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The authors present diagnostic and therapeutic documentation of a very rare case of a tumour with carcinoma embryonal in a 16-year old girl. After the surgery (total hysterectomy and adnexectomy) followed by chemotherapy, the patient survived four years.
From embryonal carcinoma cells labeled with fucose, two main classes of glycopeptide products of Pronase digestion can be distinguished by Sephadex G-50 column chromatography: one eluted near the excluded volume and a smaller one. The large fucosyl-glycopeptides are scarcely present in differentiated cells derived from embryonal carcinoma cells (i.e., fibroblastlike cells, myoblasts, and parietal yolk-sac carcinoma). During in vitro differentiation of embryonal carcinoma cells, these large glycopeptides disappear almost completely. The small glycopeptides were analyzed by paper electrophoresis, concanavalin A-Sepharose affinity chromatography, and digestion with an endoglycosidase. The major components of these glycopeptides from embryonal carcinoma cells appear to be different from complex glycopeptides known to occur in adult cells. The glycopeptide pattern of mouse preimplantation embryos resembles that of embryonal carcinoma cells. These results suggest that the carbohydrate profile changes fundamentally during early stages of mammalian development.
In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.
Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells.
Embryonal carcinoma (EC) cells provide an effective model system for studying growth factor production and regulation during mammalian embryogenesis. Our earlier data indicated that the mouse EC cell lines F9 and PC-13 and the human EC cell line NT2/D1 produce a factor with properties similar to those ascribed to members of the fibroblast growth factor (FGF) family and that production of this FGF-related factor is suppressed when all three EC cell lines are induced to differentiate. Subsequent studies suggested that NT2/D1 EC cells express transcripts for basic FGF (bFGF). The current study confirms and extends these findings using a combination of reverse transcription and polymerase chain reaction (RT-PCR). In this study, the expression of bFGF and other members of the FGF family have been examined in F9 and PC-13 cells in addition to NT2/D1 EC cells. In contrast to NT2/D1 EC cells, bFGF expression could not be detected in F9 and PC-13 EC cells. Additionally, expression of four other members of the FGF family (acidic FGF, int-2, FGF-5, and FGF-6) were not detected in NT2/D1, F9, or PC-13 EC cells. However, expression of another member of the FGF family, the k-FGF oncogene, was detected in NT2/D1, F9, and PC-13 EC cells. Moreover, the expression of this transcript is reduced dramatically when each of the three EC cell lines is induced to differentiate. Taken together, our findings argue that expression of the k-FGF oncogene is predominantly responsible for the FGF-related activity detected in EC cells and that differentiation of these EC cells results in suppression of this oncogene.
Embryonal carcinoma (EC) cells are susceptible to infection by a variety of viruses, but do not become resistant to infection by Semliki Forest virus or vesicular stomatitis virus (VSV) on treatment with interferon. These observations have led to the conclusion that interferon does not induce an antiviral state in EC cells. We report here, however, that EC cells treated with interferon become resistant to infection by two picornaviruses and two ts mutants of VSV, whereas they remain sensitive to wild-type VSV, Sindbis and influenza virus infectin. These results suggest that a partial antiviral state is induced in EC cells by interferon and that the induced antiviral protein(s) interferes with the replication of specific viruses. A significant common feature of these viruses is their replication through structures containing double-stranded RNA (dsRNA).
Embryonal carcinoma (EC) mouse cells have been shown to be resistant to infection by retroviruses and small oncogenic DNA viruses, including simian virus 40 and polyoma. When allowed to differentiate, in vitro or in vivo, EC cells become as susceptible to these viruses as differentiated mouse cell lines are. In order to study the relationships between differentiation of EC cells and viral expression, we have isolated and characterized several polyoma mutants that can express early and late functions in undifferentiated EC cells. These mutants, which arose spontaneously during high-multiplicity infection of PCD3 cells (a differentiated fibroblast-like cell line derived from PCC3 EC cells), were selected on PCC4 cells (undifferentiated EC cells) and twice plaque purified. Restriction enzyme analysis of the DNA from several mutants has shown that they all exhibit an additional sequence located in the Pvu II endonuclease fragment 4, close to the junction between Hpa II endonuclease fragments 3 and 5. The size of the insertion varies from 10 to 50 base pairs. The biological properties, including oncogenicity, transforming ability, host range, and burst size of the mutants so far analyzed are similar to those of wild-type virus.
Embryonal carcinoma (EC) cells are nonpermissive for retrovirus replication. Restriction of retroviral expression in EC cells was studied by using DNA transfection techniques. To investigate the activity of the Moloney murine leukemia virus (M-MuLV)enhancer and promoter sequences, the M-MuLV long terminal repeat and the defined long terminal repeat deletions were linked to neo structural gene sequences that encode resistance to the neomycin analog G418. Transient expression data and drug resistance frequencies support the findings that the M-MuLV enhancer is not active in EC cells but that promoter sequences are functional. In addition, a proviral DNA fragment that encodes the leader RNA sequence of a M-MuLV recombinant retrovirus was found to restrict expression specifically in EC cells. Deletion analysis of the leader fragment localized the inhibitory sequences to a region that spans the M-MuLV tRNA primer binding site. It is not known whether restriction occurs at a transcriptional or posttranscriptional level, but steady-state RNA levels in transient expression assays were significantly reduced.
Embryonal carcinoma (EC) cells are the pluripotent stem cells of teratocarcinoma. The aggregates of P19, a mouse EC cell line, undergo differentiation and rapidly lose its colony-forming ability in culture with retinoic acid (RA) or DMSO. Loss of plating efficiency is used to assess the rate of drug-induced differentiation. When exposed respectively to DMSO or RA, the isolated EC cell mutants (D3 and RAC65) did not differentiate but the gap junctions could be formed. In RA-treated co-aggregates of RAC65 and O1A1, each cell line gave responses independent of the presence of the other cell line. However, in DMSO-treated co-aggregates of P19 and D3, D3 cells remained undifferentiated and P19 cell differentiation was much poorer than that in the absence of D3 cells. The results suggest that cell-cell interaction be present in the early stage of DMSO-induced differentiation and absent in the early stage of RA-induced differentiation.
Embryonal carcinoma (EC) cells, the pluripotent stem cells of teratocarcinomas, show many similar-ities to embryonic stem (ES) cells. Since EC cells are malignant but their terminally differentiated derivatives are not, understanding the molecular mechanisms that regulate their differentiation maybe of value for diagnostic and therapeutic purposes. We have examined the expression of multiple components of two developmentally important cell-cell signalling pathways, Wnt and Notch, in the pluripotent human EC cell line, NTERA2, and the human ES cell line, H7. Both pathways have well-documented roles in controlling neurogenesis, a process that occurs largely in response to retinoicacid (RA) treatment of NTERA2 cultures and spontaneously in H7 cultures. In NTERA2, many ofthe genes tested showed altered transcriptional regulation following treatment with RA. These include members of the frizzled gene family (FZDI, FZD3, FZD4, FZD5, FZD6), encoding receptors forWnt proteins, the Frizzled Related Protein family (SFRPI, SFRP2, FRZB, SFRP4), encoding solubleWnt antagonists and also ligands and receptors of the Notch pathway (Dlkl, Jaggedl; Notchl, Notch2, Notch3). Few differences were found in the repertoire of Wnt and Notch pathway genes expressed by NTERA2 EC cells and H7 ES cells. We present a model in which interactions between and regulation of Wnt and Notch signalling are important in maintaining EC/ES stem cells and also controlling their differentiation.
Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.
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.
Human embryonal carcinoma (EC) cells represent the stem cells of testicular germ cell tumours (TGCTs) and are morphologically, antigenically and functionally related to the stem cells of early mammalian embryos. Despite the large capacity for differentiation displayed by TGCT stem cells, little is known of the factors controlling their developmental potency. We have analyzed the differentiation elicited in NT2D1 human embryonal carcinoma (EC) cells by Bone Morphogenetic Proteins (BMPs) and compared it with that elicited by retinoic acid (RA). We have found that while RA induced expression of neuronal, endodermal and epithelial markers in NT2D1 human EC cells, treatment with BMPs resulted in a predominantly epithelial phenotype. We also provide evidence to suggest that at least some of the effects elicited by RA in human EC cells might be mediated through RA-induced expression of BMP-7. Thus BMPs may play an important role in specifying the type of differentiation arising from human multipotent stem cells. The manipulation of BMP signalling in human embryonic multipotent stem cells may therefore prove a useful approach in attempts to generate specific differentiated cell types in vitro, and loss of the malignant and/or transformed phenotype.
Murine embryonal carcinoma cells are the stem cells of teratocarcinomas. They do not express H-2 antigens and previous reports have shown that these cells are not killed by major histocompatibility complex (MHC) restricted murine cytotoxic T cells. Here we report that effectors generated in MLCs between responders from a W3/13 enriched, NK-cell-depleted, rat spleen population, and spleen stimulators from 129 mice can lyse 129 embryonal carcinoma (EC) cells, but not CBA EC cells. A similar pattern of specific lysis of EC cells was seen with effectors generated in MLCs between rat lymph node and mouse spleen stimulators. Following priming in vivo with 129 EC cells, the frequency of cytotoxic T lymphocyte precursors against both 129 EC cells and 129 blast targets was increased when compared with the frequency found in age- and sex-matched unprimed animals. This response shows both specificity and memory and we conclude from this that it is due to rat T cells recognizing mouse xenogeneic antigens.
During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.
P19 embryonal carcinoma cells can be induced to differentiate into neuron-like cells by retinoic acid. P19 neurons were recently shown to express both NMDA and non-NMDA type glutamate receptor-mediated currents and be susceptible to glutamate excitotoxicity. In this study, we used RT-PCR to survey differentiated P19 cultures for glutamate receptor transcript expression. The following transcripts were detected: at least one member of the GluR1-4 family, GluR5, GluR6, GluR7, KA1, KA2, NMDAR1, and NMDAR2B. Nuclease protection assays revealed a large quantitative induction of GluR6 transcripts following retinoic acid treatment. Inotropic glutamate receptors are a fundamental and major feature of CNS neurons which are not expressed by the cell lines commonly used as experimental models for mammalian neurons. The present results show that P19 cells express multiple genes involved in glutamate receptor biology. Since the stem cells can be manipulated genetically, the system has the basic requirements for analyzing mechanisms involved in glutamate receptor gene expression.
P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.
P19 embryonal carcinoma cells are pluripotential and able to differentiate into a variety of cell types, including neurons, glia and fibroblast-like cells, upon retinoic acid treatment and cellular aggregation. Using reverse transcription-polymerase chain reaction, ligand binding and immunocytochemical methods, kappa- and delta-opioid receptors were detected in undifferentiated P19 cells. The mu-opioid receptor was not observed until one day after plating, following one essential step of differentiation, but increased in number in the four days after plating. Several different expression patterns were detected in these differentiated cells. Some cells exhibited mu- and delta-opioid receptors co-expressed, with or without K-opioid receptor; whereas some of the cells expressed only K-opioid receptor. All three opioid receptors are detected on aggregated cells which are postmitotic and also expressing neurofilaments, indicating neuronal characteristics. Furthermore, those cells expressing mu and delta-opioid receptors also expressed glutamate decarboxylase, characteristic of the GABAergic phenotype. Based on these findings, we propose that P19 cells may serve as a model system to study the developmental regulation of opioid receptors, and in particular their relationship with GABA.