 mRNA and protein. However, in differentiated cultures CD44 is not expressed in contractile cells, indicating that these P19 cells do not represent CD44-positive embryonic cardiomyocytes. Expression of CD44 is observed on fibroblast-like cells which appear to migrate over and out from the plated aggregates. Hyaluronan, the major ligand for CD44, is also associated with these CD44-positive fibroblast-like cells. It is suggested that expression of both receptor and ligand by the fibroblast cells is required for cell:matrix adhesion and cell motility. As CD44 is up-regulated in these cultures, P19 cells are now established as a useful model system to study the factors regulating expression of the CD44 gene.

Alternative Splicing↗

PubMed1993

Fibroblast growth factor-mediated growth regulation and receptor expression in embryonal carcinoma and embryonic stem cells and human germ cell tumours.

FGFs have been implicated in the induction of mesoderm in amphibian development and are present in the mouse embryo at stages that would be appropriate for a similar function in mammals. Primitive ectoderm would then be the target tissue. We have now changes in the expression of receptors for FGFs during the differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells from the mouse. These cells resemble those of the inner cell mass and later primitive ectoderm. On Northern blots of mRNA from undifferentiated cells, transcripts for FGF R1, R2 and R3 are expressed. All are upregulated during differentiation of ES cells and are upregulated or remain constant as EC cells differentiate. FGF R4 is only expressed after differentiation to derivatives resembling parietal endoderm. By contrast in human EC cells, FGF R2 is downregulated during differentiation, FGF R1 and FGF R3 are unchanged and FGF R4 is expressed before and after differentiation. In both human and mouse EC cells three members of the FGF family (a FGF, b FGF and k FGF, also known as FGFs 1,2 and 4) are mitogenic in serum-free medium and one (KGF or FGF 7) appears to have no effect on growth although cellular morphology is altered. Differences between human and mouse cells are primarily in the effects of heparin on the FGF-induced response.

Animals↗

PubMed1989

Expression of multiple heparin-binding growth factor species by murine embryonal carcinoma and embryonic stem cells.

Culture medium conditioned by P19 embryonal carcinoma (EC) cells contains potent mitogenic activity which is markedly potentiated when the medium is conditioned in the presence of heparin. Fractionation of P19 medium conditioned in the presence of heparin reveals the existence of two biochemically distinct growth factor species both of which exhibit high affinity for immobilised heparin and significant activity as amphibian mesoderm-inducing agents. One of the species is recovered as a single polypeptide of apparent Mr = 15,000. This molecule is immunologically related to the protein product of the human K-FGF proto-oncogene. Transcripts derived from the murine K-FGF gene are also expressed by both differentiated and undifferentiated EC cells and embryonic stem cells. The second heparin-binding growth factor is recovered as a complex of four polypeptides, the largest of which has an apparent Mr = 17,000. This agent is immunologically and biochemically distinct from both acidic and basic fibroblast growth factor as well as K-FGF, and represents the predominant mitogenic activity in EC-cell-conditioned medium.

Animals↗

PubMed1987

Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.

Many pluripotent embryonal carcinoma (EC) cell lines and all embryonic stem (ES) cell lines have hitherto been maintained in the undifferentiated state only by culture on feeder layers of mitomycin C-treated embryonic fibroblasts. We now demonstrate that medium conditioned by incubation with Buffalo rat liver (BRL) cells prevents the spontaneous differentiation of such cells which occurs when they are plated in the absence of feeders. This effect is not mediated via cell selection but represents a fully reversible inhibitory action ascribed to a differentiation-inhibiting activity (DIA). BRL-conditioned medium can therefore replace feeders in the propagation of homogeneous stem cell populations. Such medium also restricts differentiation in embryoid bodies formed via aggregation of EC cells and partially inhibits retinoic acid-induced differentiation. The PSA4 EC line gives rise only to extraembryonic endoderm-like cells when aggregated or exposed to retinoic acid in BRL-conditioned medium. This suggests that DIA may be lineage-specific. DIA is a dialysable, acid-stable entity of apparent molecular weight 20,000-35,000. Its actions are reproduced neither by insulin-like growth factor-II nor by transforming growth factor-beta. DIA thus appears to be a novel factor exerting a negative control over embryonic stem cell differentiation.

Animals↗

PubMed1990

Expression of transforming growth factor beta 2 during the differentiation of murine embryonal carcinoma and embryonic stem cells.

Transforming growth factor beta 2 (TGF beta 2) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF beta 2 transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, P10, and ES. Following induction of differentiation, however, TGF beta 2 became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF beta 2 transcripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF beta 2 did not stain P19 EC cells by immunofluorescence but 2-3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF beta 2, while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF beta 2 after plating on a substrate. Neither TGF beta 1 nor TGF beta 2 affected the growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF beta 2 being marginally more effective than TGF beta 1 at the same concentration.

Animals↗

PubMed1991

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos.

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

Animals↗

PubMed1992

The small heat shock protein hsp25 is accumulated in P19 embryonal carcinoma cells and embryonic stem cells of line BLC6 during differentiation.

Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.

Animals↗

PubMed1996

Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells.

A number of different cell lines that exhibit a partial neuronal phenotype have been identified, but in many cases the full extent of their neuronal differentiation has not been directly addressed by functional studies. We have used electrophysiology and immunofluorescence to examine the formation of synapses and the development of neuronal polarity by murine embryonic stem (ES) cells and the mouse P19 embryonic carcinoma cell line. Within 2-3 weeks after induction by retinoic acid, subsets of P19 and ES cells formed excitatory synapses, mediated by glutamate receptors, or inhibitory synapses, mediated by receptors for GABA or glycine. In ES-cell cultures, both NMDA and non-NMDA receptors contributed to the excitatory postsynaptic response. Staining with antibodies to growth-associated protein-43 and microtubule-associated protein-2 revealed segregation of immunoreactivity into separate axonal and somato-dendritic compartments, respectively. Consistent with our physiological evidence for synapse formation, intense punctate staining was observed with antibodies to the synaptic vesicle proteins synapsin, SV2, and synaptophysin. These results demonstrate the in vitro acquisition by pluri-potent cell lines of neuronal polarity and functional synaptic transmission that is characteristic of CNS neurons.

Action Potentials↗

PubMed1993

Type beta transforming growth factors and activins in differentiating embryonal carcinoma cells, embryonic stem cells and early embryonic development.

TGF beta was originally identified on the basis of its ability to induce phenotypic transformation of non-transformed target cells while activin was discovered as a gonadal protein. They later turned out to be related and both to have possibly crucial roles in the regulation of embryonic development. Here we review the circumstantial and direct evidence for this in the context of our own studies on their expression in and effects on murine EC and ES cells and mouse embryos. Their possible interaction in development is discussed.

Activin Receptors↗

PubMed1989

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.

Animals↗

PubMed1987

The distribution of two cell surface determinants of mouse embryonal carcinoma and early embryonic cells.

This study examines the distribution of two carbohydrate determinants recognized by monoclonal antibodies EMA-1 and EMA-6 on cells in culture, adult mouse tissues and day 0-7 embryos, and provides partial biochemical characterization of the molecules carrying the determinants for EMA-1 on embryonal carcinoma cells. Both antigens are present on primordial germ cells of day 8-day 10 mouse embryos (Hahnel and Eddy, 1986), and are expressed recurrently on pluripotent cells during earlier embryogenesis. Both antigens are also present on mouse embryonal carcinoma cells and the same set of adult tissue types. However, the distribution of the EMA-6 determinant on adult urogenital epithelia and during embryogenesis is more restricted than that of EMA-1. The determinants are detected only on lumenal surfaces of epithelial cells, suggesting that they are either not involved in processes of cell-cell or cell-substrate interaction, or are masked or altered during such interactions. Present biochemical evidence suggests that EMA-1 determinant on Nulli SCC1 cells may be on a large glycoprotein or a glycoprotein complex that is firmly attached to the cell membrane.

Animals↗

PubMed1979

Junctional modulation in mouse embryonal carcinoma cells by Fab fragments of rabbit anti-embryonal carcinoma cell serum.

Mouse embryonal carcinoma PCC4 cells are connected by extensive gap and tight junctions. When the cells are incubated in a medium containing Fab fragments against embryonal carcinoma F9 cells, they round up and a process of junctional removal is initiated. In particular, gap junctions are internalized and after 30 hr of incubation with the anti-F9 Fab fragments both tight and gap junctions are no longer present at the cell surface; however, the cells are still in contact by means of small attachment sites.

Animals↗

PubMed1988

Characterization of neurotransmitter phenotype during neuronal differentiation of embryonal carcinoma cells.

Embryonal carcinoma cells are useful in the study of embryogenesis and development, and their differentiation into neurons serves as a model of neuronal development. Retinoic acid was used to differentiate P19S18O1A1 embryonal carcinoma cells into neuronal, glial, and fibroblast-like cells and the phenotype of the neuronal population was examined. Neuron-specific enolase was present in the neuronal cells, suggesting that these neurons had reached some degree of maturity. A population (approximately 70%) of the neurons showed positive immunocytochemistry for tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, three enzymes in the pathway of catecholamine synthesis. Therefore a population of the neurons appeared to be adrenergic. These neurons also showed a low level of histofluorescence for endogenous catecholamines and exhibited an exogenous catecholamine reuptake system. In order to determine the phenotype of other neuron-like cells found to be negative for the adrenergic properties examined, immunocytochemistry for neuropeptides and neurotransmitters known to coexist within central neurons was performed. Serotonin, vasoactive intestinal peptide, glutamic acid decarboxylase, and choline acetyltransferase were all absent from retinoic acid-treated P19S18O1A1 neuronal cultures. These studies, along with those that compare the effects of retinoic acid and other growth modulators on neuronal differentiation of embryonal carcinoma cells, should aid in the understanding of neuronal induction and development in vivo.

Animals↗

PubMed1994

Expression of retinoid X receptors in P19 embryonal carcinoma cells and embryonic stem cells.

The pluripotent mouse embryonal carcinoma cell line P19 provides an excellent model system to study the mechanisms by which retinoic acid (RA) exerts its biological effects. When aggregated and exposed to low concentrations of RA, P19 cells differentiate into neuron- and glial-like cells. The diverse biological effects of RA are mediated by two families of receptors localized in the cell nucleus, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Each family consists of three members designated alpha, beta and gamma. While the patterns of expression of the RARs have been studied in P19 cells, similar data for the RXRs have not been available. We demonstrate here that these receptors are expressed in P19 and are regulated during RA-induced differentiation. We also show that pluripotent mouse embryonic stem cells express the RXRs as well.

Animals↗