F9 Teratocarcinoma Cells Research Articles

Frizzled gene expression and negative regulation of canonical WNT-β-catenin signaling in mouse F9 teratocarcinoma cells.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-β-catenin signaling, thereby allowing PE cells to differentiate.

Interleukin-2 as anticancer immunotherapy in the age of checkpoint inhibition

Rekers and coworkers recently published their study on anti-tumor effects of recombinant interleukin-2 (IL2) and the immunoconjugate L19-IL2 with and without combined radiotherapy. Immunocompetent mice were injected with F9 teratocarcinoma cells. As soon as an average tumor volume of 200 mm³ resulted, different therapy schedules were administered including IL2, L19-IL2 and radiotherapy. Tumor growth was monitored until the tumor volume reached 4 times the volume at the start of therapy (1). This study raises the question on the role of immunotherapy with IL2 and IL2-related immunocytokines, after immunotherapy with checkpoint inhibitors has been widely established.

IGF antagonizes the Wnt/β-Catenin pathway and promotes differentiation of extra-embryonic endoderm

Mouse F9 teratocarcinoma cells are an established model for the differentiation of extra-embryonic endoderm (ExEn). Primitive endoderm, parietal and visceral endoderm can be generated by stimulation of F9 cells with retinoic acid and dibutyryl cyclic adenosine monophosphate. Here we show that Wnt/β-Catenin signaling is down-regulated during ExEn differentiation in F9 cells and that the inhibition of the Wnt pathway promotes differentiation of the three extra-embryonic endoderm lineages. Wnt inhibition is achieved through the IGF pathway, which is up-regulated during differentiation. IGF signaling antagonizes the Wnt pathway by stimulating transcription of axin2 and by stabilizing Axin1 protein. Both Axin1 and Axin2 are components of the β-Catenin destruction complex and act as intra-cellular inhibitors of the Wnt/β-Catenin pathway. The data presented reveal a mechanism which restricts pluripotency of undifferentiated cells and directs them toward extra-embryonic lineages.

Extracellular syntaxin4 triggers the differentiation program in teratocarcinoma F9 cells that impacts cell adhesion properties

The proteins in the syntaxin family are known to mediate fusion of cytoplasmic vesicles to the target membrane, yet subpopulations of certain syntaxins, including syntaxin4, translocate across the cell membrane in response to external stimuli. Here, we show that extracellularly presented syntaxin4 impacts cell behavior and differentiation in teratocarcinoma F9 cells. While undifferentiated F9 cells extruded a small subpopulation of extracellular syntaxin4 at the lateral cell membrane, the induction of differentiation with all-trans retinoic acid (RA) abolished this localized expression pattern. We found that the cells that were stimulated in a non-directional fashion by extracellular syntaxin4 displayed a flattened shape and retained a substrate-bound morphology even under a long-term, serum-starved cultivation. Such a cellular response was also elicited by a circular peptide composed of the potential functional core of syntaxin4 (AIEPQK; amino acid residues 103~108) (ST4n1). While the proliferation and metabolism were not affected in these cells, cell-cell interaction became weakened and the expression of vinculin, a regulator of both intercellular and cell-substrate adhesion molecules, was altered. We also found that the expressions of several differentiation markers were up-regulated in cells stimulated with extracellular syntaxin4 and that syntaxin3, another family member, was most prominent. Intriguingly, forced expression of syntaxin3 induced the spread morphology in F9 cells, indicating that syntaxin3 partly mediates the function of extracellular syntaxin4. These results demonstrate the involvement of a non-directional stimulation of extracellular syntaxin4 in the functional and morphological differentiation of F9 cells.

Dishevelled3 is a novel arginine methyl transferase substrate

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Reactive oxygen species and Wnt signalling crosstalk patterns mouse extraembryonic endoderm

Primitive endoderm formation from the inner cell mass is one of the earliest known cell fate decisions made in the mouse embryo. The mechanisms involved in orchestrating this process are not fully understood and are difficult to study in vivo. The F9 teratocarcinoma cell line is an in vitro model used to circumvent many technical problems surrounding the study of extraembryonic endoderm differentiation. F9 cells treated with retinoic acid differentiate to primitive endoderm and this is accompanied by the activation of canonical Wnt–β-catenin signalling. Reactive oxygen species can modulate this signalling pathway [1], but whether they are sufficient to induce extraembryonic endoderm in vitro is not known. In the present study, a sustained increase in ROS levels was found in retinoic acid-treated F9 cells. An increase in Tcf-Lef transcriptional activity, a read out of Wnt–β-catenin signalling, was also seen in response to exogenous H2O2. Analysis from immunoblots, immunocytochemistry and real time PCR revealed the presence of markers of differentiation and a reduction in the expression of a marker of proliferation, confirming that H2O2-treated F9 cells developed into primitive endoderm. In contrast, exposing retinoic acid-treated cells to antioxidants impeded differentiation. Real time PCR was also used to identify candidates responsible for the observed elevation in ROS production. Results indicated that the NADPH oxidase 1, 2, 3 and 4 and Duox2 genes were RA responsive. Furthermore, the NADPH oxidase inhibitor, diphenyleneiodonium chloride was shown to attenuate primitive endoderm formation. Together, these results shed new light on how early mouse embryogenesis might be influenced by the crosstalk involving ROS and the Wnt–β-catenin signalling pathway.

Assembly of Dishevelled 3-based supermolecular complexes via phosphorylation and Axin

BackgroundDishevelled-3 (Dvl3) is a multivalent scaffold essential to cell signaling in development. Dsh/Dvls enable a myriad of protein-protein interactions in Wnt signaling. In the canonical Wnt/β-catenin pathway specifically, Dvl3 polymerizes to form dynamic protein aggregates, so-called “signalsomes”, which propagate signals from the Wnt receptor Frizzled to downstream elements.ResultsVery large Dvl3-based supermolecular complexes form in response to Wnt3a. These complexes are identified by steric-exclusion chromatography, affinity pull-downs, proteomics, and fluorescence correlation microscopy (fcs). In the current work, the roles of Dvl3 phosphorylation and of Axin in the assembly of Dvl3-based supermolecular complexes in response to Wnt3a are probed in totipotent mouse F9 teratocarcinoma cells. Point mutations of phosphorylation sites of Dvl3 which interfere with Lef/Tcf-sensitive transcriptional activation by Wnt3a are shown to interfere more proximally with the assembly of Dvl3-based supermolecular complexes. Axin, a Dvl-interacting protein, plays a central role in organizing the beta-catenin destruction complex. The assembly of Dvl3-based supermolecular complexes is blocked either by depletion of Axin or by mutation of Axin sites necessary for polymerization in response to Wnt3a.ConclusionThese data demonstrate that Wnt3a activation of the canonical pathway requires specific phosphorylation events as well as Axin to assemble very large, Dvl3-based supermolecular complexes; these complexes are a prerequisite to activation of Lef/Tcf-sensitive transcription.

GATA6 and FOXA2 RegulateWnt6Expression During Extraembryonic Endoderm Formation

One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis involves the differentiation of inner cell mass cells into primitive and then into parietal endoderm. These processes can be recapitulated in vitro using F9 teratocarcinoma cells, which differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm with subsequent treatment with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on how primitive endoderm develops revealed that the Wnt6 gene is upregulated by RA, leading to the activation of the canonical WNT-β-catenin pathway. The mechanism by which Wnt6 is regulated was not determined, but in silico analysis of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 transcription factors might be involved [1]. Subsequent analysis determined that both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced molecular and morphological markers of primitive endoderm, which occurred concomitantly with the upregulation of the Wnt6 gene. Gata6- or Foxa2-overexpressing cells were also found to have increased levels in T-cell factor (TCF)-dependent transcription, and when these cells were treated with db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and overexpression studies showed that these transcription factors were sufficient to switch on the gene expression of a Wnt6 reporter construct. Together, these results provide evidence for the direct regulation of Wnt6 that leads to the activation of the canonical WNT-β-catenin pathway and subsequent induction of primitive extraembryonic endoderm.

Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin

Canonical Wnt signalling requires caveolin-dependent internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Here we report that the tumour suppressor and endocytic adaptor disabled-2 (Dab2), previously described as an inhibitor of Wnt/β-catenin signalling, selectively recruits LRP6 to the clathrin-dependent endocytic route, thereby sequestering it from caveolin-mediated endocytosis. Wnt stimulation induces the casein kinase 2 (CK2)-dependent phosphorylation of LRP6 at S1579, promoting its binding to Dab2 and internalization with clathrin. LRP6 receptor mutant (S1579A), deficient in CK2-mediated phosphorylation and Dab2 binding, fails to associate with clathrin, and thus escapes the inhibitory effects of Dab2 on Wnt/β-catenin signalling. Our data suggest that the S1579 site of LRP6 is a negative regulatory point during LRP6-mediated dorsoventral patterning in zebrafish and in allograft mouse tumour models. We conclude that the tumour suppressor functions of Dab2 involve modulation of canonical Wnt signalling by regulating the endocytic fate of the LRP6 receptor.

The Functional Link Between CDK1 and Retinoic Acid Receptor γ (RARγ) in Response to Treatment with All-Trans Retinoic Acid

Abstract 2485Alterations in cell cycle pathways and retinoic acid signaling are implicated in the pathogenesis of leukemia and treatment response. The retinoic acid receptors (RARα, RARβ and RARγ) mediate the differentiation, cell cycle arrest and cellular response to all-trans retinoic acid (ATRA). However, it is largely unknown whether the cyclin dependent kinases (CDK1 and CDK2) mediate the ATRA effects, also whether and how CDK1 and CDK2 are functional linked to RARs. Therefore in the present study, we examined the function of CDKs and their regulation in relationship to the RAR α, β and γ.The use of siRNA mediated knockdown of CDK1 in human leukemic U-937 cells affected cell cycle distribution, resulting in a decreased fraction of G0/G1 cells which coincided with the reduced expression of p27 protein. Treatment of U-937 si-CDK1 cells with ATRA demonstrated reduced terminal differentiation and decreased cell cycle arrest at G0/G1 phases in comparison to U-937 siRNA control cells. We observed a reduced responsiveness to ATRA treatment due to the loss of CDK1 function, and this was accompanied with the alterations in the expression of RARγ and the increased phosphorylation of Akt. However, loss of CDK2 function showed no effects on ATRA-mediated growth arrest and differentiation in U-937 cells.We also found that nuclear CDK1 was actively phosphorylated and was rapidly degraded upon the induction of ATRA, in contrast to its cytoplasmic counterpart in human leukemic U-937 cells. When examining the functional link between RARs and the regulation of CDKs in response to ATRA, we found that RARβ and RARγ, but not RARα, participated in ATRA-mediated protein degradation of CDK1 and CDK2. Moreover, we compared the effects of ATRA on ubiquitin-mediated protein degradation in the F9 teratocarcinoma cells and in F9 cells harboring targeted deletions of the RARα, RARβ and RARγ genes. ATRA induced ubiquitin conjugates showed striking changes in patterns in cells lacking RARγ. This indicates that the blockage in protein degradation of CDK1 may be due to the loss of RARγ function. Among the RAR isoforms, RARγ co-localized with CDK1 predominantly in the nuclear bodies. There was no detectable complex formation between RARβ and CDK1. This suggests that the phosphorylation and degradation of the nuclear CDK1 and its interaction with RARγ may be important events in mediating ATRA-induced effects on cell cycle and differentiation.Our study has revealed a novel mechanism by which CDK1 cooperates with RARγ to mediate the effects of all-trans retinoic acid. Disclosures:No relevant conflicts of interest to declare.

Abstract 2106: The role of KSRP in non-small cell lung cancer

Abstract Precedent has established the role of the Wnt/β-catenin pathway in oncogenesis. Several members of the Wnt family are known to be tumor promoting in non-small cell lung cancer, NSCLC. In this pathway β-catenin, a downstream component, accumulates and serves as a T-cell transcription /lymphoid enhancer factor. We previously identified the Wnt ligand in the lung, Wnt 7a, as a member of the non-canonical pathway/ β-catenin independent pathway, which inhibits transformed cell growth. Currently, we hypothesize that the regulatory protein, KSRP, holds a switch between the canonical and non-canonical pathway. K-homology splicing regulatory protein (KSRP) is a member of the family of AU rich element binding proteins. It possesses four K-homology motifs and causes decay of specific mRNA transcripts in different cell types. In mouse F9 teratocarcinoma cells KSRP negatively regulates β-catenin. The role of KSRP in the non-canonical Wnt pathway in the lung has previously been unknown. Using NSCLC cell lines and nontransformed lung cells we show a correlation between Wnt 7a expression and a positive regulation of KSRP. KSRP expression is elevated in nontransformed cells and alternatively, lower levels of KSRP expression in NSCLC. Over expression of KSRP in NSCLC cells decreases cell proliferation in MTS assay as well as reducing colony formation in clonogenic assays. The silencing of KSRP in nontransformed cells results in an increase in cell proliferation. The positive regulation of KSRP by Wnt 7a positively regulates this mRNA regulatory protein in the noncanonical Wnt pathway as an important factor in tumor suppression. The canonical pathway negatively regulates KSRP, which allows the stabilization of β-catenin mRNA thereby promoting progression of the tumor-promoting pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2011-2106

Microvascular biodistribution of L19-SIP in angiogenesis targeting strategies

IntroductionVarious strategies using L19-mediated fibronectin targeting have become useful clinical tools in anti-tumour therapy and diagnostics. The aim of our study was to characterise the microvascular biodistribution and binding process during tumour angiogenesis and after anti-angiogenic therapy. Materials and methodsSF126 glioma and F9 teratocarcinoma cells were implanted into dorsal skin fold chambers (SF126: n=4; F9: n=6). Using fluorescence and confocal intravital microscopy the biodistribution process was assessed at t=0h, t=4h and t=24h after intravenous application of Cy3-L19-SIP. Sunitinib treatment was applied for six days and microscopy was performed 2 and 6days after treatment initiation. Analysed parameters included: vascular and interstitial binding, preferential binding sites of L19-SIP, microvascular blood flow rate, microvascular permeability. Histological analysis included CD31 and DAPI. ResultsL19-SIP showed a specific and time-dependent neovascular binding with a secondary extravasation process reaching optimal vascular/interstitial binding ratio 4hours after iv administration (F9: L19-SIP: vascular binding: 74.6±14.5; interstitial binding: 46.8±12.1; control vascular: 22,2±16.6). Angiogenic sprouts were preferred binding sites (F9: L19-SIP: 188±15.5; RTV: 90.6±13.5). Anti-angiogenic therapy increased microvascular hemodynamics (SF126: Su: 106.6±13.3μl/sec; Untreated: 19.7±9.1μl/sec) and induced increased L19-SIP accumulation (SF 126: t24; Su: 92.6±2.7; Untreated: 71.9±5.9) in therapy resistant tumour vessels. ConclusionL19-SIP shows a time and blood-flow dependent microvascular biodistribution process with angiogenic sprouts as preferential binding sites followed by secondary extravasation of the antibody. Microvascular biodistribution is enhanced in anti-angiogenic-therapy resistant tumour vessels.

Probing the physical nature and composition of signalsomes

BackgroundRecent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules?ResultsSteric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr (0.5 and 2 MDa). Activation of the Wnt canonical β-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells.ConclusionDvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome Mr/MW, molecular composition, and intracellular locale.

Defining the Roles of .ALPHA.-Catenin in Cell Adhesion and Cytoskeleton Organization: Isolation of F9 Cells Completely Lacking Cadherin-catenin Complex

To define the roles of α-catenin in cell-cell adhesion, the E-cadherin, α-catenin, β-catenin, and/or plakoglobin genes were inactivated in F9 teratocarcinoma cells. An E-cadherin-α-catenin fusion protein (Eα) restored full cell-adhesion function and organized the actin-based cytoskeleton and ZO-1, an actin filament binding protein, in F9 cells lacking all endogenous cadherin-catenin complex components. There were two types of cadherin-based cell-adhesion junctions in parental F9 cells, those with ZO-1 and those without ZO-1, and only junctions with ZO-1 were associated with thick actin bundles. Additionally, ZO-1 localized to most Eα-based cell-adhesion junctions. These data demonstrated that Eα supported cadherin-based cell adhesion and recruited actin bundles and ZO-1 to cell-cell contact sites in the absence of cytoplasmic α-catenin. Moreover, the C-terminal half of α-catenin was involved in the formation of cell-adhesion junctions with ZO-1.

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

Dishevelled-2 docks and activates Src in a Wnt-dependent manner

Wnt3a activates the ;canonical' signaling pathway, stimulating the nuclear accumulation of beta-catenin and activation of Lef/Tcf-sensitive transcription of developmentally important genes. Using totipotent mouse F9 teratocarcinoma cells expressing frizzled-1 (Fz1), we investigated roles of tyrosine kinase activity in Wnt/beta-catenin signaling. Treatment with either genistein or Src family kinase inhibitor PP2 attenuates Wnt3a-stimulated Lef/Tcf transcription activation and primitive endoderm formation. siRNA-induced knockdown of Src likewise attenuates Lef/Tcf transcription and primitive endoderm formation in response to Wnt3a, implicating Src as a positive regulator of Wnt/beta-catenin signaling. We discovered that Src binds dishevelled-2 (Dvl2), a key phosphoprotein in Wnt signaling, at two positions: an SH3-binding domain and a C-terminal domain. The Y18F mutant of Dvl2 attenuates the Wnt3a-stimulated Lef/Tcf-sensitive transcriptional response. Wnt3a stimulates Src docking to Dvl2 and activation of this tyrosine kinase. Activated Src, in turn, enhances Wnt activation of the canonical pathway. We show that Dvl2 and beta-catenin are crucially important substrates for tyrosine phosphorylation in the canonical Wnt/beta-catenin pathway.

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

BackgroundMultipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs).ResultsWe have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide.ConclusionPluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells.ReviewersThis article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter.

P38 mitogen-activated protein kinase regulates canonical Wnt–β-catenin signaling by inactivation of GSK3β

The Wnt-beta-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-beta-catenin signaling pathway in response to Wnt3a, we observed a strong activation of p38 MAPK in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Galpha(q) or Galpha(s), but not against either Galpha(o) or Galpha(11). Activation of p38 MAPK is critical for canonical Wnt-beta-catenin signaling. Chemical inhibitors of p38 MAPK (SB203580 or SB239063) and expression of a dominant negative-version of p38 MAPK attenuate Wnt3a-induced accumulation of beta-catenin, Lef/Tcf-sensitive gene activation, and primitive endoderm formation. Furthermore, epistasis experiments pinpoint p38 MAPK as operating downstream of Dishevelleds. We also demonstrate that chemical inhibition of p38 MAPK restores Wnt3a-attenuated GSK3beta kinase activity. We demonstrate the involvement of G-proteins and Dishevelleds in Wnt3a-induced p38 MAPK activation, highlighting a critical role for p38 MAPK in canonical Wnt-beta-catenin signaling.

Gαo mediates WNT-JNK signaling through Dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-beta-catenin signaling pathway requires both G alpha o and G alpha q heterotrimeric G-proteins, whereas we show that WNT-JNK signaling requires only G alpha o protein. G alpha o propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-beta-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-beta-catenin signaling.

Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by β-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6–β-catenin–Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.