P19 Mouse Embryonal Carcinoma Cells Research Articles

Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression

NELL2 was first identified as a mammalian homolog of chick NEL (Neural EGF-like) protein. It is almost exclusively expressed in neurons of the rat brain and has been suggested to play a role in neural differentiation. However, there is still no clear evidence for the detailed function of NELL2 in the differentiation of neurons. In this study, we identified NELL2 function during neural differentiation of mouse embryonic carcinoma P19 cells. Endogenous expression of NELL2 in the P19 cells increased in parallel with the neuronal differentiation induced by retinoic acid (RA). We found that the mouse NELL2 promoter contains RA response elements (RAREs) and that treatment with RA increased NELL2 promoter activity. Transfection of P19 cells with NELL2 expression vectors induced a dramatic increase in cell aggregation, resulting in the facilitation of neural differentiation. Moreover, NELL2 significantly increased N-cadherin expression in the P19 cell. These data suggest that NELL2 plays an important role in the regulation of neuronal differentiation via control of N-cadherin expression and cell aggregation.

Synergistic action in P19 pluripotential cells of retinoic acid and Wnt3a on Cdx1 enhancer elements

Cdx1 encodes a homeodomain protein that regulates expression of some Hox genes. Cdx1 itself is known to be regulated in the primitive streak/tailbud by both retinoic acid (RA) and Wnt3a. Cdx1 in eutherian mammals has two retinoic acid response elements (RAREs), located upstream and in the first intron, and each is adjacent to structural Lef/Tcf motifs. Upstream Lef/Tcf motifs respond to canonical Wnt signalling to activate Cdx1 synergistically with RA. By combined use of reporter assays, immunofluorescence and flow cytometry in mouse P19 embryonal carcinoma cells we show that the Cdx1 intron Lef/Tcf motif also responds to Wnt3a signalling. Synergy between individual Cdx1 RARE and Lef/Tcf motifs can occur whether they are adjacent or distant in the gene. Part, though not all, of the Cdx1 stimulation by RA (in absence of added Wnt3a) likely depends upon Wnt protein produced by the cells themselves, since it is inhibited by mutation of Lef/Tcf motifs, or by IWP-2, an inhibitor of Wnt production. RA and Wnt3a stimulate Cdx1 by increasing both the proportion of P19 cells that are expressing and also their mean level of expression. The expressing/non-expressing sub-populations do not simply correspond with those that express a marker of pluripotentiality, Nanog. We conclude that RA and Wnt3a activate Cdx1 synergistically by overlapping use of both upstream and intron enhancers, and that mouse embryonal carcinoma cell populations display heterogeneity in their response to these activators.

AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from −83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at −29/−17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity.In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity.

Increased Resistance of Breast, Prostate, and Embryonic Carcinoma Cells against Herpes Simplex Virus in Three-Dimensional Cultures

In previous studies we found that uveal melanoma cells grown in extracellular matrix (ECM)-containing three-dimensional (3D) cultures have increased resistance against herpes simplex virus type 1 (HSV-1)-mediated destruction relative to cells cultured without ECM. Using additional tumor cell types including MB-231 human breast cancer cells, PC-3 human prostate cancer cells, and P19 mouse embryonal carcinoma cells, we show here that tumor cell lines other than melanoma are also more resistant to HSV-1-mediated destruction in 3D cultures than cells grown in 2D. We also demonstrate here that one mechanism responsible for the increased resistance of tumor cells to HSV-1 infection in 3D cultures is an ECM-mediated inhibition of virus replication following virus entry into cells. These findings confirm and extend previous observations related to the role of the ECM in tumor resistance against HSV-1 and may lead to improved strategies of oncolytic virotherapy.

Identification of DPPA4 and DPPA2 as a novel family of pluripotency-related oncogenes

In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.

Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 μM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm(2). The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.

Involvement of Crosstalk between Oct4 and Meis1a in Neural Cell Fate Decision

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6–12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.

Sperm Performance Better on Diamond than on Polystyrene

ABSTRACTRecently we postulated that polystyrene Petri dishes become soft when in contact with an aqueous milieu. Specifically, we assumed that the effect is restricted to a superficial nanolayer, a condition presumably favoring the establishment of a stable nanolayer of reactive oxygen species (ROS) at the liquid/solid-interface. Cells are known to be hypersensitive to ROS. Previously we used P19 mouse embryonal carcinoma cells and systematically analyzed their capability to climb different substrates placed vertically into a Petri dish. The worst and best performance was found on polystyrene (Petri dish material) and nanocrystalline diamond, respectively. Polystyrene Petri dishes are today standard in laboratories conducting in vitro fertilization (IVF). Here we proceed and extend the investigation to human spermatozoa and show that their performance (vitality) on polystyrene Petri dishes is low compared to that on diamond Petri dishes. This work may propel further research and inspire the development of a new generation of cell-friendly Petri dishes.

ADAM23 knockdown promotes neuronal differentiation of P19 embryonal carcinoma cells by up‐regulating P27KIP1 expression

ADAM23 (a disintegrin and metalloprotease 23), a member of brain MDC (macrophage-derived chemokine) family, is important for the development of CNS (central nervous system). P19 mouse embryonal carcinoma cells can differentiate into neurons when cultured in aggregates and induced with RA (retinoic acid). We have found that under conditions without RA induction, knocking down ADAM23 with RNAi (RNA interference) promoted neuronal differentiation, and similarly recombinant GST (glutathione transferase)-ADAM23-DIS protein inhibited neuronal differentiation of P19/ADAM23KD (P19/ADAM23-knockdown) cells. In P19/ADAM23KD, there were more cells arrested in G1 phase than normal P19 cells, due to the up-regulation of P57KIP2 and P27KIP1 expression. P27KIP1 was up-regulated during the differentiation process of both P19/ADAM23KD cells without RA induction, and P19 cells with RA induction. Transient overexpression of P27KIP1 in P19 cells also promoted neuronal differentiation of P19 cells. The findings indicate that ADAM23 suppresses neuronal differentiation through its disintegrin domain, and Adam23 KD up-regulates P27KIP1 in P19/ADAM23KD cells, one reason that P19/ADAM23KD cells can differentiate into neurons without RA induction.

Differential Expression Profile of MicroRNAs during Differentiation of Cardiomyocytes Exposed to Polychlorinated Biphenyls

Exposure to persistent environmental pollutants, such as polychlorinated biphenyls (PCBs), is a risk factor for the development of congenital heart defects. MicroRNAs (miRNAs) have been shown to be involved in cardiac development. The objective of this study was to investigate changes in miRNA expression profiles during the differentiation of cardiomyocytes exposed to PCBs. For that purpose, PCBs (Aroclor 1254) at a concentration of 2.5 μmol/L were added on day 0 of differentiation of P19 mouse embryonal carcinoma cells into cardiac myocytes. The relative expression of miRNA genes was determined by miRNA microarray and real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) analyses. The microarray results revealed that 45 miRNAs, of which 14 were upregulated and 31 were downregulated, were differentially expressed in P19 cells treated with PCBs compared with control cells. The miRNA expression data was validated with real-time RT-PCR. The expression of certain potential target genes (Wnt1) was found to be reduced in P19 cells treated with PCBs, whereas the expression of other potential predicted target genes (GSK3β) was increased. Our results demonstrate a critical role of miRNAs in mediating the effect of PCBs during the differentiation of P19 cells into cardiac myocytes.

Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

Altered RNA processing is an underlying mechanism of amyotrophic lateral sclerosis (ALS). Missense mutations in a number of genes involved in RNA function and metabolisms are associated with ALS. Among these genes is angiogenin (ANG), the fifth member of the vertebrate-specific, secreted ribonuclease superfamily. ANG is an angiogenic ribonuclease, and both its angiogenic and ribonucleolytic activities are important for motor neuron health. Ribonuclease 4 (RNASE4), the fourth member of this superfamily, shares the same promoters with ANG and is co-expressed with ANG. However, the biological role of RNASE4 is unknown. To determine whether RNASE4 is involved in ALS pathogenesis, we sequenced the coding region of RNASE4 in ALS and control subjects and characterized the angiogenic, neurogenic, and neuroprotective activities of RNASE4 protein. We identified an allelic association of SNP rs3748338 with ALS and demonstrated that RNASE4 protein is able to induce angiogenesis in in vitro, ex vivo, and in vivo assays. RNASE4 also induces neural differentiation of P19 mouse embryonal carcinoma cells and mouse embryonic stem cells. Moreover, RNASE4 not only stimulates the formation of neurofilaments from mouse embryonic cortical neurons, but also protects hypothermia-induced degeneration. Importantly, systemic treatment with RNASE4 protein slowed weight loss and enhanced neuromuscular function of SOD1 (G93A) mice.

Promotion of Both Proliferation and Neuronal Differentiation in Pluripotent P19 Cells with Stable Overexpression of the Glutamine Transporter slc38a1

BackgroundWe previously demonstrated the functional expression in newborn rat neocortical astrocytes of glutamine transporter (GlnT = slc38a1) believed to predominate in neurons over astroglia in the brain. In order to evaluate the possible role of this transporter in neurogenesis, we attempted to establish stable transfectants of GlnT in mouse embryonal carcinoma P19 cells endowed to proliferate for self-renewal and differentiate into progeny cells such as neurons and astroglia, in addition to in vitro pharmacological profiling of the green tea ingredient theanine, which is shown to be a potent inhibitor of glutamine transport mediated by GlnT in cultured neurons and astroglia.Methodology/Principal FindingsThe full-length coding region of rat GlnT was inserted into a vector for gene transfection along with selection by G418, followed by culture with all-trans retinoic acid under floating conditions and subsequent dispersion for spontaneous differentiation under adherent conditions. Stable overexpression of GlnT led to marked increases in the size of round spheres formed during the culture for 4 days and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction, with concomitant promotion of subsequent differentiation into cells immunoreactive for a neuronal marker protein. In these stable GlnT transfectants before differentiation, drastic upregulation was seen for mRNA expression of several proneural genes with a basic helix-loop-helix domain such as NeuroD1. Although a drastic increase was seen in NeuroD1 promoter activity in stable GlnT transfectants, theanine doubled NeuroD1 promoter activity in stable transfectants of empty vector (EV), without affecting the promoter activity already elevated in GlnT transfectants. Similarly, theanine promoted cellular proliferation and neuronal differentiation in stable EV transfectants, but failed to further stimulate the acceleration of both proliferation and neuronal differentiation found in stable GlnT transfectants.Conclusions/SignificanceGlnT would promote both proliferation and neuronal differentiation through a mechanism relevant to the upregulation of particular proneural genes in undifferentiated P19 cells.

Corrigendum] Effects of miR-19b knockdown on the cardiac differentiation of P19 mouse embryonic carcinoma cells.

Mol Med Rep 11: [Related article:] 2504–2512, 2015; DOI: 10.3892/mmr.2014.3037 Following the publication of this article, an interested reader drew to our attention some anomalies associated with the presentation of Fig. 7. In examining our original data, we identified that the upper and lower panels of 'D2' in Fig. 7A have been erroneously reproduced from the same image, which was derived from the negative control of the P19 cells ('vector'). A corrected version of the Figure is presented below, featuring the data which correctly correspond to day 2 ('D2') for the microRNA-19b knockdown experiment. We also checked the other figures very carefully, and failed to identify any further errors. We thank the reader of our article for drawing this matter to our attention, and we would like to offer our sincere apologies for this mistake, and our regrets for any inconvenience caused by this error.

New views and new tools for complex diseases

The non-transmittable chronic diseases such as cancer, cardiovascular, metabolic and neurodegeneration diseases already become the major threats to the mankind. All of these chronic diseases are recognized as complex diseases, which involve in multiple genetic factors, environment factors and the interaction of these two kinds of the factors, and the complicated molecular networks consisting of huge number of the genes and proteins. Therefore, it is clear that the conventional experimental biology that focuses on individual gene or protein is not enough to reveal the molecular mechanisms of complex diseases. The collection of six papers in this issue deliver new views to understand the molecular mechanisms of the initiation and progression of the complex diseases, and might provide new powerful tools to fight these diseases. Conventional way often labels particular genes as ‘oncogene’ or ‘tumor suppressor gene’ for their tumorigenic potential. Wu’s group revealed that a famous tumor suppressor gene p53 activated by 5-aza-2 ′ -deoxycytidine (ZdCyd) resulted in high protumorigenicity of mouse P19 embryonic carcinoma cells that are growing in suspension condition, whereas the ZdCyd-induced p53 activation resulted in suppression of tumorigenic potential of P19 EC cells growing on adherent condition. Based on their developed computational method, the authors revealed two different p53-dominant gene-regulatory networks of ZdCyd-treated P19 EC cells at adherent and non-adherent growth conditions, respectively. This study implies that changing cellular contexts of mammalian cells from adherent to non-adherent growth conditions rewires p53-regulating networks that could convert the tumor suppression to tumor-promoting activity. In addition, Auberger’s group reported that Imatinib, an inhibitor of a chimeric tyrosine kinase BCR-ABL, as the leading compound to treat chronic myelogenous leukemia (CML), presented various inhibitory effects on different CML cells. They also found that the variation in the drug effects was derived from different molecular contexts in the CML subpopulations. Regenerative medicine is an important way to treat the chronic diseases such as heart disease, diabetes and Alzheimer’s disease. Stem cells and the related molecular mechanisms of cell reprogramming have been recognized as potential tools for regenerative medicine. Asuelime and Shi reviewed the recent progress on cell-lineage reprogramming and its potential in regenerative medicine. Although the researchers have achieved significant progress, these authors conclude that more studies on this area need to be done before the technology based on lineage reprogramming is ready to the clinical practice. In addition, Kumar’s group in a letter reported that a transcription factor, zinc-finger transcription factor in cerebellum-3 (Zic3), combined with other three transcriptional factors, could induce the conversion of human fibroblasts to neuroprogenitorlike cells in a rapid way; and they suggest that this new approach to reprogramming cells could be a potential tool for screening chemical compounds against neural diseases. Diabetes is a typical age-dependent chronic disease. In this issue, Lammert’s group reported that the up-regulation of an antioxidant protein, DJ-1, in mouse and human islets during aging with non-diabetic conditions, whereas the expression of DJ-1 could be reduced in pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Their further experimental data suggest that DJ-1 might serve as a novel drug target for T2DM. In another paper in this issue, Jia’s group showed that ficolin-3 as an innate immune protein presented a low serum level in the population with insulin-resistance based on a human cross-sectional study. The authors further proposed that serum ficolin-3 could be used as a biomarker for predicting the development of T2DM and even as a potential drugtarget for the treatment of T2DM. We hope that these papers will provide different angles from conventional views to understand the molecular mechanism of complex diseases and new potential tools against these diseases.

ShRNA-mediated gene silencing of AHR promotes the differentiation of P19 mouse embryonic carcinoma cells into cardiomyocytes

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix (bHLH) transcription factor that is activated by environmental contaminants including polychlorinated biphenyls (PCBs). The AHR affects a variety of processes that are involved in cell growth and differentiation. In this study, we constructed a P19 embryonic carcinoma cell line with AHR gene silencing using the vector-based approach of short hairpin (sh)RNA interference that allows cells to differentiate into cardiac myocytes when treated with dimethyl sulfoxide (DMSO). The expression levels of the cardiac development-specific GATA4 and Nkx2.5 genes were measured using real-time quantitative polymerase chain reaction (qPCR). Our data showed that the expression levels of the GATA4 and Nkx2.5 genes were increased in the AHR-silenced P19 cells compared with the control groups. Four critical genes (ARNT, CYP1A1, GSK3β and β-catenin) expressed in the AHR and in the Wnt signaling pathway were also measured by qPCR. We found that the expression levels of ARNT, CYP1A1 and β-catenin were suppressed, whereas GSK3β expression was elevated in the AHR-silenced P19 cells. Therefore, it is possible that the silencing of AHR promotes the differentiation of P19 cells through the AHR and Wnt signal transduction pathway.

Rewiring drug-activated p53-regulatory network from suppressing to promoting tumorigenesis

Many of oncogenes and tumor suppressor genes have been found to exert variable and even opposing roles in different kinds of tumors or at different stages of cancer development. Here we showed that tumorigenic potential of mouse embryonic carcinoma P19 cells cultured in adherent plates (attached-P19-cells) was suppressed by a chemotherapeutic agent, 5-aza-2'-deoxycytidine (ZdCyd), whereas the higher pro-tumorigenicity of P19 cells growing in suspension (detached-P19-cells) was generated by the ZdCyd treatment. Surprisingly, p53 activity was highly up-regulated by ZdCyd in both growing conditions. By our developed computational approaches, we revealed that there was a significant enrichment of apoptotic pathways in the ZdCyd-induced p53-dominant gene-regulatory network in attached P19 cells, whereas the pro-survival genes were significantly enriched in the ZdCyd-induced p53 network in detached P19 cells. The protein-protein interaction network of the ZdCyd-treated detached P19 cells was significantly different from that of ZdCyd-treated attached P19 cells. On the other hand, inhibition of p53 expression by siRNA suppressed the ZdCyd-induced tumorigenesis of detached P19 cells, suggesting that the ZdCyd-activated p53 plays oncogenic function in detached P19 cells. Taken together, these results indicate a context-dependent role for the ZdCyd-activated p53-dominant network in tumorigenesis.

Caudal-related Homeobox (Cdx) Protein-dependent Integration of Canonical Wnt Signaling on Paired-box 3 (Pax3) Neural Crest Enhancer

One of the earliest events in neural crest development takes place at the neural plate border and consists in the induction of Pax3 expression by posteriorizing Wnt·β-catenin signaling. The molecular mechanism of this regulation is not well understood, but several observations suggest a role for posteriorizing Cdx transcription factors (Cdx1/2/4) in this process. Cdx genes are known as integrators of posteriorizing signals from Wnt, retinoic acid, and FGF pathways. In this work, we report that Wnt-mediated regulation of murine Pax3 expression is indirect and involves Cdx proteins as intermediates. We show that Pax3 transcripts co-localize with Cdx proteins in the posterior neurectoderm and that neural Pax3 expression is reduced in Cdx1-null embryos. Using Wnt3a-treated P19 cells and neural crest-derived Neuro2a cells, we demonstrate that Pax3 expression is induced by the Wnt-Cdx pathway. Co-transfection analyses, electrophoretic mobility shift assays, chromatin immunoprecipitation, and transgenic studies further indicate that Cdx proteins operate via direct binding to an evolutionarily conserved neural crest enhancer of the Pax3 proximal promoter. Taken together, these results suggest a novel neural function for Cdx proteins within the gene regulatory network controlling neural crest development.

Nkx1-2 is a transcriptional repressor and is essential for the activation of Brachyury in P19 mouse embryonal carcinoma cell

Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.