Teratocarcinoma Differentiation Research Articles

Characterization of conventional protein kinase C (PKC) isotype expression during F9 teratocarcinoma differentiation. Overexpression of PKC alpha alters the expression of some differentiation-dependent genes.

F9 teratocarcinoma is a useful model for studying early embryogenesis since these cells can differentiate into primitive or parietal endoderm under the influence of retinoic acid or retinoic acid and cyclic AMP, respectively. We have found that three isoforms of protein kinase C (PKC alpha, -beta, and -gamma) were expressed in undifferentiated stem cells. When the cells were treated with retinoic acid either alone or in the presence of cAMP for 120 h, PKC alpha mRNA and protein levels increased, whereas those of PKC beta and PKC gamma became undetectable. These changes began within 24 h of drug treatment and were complete by 48-72 h. In order to determine the functional significance of the induction of PKC alpha during F9 differentiation, we established two stable transfectants that overexpressed PKC alpha protein between 4- and 5-fold compared to wild type cells. Characterization of these cell lines revealed an altered pattern of expression of some of the markers of F9 differentiation. The clone that had the highest amount of PKC alpha protein constitutively expressed mRNA for type IV collagen and c-Jun, which are not normally expressed until 24-48 h of treatment with differentiation agents. In the other overexpressing clone, these markers were induced much faster than in wild type cells. The growth rate of both overexpressing clones was less than wild type cells, while the expression of the PKC beta protein in these clones was similar to the levels found in differentiated F9 cells. However, other markers of differentiation, including the cellular morphology and levels of pST6-135 and c-myc RNA, responded to agents identically in both wild type and PKC-alpha-overexpressing clones. Therefore, overexpression of PKC alpha is not sufficient to induce full differentiation of F9 cells. However, our data suggest that certain pathways that lead to the expression of differentiation-dependent genes are regulated by PKC alpha protein levels.

Retinoic acid stimulates protein kinase A-associated G proteins during human teratocarcinoma differentiation

Retinoic acid (RA) treatment of F9 murine teratocarcinoma (TC) cells reduces the expression of the protein kinase A (PKA)-associated G protein, G αi2. The present study reveals interactions between the RA and PKA pathways during differentiation of the multipotent human TC cell line NTERA-2 clone D1 (abbreviated NT2/D1) which differ from prior reports in F9 TC cells. Compared to untreated NT2/D1 cells, differentiated NT2/D1 cells expressed increased levels of G αs and G αi1,2 proteins as shown by both immunoblot analysis and cholera toxin- and pertussis toxin-induced ADP ribosylation. To further explore cooperation between these pathways during human TC differentiation, we examined the effects of cyclic adenosine monophosphate (cAMP) on RA-responsive genes and of RA treatment on the transcriptional activation of a cAMP response element (CRE). Compared to RA alone, combined treatment with RA and cAMP augmented the expression of the RA nuclear receptor-β (RAR-β). Also, transient transfection assays revealed that cAMP and RA cooperated to enhance CRE transcriptional activation. The cAMP-induced enhancement of RA actions in NT2/D1 cells extended to immunophenotypic changes typical of the neuronal differentiation program induced by RA. In contrast to these findings in NT2/D1 cells, prior work in F9 TC cells showed that cAMP inhibits the RA-mediated augmentation of RAR-β expression and switches the differentiation program from visceral to parietal endoderm. Thus, unlike murine TC cells, in human NT2/D1 cells RA stimulates PKA-associated G proteins and PKA pathway activation enhances RA-mediated TC differentiation.

Retinoic acid stimulates the protein kinase C pathway before activation of its β-nuclear receptor during human teratocarcinoma differentiation

We previously reported that protein kinase C (PKC) stimulation through phorbol ester (TPA) treatment enhances the effects of all- trans retinoic acid (RA) on immunophenotypic differentiation and RA nuclear receptor (RAR) activation in the multipotential human teratocarcinoma (TC) cell line NTera-2 clone D1 (abbreviated NT2 D1 ). This study extends prior work in NT2 D1 cells by demonstrating that PKC pathway activation is an early effect of RA treatment which regulates RAR transcriptional activity. RA activated the PKC pathway prior to induction of RAR-β expression at 6 h, which is an established early marker of RAR activation in NT2 D1 cells. RA caused a transient 1.3-fold increase in intracellular diacylglycerol (DG) at 2 min and a translocation of the gamma isozyme of PKC (PKC-γ) within 5 min. Transient co-transfection studies provided evidence that PKC pathway activation plays a role in the regulation of RAR-β expression. In these studies a constitutively active PKC-γ augmented the RA-mediated transactivation of a luciferase reporter containing the native RAR-β promoter which has a retinoic-acid-response element (RARE). These findings reveal that PKC pathway activation is an early step in RA-mediated human TC differentiation and that PKC-γ can potentiate the effects of RA on RAR transcriptional activation.

Cooperation between retinoic acid and phorbol esters enhances human teratocarcinoma differentiation

Abstract This study explored cooperation between the retinoic acid (RA) and protein kinase C (PKC) pathways during differentiation of the multipotential human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1). We report here that, compared to RA treatment alone, RA combined with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the regulated expression of the immunophenotypic differentiation markers SSEA-3, a globo-series carbohydrate, and the ganglio-series carbohydrate antigens GD2 and GD3. Northern analysis and transient transfection assays revealed that TPA co-treatment augmented the RA-induced expression and activation of the RA nuclear receptor-β (RAR-β), one early marker of RA response in NT2/D1 cells. This finding was extended with transient co-transfection experiments using a PKC-α expression vector which revealed that the PKC pathway can augment the activation of RAR-β by RA. These experiments establish PKC as a modulator of RAR-β expression in NT2/D1 cells. Similarly, experiments showed that RA can modulate activation of the PKC-responsive AP- 1 complex, a transcription factor rapidly activated by TPA. Northern analysis and transient transfection assays revealed that, compared to TPA treatment alone, RA and TPA augmented the expression and transcriptional activity of AP-1 in NT2/D1 cells. In contrast, transient transfection assays revealed no cooperative effect between RA and TPA in HeLa cells, indicating that this effect in NT2/D1 cells is cell type-specific. In summary, these studies show that stimulation of the PKC second messenger pathway can modulate tumor differentiation and transcriptional activation of a retinoid receptor associated with RA response.

Cooperation between retinoic acid and phorbol esters enhances human teratocarcinoma differentiation

Abstract This study explored cooperation between the retinoic acid (RA) and protein kinase C (PKC) pathways during differentiation of the multipotential human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1). We report here that, compared to RA treatment alone, RA combined with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the regulated expression of the immunophenotypic differentiation markers SSEA-3, a globo-series carbohydrate, and the ganglio-series carbohydrate antigens GD2 and GD3. Northern analysis and transient transfection assays revealed that TPA co-treatment augmented the RA-induced expression and activation of the RA nuclear receptor-β (RAR-β), one early marker of RA response in NT2/D1 cells. This finding was extended with transient co-transfection experiments using a PKC-β expression vector which revealed that the PKC pathway can augment the activation of RAR-β by RA. These experiments establish PKC as a modulator of RAR-β expression in NT2/D1 cells. Similarly, experiments showed that RA can modulate activation of the PKC-responsive AP-1 complex, a transcription factor rapidly activated by TPA. Northern analysis and transient transfection assays revealed that, compared to TPA treatment alone, RA and TPA augmented the expression and transcriptional activity of AP-1 in NT2/D1 cells. In contrast, transient transfection assays revealed no cooperative effect between RA and TPA in HeLa cells, indicating that this effect in NT2/D1 cells is cell type-specific. In summary, these studies show that stimulation of the PKC second messenger pathway can modulate tumor differentiation and transcriptional activation of a retinoid receptor associated with RA response.

Cyclic adenosine monophosphate‐mediated induction of F9 teratocarcinoma differentiation in the absence of retinoic acid

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.

Basement membrane matrices in mouse embryogenesis, teratocarcinoma differentiation and in neuromuscular maturation.

In this paper we discuss studies on basement membrane and interstitial matrix molecules in early development and teratocarcinoma differentiation. In the early embryo a compartmentalization of newly formed cell types takes place immediately by formation of basement membranes. The stage-specific developmental appearance of extracellular matrix molecules such as type IV collagen, laminin, entactin, fibronectin and proteoglycans seems to reflect a diversified role of extracellular matrices already in the earliest stages of development. In teratocarcinoma cultures the appearance and composition of extracellular matrices during the differentiation of endoderm cells closely resembles that found in the early embryo. Also in this respect the teratocarcinoma system can be used as a model for studies on early development. In later developmental phenomena other matrix molecules can also be of importance. Merosin, a novel tissue-specific basement membrane-associated protein that appears during muscle and nerve maturation is an example of such molecules.

In vitro Differentiation of Mouse Teratocarcinoma Cells Monitored by Intermediate Filament Expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.

Protein synthesis and differentiation in a clonal line of teratocarcinoma and in preimplantation mouse embryo

Undifferentiated cells of a clonal line of teratocarcinoma can differentiate in vitro into embryoid bodies with morphological and biochemical features of early mouse embryo. During the first step of differentiation protein synthesis has been analysed by 2 dimensional gel electrophoresis. While new proteins are synthesized, the synthesis of others is turned off with the appearance of endodermal cells in embryoid bodies. We have compared protein synthesis during teratocarcinoma differentiation and during early mouse embryogenesis at three stages of mouse preimplantation embryo. The results demonstrate that only the late blastocyst protein synthesis pattern shows most of the polypeptides identified in the differentiated protein synthesis pattern of teratocarcinoma. In contrast, protein synthesis during the early stages of mouse embryonic development is very different from protein synthesis in undifferentiated teratocarcinoma.

Teratocarcinoma differentiation: Plasminogen activator activity associated with embryoid body formation

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activator activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.