F9 Teratocarcinoma Stem Cells Research Articles

Expression of Chrna9 is regulated by Tbx3 in undifferentiated pluripotent stem cells

It was reported that nicotinic acetylcholine receptor (nAChR)-mediated signaling pathways affect the proliferation and differentiation of pluripotent stem cells. However, detail expression profiles of nAChR genes were unrevealed in these cells. In this study, we comprehensively investigated the gene expression of α subunit of nAChRs (Chrna) during differentiation and induction of pluripotent stem cells. Mouse embryonic stem (ES) cells expressed multiple Chrna genes (Chrna3-5, 7 and 9) in undifferentiated status. Among them, Chrna9 was markedly down-regulated upon the differentiation into mesenchymal cell lineage. In mouse tissues and cells, Chrna9 was mainly expressed in testes, ES cells and embryonal F9 teratocarcinoma stem cells. Expression of Chrna9 gene was acutely reduced during differentiation of ES and F9 cells within 24 h. In contrast, Chrna9 expression was increased in induced pluripotent stem cells established from mouse embryonic fibroblast. It was shown by the reporter assays that T element-like sequence in the promoter region of Chrna9 gene is important for its activities in ES cells. Chrna9 was markedly reduced by siRNA-mediated knockdown of Tbx3, a pluripotency-related transcription factor of the T-box gene family. These results indicate that Chrna9 is a nAChR gene that are transcriptionally regulated by Tbx3 in undifferentiated pluripotent cells.

Biocompatible Gold Nanoparticles Ameliorate Retinoic Acid-Induced Cell Death and Induce Differentiation in F9 Teratocarcinoma Stem Cells.

The unique properties of gold nanoparticles (AuNPs) have attracted much interest for a range of applications, including biomedical applications in the cosmetic industry. The current study assessed the anti-oxidative effect of AuNPs against retinoic acid (RA)-induced loss of cell viability; cell proliferation; expression of oxidative and anti-oxidative stress markers, pro- and anti-apoptotic genes, and differentiation markers; and mitochondrial dysfunction in F9 teratocarcinoma stem cells. AuNPs were prepared by reduction of gold salts using luteolin as a reducing and stabilizing agent. The prepared AuNPs were spherical in shape with an average diameter of 18 nm. F9 cells exposed to various concentrations of these AuNPs were not harmed, whereas cells exposed to RA exhibited a dose-dependent change in cell viability and cell proliferation. The RA-mediated toxicity was associated with increased leakage of lactate dehydrogenase, reactive oxygen species, increased levels of malondialdehyde and nitric oxide, loss of mitochondrial membrane potential, and a reduced level of ATP. Finally, RA increased the level of pro-apoptotic gene expression and decreased the expression of anti-apoptotic genes. Interestingly, the toxic effect of RA appeared to be decreased in cells treated with RA in the presence of AuNPs, which was coincident with the increased levels of anti-oxidant markers including thioredoxin, glutathione peroxidases, glutathione, glutathione disulfide, catalase, and superoxide dismutase. Concomitantly, AuNPs ameliorated the apoptotic response by decreasing the mRNA expression of p53, p21, Bax, Bak, caspase-3, caspase-9, and increasing the expressions of Bcl-2 and Bcl-Xl. Interestingly, AuNPs not only ameliorated oxidative stress but also induced differentiation in F9 cells by increasing the expression of differentiation markers including retinoic acid binding protein, laminin 1, collagen type IV, and Gata 6 and decreasing the expressions of markers of stem cell pluripotency including Nanog, Rex1, octamer-binding transcription factor 4, and Sox-2. These consistent cellular and biochemical data suggest that AuNPs could ameliorate RA-induced cell death and facilitate F9 cell differentiation. AuNPs could be suitable therapeutic agents for the treatment of oxidative stress-related diseases such as atherosclerosis, cancer, diabetes, rheumatoid arthritis, and neurodegenerative diseases.

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

BackgroundSilver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells.Materials and methodsAgNPs were synthesized and characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively.ResultsThe AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm). High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation.ConclusionThe results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner. Therefore, AgNPs can be used for differentiation therapy, along with chemotherapeutic agents, for improving cancer treatment by targeting specific chemotherapy-resistant cells within a tumor. Furthermore, understanding the molecular mechanisms of apoptosis and differentiation in stem cells could also help in developing new strategies for cancer stem cell (CSC) therapies. The findings of this study could significantly contribute to the nanomedicine because this study is the first of its kind, and our results will lead to new strategies for cancer and CSC therapies.

Sema3F (Semaphorin 3F) Selectively Drives an Extraembryonic Proangiogenic Program.

Molecular pathways governing blood vessel patterning are vital to vertebrate development. Because of their ability to counteract proangiogenic factors, antiangiogenic secreted Sema3 (class 3 semaphorins) control embryonic vascular morphogenesis. However, if and how Sema3 may play a role in the control of extraembryonic vascular development is presently unknown. By characterizing genetically modified mice, here, we show that surprisingly Sema3F acts instead as a selective extraembryonic, but not intraembryonic proangiogenic cue. Both in vivo and in vitro, in visceral yolk sac epithelial cells, Sema3F signals to inhibit the phosphorylation-dependent degradation of Myc, a transcription factor that drives the expression of proangiogenic genes, such as the microRNA cluster 17/92. In Sema3f-null yolk sacs, the transcription of Myc-regulated microRNA 17/92 cluster members is impaired, and the synthesis of Myc and microRNA 17/92 foremost antiangiogenic target Thbs1 (thrombospondin 1) is increased, whereas Vegf (vascular endothelial growth factor) signaling is inhibited in yolk sac endothelial cells. Consistently, exogenous recombinant Sema3F inhibits the phosphorylation-dependent degradation of Myc and the synthesis of Thbs1 in mouse F9 teratocarcinoma stem cells that were in vitro differentiated in visceral yolk sac epithelial cells. Sema3f-/- mice placentas are also highly anemic and abnormally vascularized. Sema3F functions as an unconventional Sema3 that promotes extraembryonic angiogenesis by inhibiting the Myc-regulated synthesis of Thbs1 in visceral yolk sac epithelial cells.

Potential Significance of EMP3 in Patients with Upper Urinary Tract Urothelial Carcinoma: Crosstalk with ErbB2-PI3K-Akt Pathway

Upper urinary tract (pyelocalyceal cavities and ureter) urothelial carcinoma is a relatively rare neoplastic disease. Although diagnosis and treatment of this tumor variant have improved significantly, accurate risk stratification remains a challenge. To identify the putative oncogene involved in urothelial carcinoma progression we performed bioinformatics guided experimental investigation targeting chromosome 19q13. We investigated the effects of EMP3 on cancer cell growth, migration and adhesion in transfection and siRNA experiments in vitro. Crosstalk of integrins or ErbB2 with EMP3 was examined by reverse transcriptase-polymerase chain reaction and immunoblot. The potential involvement of epigenetic alterations of EMP3 in vitro and in vivo was analyzed by methylation specific polymerase chain reaction. To validate clinical relevance we measured EMP3 expression at the mRNA and protein levels in a cohort of 77 patients with upper urinary tract urothelial carcinoma and compared prognostic significance in relation to that of ErbB2 expression. We noted functional crosstalk between ErbB2 and EMP3 in vitro. EMP3 over expression promoted cancer cell proliferation and migration but suppressed cell adhesion in vitro. EMP3 activated the ErbB2-PI3K-AKT pathway to increase cell growth in vitro. In the clinical cohort Kaplan-Meier survival estimates showed that ErbB2 and EMP3 co-expression was the most important indicator of progression-free and metastasis-free survival in patients with upper urinary tract urothelial carcinoma (log rank test p = 0.018 and 0.04, respectively). EMP3 is an important prognostic indicator for selecting patients with upper urinary tract urothelial carcinoma for more intensive therapy. EMP3 is an innovative co-targeting candidate for designing ErbB2 based cancer therapy.

Epigenetic Regulatory Mechanisms Distinguish Retinoic Acid-mediated Transcriptional Responses in Stem Cells and Fibroblasts

Retinoic acid (RA), a vitamin A metabolite, regulates transcription by binding to RA receptor (RAR) and retinoid X receptor (RXR) heterodimers. This transcriptional response is determined by receptor interactions with transcriptional regulators and chromatin modifying proteins. We compared transcriptional responses of three RA target genes (Hoxa1, Cyp26a1, RARbeta(2)) in primary embryo fibroblasts (mouse embryonic fibroblasts), immortalized fibroblasts (Balb/c3T3), and F9 teratocarcinoma stem cells. Hoxa1 and Cyp26a1 transcripts are not expressed, but RARbeta(2) transcripts are induced by RA in mouse embryonic fibroblasts and Balb/c3T3 cells. Retinoid receptors (RARgamma, RXRalpha), coactivators (pCIP (NCOA3, SRC3)), and p300 and RNA polymerase II are recruited only to the RARbeta(2) RA response element (RARE) in Balb/c3T3, whereas these proteins are recruited to RAREs of all three genes by RA in F9 cells. In F9, RA reduces polycomb (PcG) protein Suz12 and the associated H3K27me3 repressive epigenetic modification at the RAREs of all three genes. In contrast, in Balb/c3T3 cells cultured in the +/-RA, Suz12 is not associated with the Hoxa1, RARbeta(2), and Cyp26a1 RAREs, whereas slow levels of the H3K27me3 mark are seen at these RAREs. Thus, Suz12 is not required for gene repression in the absence of RA. Even though the Hoxa1 RARE and proximal promoter show high levels of H3K9,K14 acetylation in Balb/c3T3, the Hoxa1 gene is not transcriptionally activated by RA. In Balb/c3T3, CpG islands are methylated in the Cyp26a1 promoter region but not in the Hoxa1 promoter or in these promoters in F9 cells. We have delineated the complex mechanisms that control RA-mediated transcription in fibroblasts versus stem cells.

Transcriptional regulation of Rex1 (zfp42) in normal prostate epithelial cells and prostate cancer cells

Rex1 (zfp42) was identified by our laboratory because of its reduced expression in F9 teratocarcinoma stem cells after retinoic acid (RA) treatment. The Rex1 (Zfp42) gene is currently widely used as a marker of embryonic stem cells. We compared the transcriptional regulation of the human Rex1 gene in NTera-2 (NT-2) human teratocarcinoma, normal human prostate epithelial cells (PrEC), and prostate cancer cells (PC-3) by promoter/luciferase analyses. Oct4, Sox2, Nanog, and Dax1 transcripts are expressed at higher levels in NT-2 and PrEC cells than in PC-3 cells. Co-transfection analyses showed that YY1 and Rex1 are positive regulators of hRex1 transcription in NT-2 and PrEC cells, whereas Nanog is not. Serial deletion constructs of the hRex1 promoter were created and analyzed, by which we identified a potential negative regulatory site that is located between -1 and -0.4 kb of the hRex1 promoter. We also delineated regions of the hRex1 promoter between -0.4 kb and the TSS that, when mutated, reduced transcriptional activation; these are putative Rex1 binding sites. Mutation of a putative Rex1 binding site in electrophoretic mobility shift assays (EMSA) resulted in reduced protein binding. Taken together, our results indicate that hRex1 binds to the hRex1 promoter region at -298 bp and positively regulates hRex1 transcription, but that this regulation is lost in PC-3 human prostate cancer cells. This lack of positive transcriptional regulation by the hRex1 protein may be responsible for the lack of Rex1 expression in PC-3 prostate cancer cells.

Retinoic acid receptor γ activates receptor tyrosine kinase Tie1 gene transcription through transcription factor GATA4 in F9 stem cells

The retinoic acid receptors (RARs) alpha, beta2, and gamma regulate specific subsets of target genes during all-trans retinoic acid (RA) induced differentiation of F9 teratocarcinoma stem cells. The Tie1 gene exhibited reduced expression in RA-treated F9 RARgamma-/- cells as compared to wild-type (WT) by microarray analysis. Our goal was to analyze the Tie1 gene, which encodes a surface receptor tyrosine kinase expressed in the hematovascular system. We assessed Tie1, Tie2, Flk1, Runx1, Peg/Mest2, and angiopoietin-1 and 2 mRNA levels and Tie1 promoter activity. We showed that RARgamma, but not RARalpha or RARbeta2, is required for Tie1 promoter activation by RA. Treatment with a RARgamma selective agonist plus a retinoid X receptor agonist (LGD1069) increased Tie1 mRNA levels by 11- +/- 2.5-fold 48 hours after RA addition in F9 WT, but not in F9 RARgamma-/- cells, by quantitative reverse transcription polymerase chain reaction. Multiple putative GATA elements were identified in the Tie1 proximal promoter. RA increased GATA4 transcripts by 12- +/- 1-fold in F9 WT at 48 hours, but not in F9 RARgamma-/- cells. In addition, transfection of a GATA4 expression vector increased Tie1 promoter/luciferase activity in both RA-treated F9 WT and RARgamma-/- cells. Tie1 promoter deletion analyses indicated that a region of the promoter that possessed multiple GATA sites mediated the RA-associated Tie1 transcriptional increase. Our results indicate that GATA4 plays a role in the RA/RARgamma-associated transcriptional activation of the Tie1 promoter. An understanding of RAR specificity in RA signaling should result in insights into hematopoietic stem cell signaling and potentially in improved therapies for several human diseases.

Transcriptional Activation of the Suppressor of Cytokine Signaling-3 (SOCS-3) Gene via STAT3 Is Increased in F9 REX1 (ZFP-42) Knockout Teratocarcinoma Stem Cells Relative to Wild-type Cells

Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent embryonic stem cells. We identified Suppressor of Cytokine Signaling-3 ( SOCS-3) as a gene that exhibits greatly increased transcriptional activation in RA, cAMP, and theophylline (RACT)-treated F9 Rex1 −/− cells (∼ 25-fold) as compared to wild-type (WT) cells (∼ 2.5-fold). By promoter deletion, mutation, and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA-binding elements located between − 99 to − 60 in the SOCS-3 promoter. Overexpression of STAT3 dominant-negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1 −/− cells. This increase in SOCS-3 transcription is associated with a four- to fivefold higher level of tyrosine-phosphorylated STAT3 in the RACT-treated F9 Rex1 −/− cells as compared to WT. Dominant-negative Src tyrosine kinase, Jak2, and protein kinase A partially reduce the transcriptional activation of the SOCS 3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1 −/− cells, but not in F9 WT. Thus, Rex1, which is highly expressed in stem cells, inhibits signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, thereby modulating the differentiation of F9 cells.

Gene expression profiling elucidates a specific role for RARγ in the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

The biological effects of all- trans-retinoic acid (RA), a major active metabolite of retinol, are mainly mediated through its interactions with retinoic acid receptor (RARs α, β, γ) and retinoid X receptor (RXRs α, β, γ) heterodimers. RAR/RXR heterodimers activate transcription by binding to RA-response elements (RAREs or RXREs) in the promoters of primary target genes. Murine F9 teratocarcinoma stem cells have been widely used as a model for cellular differentiation and RA signaling during embryonic development. We identified and characterized genes that are differentially expressed in F9 wild type (Wt) and F9 RARγ −/− cells, with and without RA treatment, through the use of oligonucleotide-based microarrays. Our data indicate that RARγ, in the absence of exogenous RA, modulates gene expression. Genes such as Sfrp2, Tie1, Fbp2, Emp1, and Emp3 exhibited higher transcript levels in RA-treated Wt, RARα −/− and RARβ 2 −/− lines than in RA-treated RARγ −/− cells, and represent specific RARγ targets. Other genes, such as Runx1, were expressed at lower levels in both F9 RARβ 2 −/− and RARγ −/− cell lines than in F9 Wt and RARα −/−. Genes specifically induced by RA at 6 h with the protein synthesis inhibitor cycloheximide in F9 Wt, but not in RARγ −/− cells, included Hoxa3, Hoxa5, Gas1, Cyp26a1, Sfrp2, Fbp2, and Emp1. These genes represent specific primary RARγ targets in F9 cells. Several genes in the Wnt signaling pathway were regulated by RARγ. Delineation of the receptor-specific actions of RA with respect to cell proliferation and differentiation should result in more effective therapies with this drug.

Retinoic Acid Receptor Isotype Specificity in F9 Teratocarcinoma Stem Cells Results from the Differential Recruitment of Coregulators to Retinoic Acid Response Elements

The retinoic acid receptor (RAR) alpha, beta(2), and gamma isotypes each regulate specific subsets of target genes in F9 teratocarcinoma stem cells. We used chromatin immunoprecipitation assays to monitor the association of RARgamma, retinoic X receptor (RXR) alpha, and coregulators with the RARbeta(2), Hoxa1, and Cyp26A1 retinoic acid response elements (RAREs) in F9 wild type and RARalpha, -beta(2), and -gamma null cells. Additionally we quantitatively monitored expression of the corresponding mRNAs. We demonstrated that the association of RARgamma and/or RXRalpha with a RARE was not sufficient for retinoic acid (RA)-mediated transcription of the corresponding target gene. However, the ability of RARgamma and/or RXRalpha to recruit pCIP (AIB1/ACTR/RAC-3/TRAM-1/SRC-3) and p300 to a RARE did correlate with RA-associated transcription of target mRNAs. Therefore, the specific functions of the RAR isotypes do not manifest at the level of their DNA binding but rather from a differential ability to recruit specific components of the transcriptional machinery. We also demonstrated that RA-mediated displacement of the polycomb group protein SUZ12 from a RARE was inhibited in the absence of RARgamma. Thus, transcriptional components of the RAR signaling pathway are specifically required for displacement of SUZ12 from RAREs during RA-mediated differentiation of F9 cells.

A conserved organization of transcription during embryonic stem cell differentiation and in cells with high C value.

Although we have detailed information on the alterations occurring in steady-state levels of all cellular mRNAs during differentiation, we still know little about more global changes. Therefore, we investigated the numbers of molecules of RNA polymerase II that are active--and the way those molecules are organized--as two mouse cells (aneuploid F9 teratocarcinoma, and euploid and totipotent embryonic stem cells) differentiate into parietal endoderm. Quantitative immunoblotting shows the number of active molecules roughly halves. Transcription sites (detected by light and electron microscopy after allowing engaged polymerases to extend nascent transcripts in bromouridine-triphosphate) are uniformly distributed throughout the nucleoplasm. The numbers of such sites fall during differentiation as nuclei become smaller, but site density and diameter remain roughly constant. Similar site densities and diameters are found in salamander (amphibian) cells with 11-fold larger genomes, and in aneuploid HeLa cells. We conclude that active polymerases and their nascent transcripts are concentrated in a limited number of discrete nucleoplasmic sites or factories, and we speculate that the organization of transcription is conserved during both differentiation and evolution to a high C value.

Retinoic acid induces parietal endoderm but not primitive endoderm and visceral endoderm differentiation in F9 teratocarcinoma stem cells with a targeted deletion of the Rex-1 (Zfp-42) gene

Cultured murine F9 teratocarcinoma stem cells resemble pluripotent stem cells of the inner cell mass of the mouse blastocyst and, depending upon their treatment, can be induced to differentiate along the primitive endoderm, the parietal endoderm (PE), or the visceral endoderm (VE) pathway. The Rex-1 gene encodes a zinc finger family transcription factor which is expressed at high levels in undifferentiated F9 stem cells, embryonic stem cells, and in other types of stem cells. To examine whether the Rex-1 protein plays a role in F9 cell differentiation, homologous recombination was employed to generate F9 cell lines which lack both alleles of Rex-1. F9 wild type cells in monolayer culture require both retinoic acid and cyclic AMP analogs to differentiate into PE, whereas the F9 Rex-1 −/− cells differentiate into PE, as assessed by several molecular markers, including thrombomodulin and laminin B1, in the presence of RA alone. The F9 Rex-1 −/− cells do not completely differentiate into VE after RA treatment in aggregate culture; they do not express alpha-fetoprotein, a definitive marker of VE differentiation. These results indicate that the Rex-1 transcription factor regulates the differentiation of F9 stem cells along several distinct cell lineages found in the early embryo.

Molecular Cloning and Analysis of a Group of Genes Differentially Expressed in Cells Which Overexpress the Hoxa-1 Homeobox Gene

The homeobox gene Hoxa-1 is transcriptionally regulated by retinoic acid (RA) and encodes a transcription factor which has been shown to play important roles in cell differentiation and embryogenesis. In order to clone and characterize target genes of Hoxa-1, we utilized differential hybridization screening and cDNA subtractive hybridization methods to identify genes which are differentially expressed in F9-10, a murine F9 teratocarcinoma stem cell line which expresses high levels of exogenous Hoxa-1, compared to F9 wild-type stem cells, which do not express endogenous Hoxa-1 mRNA in the absence of RA. Twenty-eight candidate genes were identified; these genes encode very diverse proteins, including signaling molecules such as BMP-4, the enzyme superoxide dismutase, the cell adhesion molecule cadherin-6, proteins involved in gene transcription such as HMG-1 and SAP18, homeodomain-containing proteins Gbx-2 and Evx-2, and cell cycle regulatory proteins such as the retinoblastoma binding protein-2. Clone 104 encodes a novel protein; the expression of the clone 104 mRNA is also regulated in a fashion very similar to that of the exogenous Hoxa-1 gene in another F9 cell line, called F9-tet-Hoxa1-8, in which the exogenous Hoxa-1 mRNA expression is tightly regulated by a Tet-off gene expression system. These data strongly suggest that clone 104 is a direct downstream target of the transcription factor Hoxa-1. The cDNA sequence of clone 104 is related to that of human ubiquitin carboxyl-terminal hydrolase T. Further characterization of these putative Hoxa-1 target genes will aid in delineating the functions of the Hoxa-1 protein in the differentiation processes which occur during embryogenesis.

Polyamine depletion up-regulates c-Myc expression, yet induces G(1) arrest and terminal differentiation of F9 teratocarcinoma stem cells.

The ornithine decarboxylase (ODC) gene is a transcriptional target of c-Myc. Exponentially growing cells usually exhibit high c-Myc levels and high ODC levels, whereas stationary phase cells and terminally differentiated cells have low levels of both proteins. Therefore, we were surprised to find that when F9 teratocarcinoma stem cells were blocked in the G(1) phase of their cell cycle and induced to differentiate by irreversible inhibition of the ODC activity, the expression of c-Myc was up-regulated instead of being down-regulated. During the course of differentiation, the c-myc gene was constitutively expressed, and c-Myc protein accumulated. In transfection experiments, using ODC promoter-reporter gene fusion constructs, the accumulation of c-Myc protein, resulting from polyamine depletion, led to increased reporter gene expression. This finding is consistent with the view that depletion of polyamines relieves the suppression that they exert on c-myc mRNA translation, causing an accumulation of c-Myc protein, which in turn transactivates its target gene, the bona fide ODC gene. Thus, the accumulation of an active c-Myc protein does not preclude differentiative events, nor does it override the growth arrest caused by polyamine depletion. These results suggest a new role for polyamines-as negative regulators of c-Myc expression.

Activation of Rat Frizzled-1 Promotes Wnt Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Pathways That Require Gαq and Gαo Function

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.

Involvement of glucocorticoid receptor in the induction of differentiation by ginsenosides in F9 teratocarcinoma cells

We have previously reported that ginsenosides Rh1 and Rh2 induced the differentiation of F9 teratocarcinoma stem cells [Lee, Y. N., Lee, H. Y., Chung, H. Y., Kim, S. I., Lee, S. K., Park, B. C. and Kim, K. W., In vitro induction of differentiation by ginsenosides in F9 teratocarcinoma cells. Eur. J. Cancer 1996, 32, 1420–1428.]. Since the chemical structure of Rh1 and Rh2 is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether Rh1 and Rh2 act through the glucocorticoid receptor (GR). Immunocytochemistry showed that Rh1 or Rh2 increased the nuclear translocation of GR in the same manner of dexamethasone. In the gel shift assay, glucocorticoid response element (GRE) binding protein in F9 cells was increased by Rh1 or Rh2. To confirm whether the increased binding protein is GR, we performed the competition assay with unlabeled GRE as a specific competitor. Moreover, supershift assay with the GR antibody showed that the binding proteins are GR. In addition, to confirm the Rh1 or Rh2-induced transactivation of GRE promoter, we cotransfected GR expression vector and GRE-luciferase vector. In the luciferase assay, Rh1 or Rh2 potently induced luciferase activity and this induction was blocked by RU486, a potent GR antagonist. Taken together, we suggest that ginsenosides Rh1 and Rh2 may induce the differentiation of F9 cells by stimulating the nuclear translocation of GR.

Insulin receptor substrate‐1 is expressed at high levels in all cells of the peri‐implantation mouse embryo

Insulin and insulinlike growth factors are important for embryonic growth and metabolism. Intracellular transduction for these factors has not been studied in the preimplantation mouse embryo. Peri-implantation mouse embryos synthesize insulinlike growth factor (IGF)-II ligand, insulin receptor, IGF-I receptor, and IGF-II receptor and respond to IGF-II, IGF-I, and insulin metabolically and mitogenically. Maternal tissues in the oviduct and uterus are also sources of IGF-I and insulin. Signaling of IGFs occurs through insulin receptor substrate (IRS)-1 and IRS-2. This paper shows that IRS-1 mRNA and protein are highly expressed in preimplantation mouse embryos, in embryonic cell lines, and in cultured blastocyst outgrowths. IRS-1 mRNA and protein are detected in embryo-derived cell lines cultured to produce the three cell lineages (stem cells, endoderm, and trophoblast cells). IRS-1 mRNA is detected by reverse transcription-polymerase chain reaction (RT-PCR) in the E3.5 blastocyst before implantation and in F9 teratocarcinoma stem cells and parietal endoderm cells. IRS-1 mRNA is detected by Northern blot hybridization at high levels in stem cells and in differentiated progeny of F9 cells and C3H/NE trophectoderm cells. IRS-1 protein was detected in these cell lines and in an overexpressing CHO-IRS-1 fibroblast cell line by immunocytochemistry. Cultured blastocyst outgrowths are a model for implantation events of the trophoblast/placenta lineage and endoderm/yolk sac lineage. In the blastocyst outgrowth, IRS-1 protein is detected in inner cell mass cells (ICM cells), primitive endoderm, parietal endoderm, and trophectoderm cells. These data suggest that IRS-1 is expressed in all cell lineages of the peri-implantation mouse embryo and mediates some effects of insulin and IGFs at this stage. Mol. Reprod. Dev. 49:386–393, 1998. © 1998 Wiley-Liss, Inc.

Insulin receptor substrate-1 is expressed at high levels in all cells of the peri-implantation mouse embryo.

Insulin and insulinlike growth factors are important for embryonic growth and metabolism. Intracellular transduction for these factors has not been studied in the preimplantation mouse embryo. Peri-implantation mouse embryos synthesize insulinlike growth factor (IGF)-II ligand, insulin receptor, IGF-I receptor, and IGF-II receptor and respond to IGF-II, IGF-I, and insulin metabolically and mitogenically. Maternal tissues in the oviduct and uterus are also sources of IGF-I and insulin. Signaling of IGFs occurs through insulin receptor substrate (IRS)-1 and IRS-2. This paper shows that IRS-1 mRNA and protein are highly expressed in preimplantation mouse embryos, in embryonic cell lines, and in cultured blastocyst outgrowths. IRS-1 mRNA and protein are detected in embryo-derived cell lines cultured to produce the three cell lineages (stem cells, endoderm, and trophoblast cells). IRS-1 mRNA is detected by reverse transcription-polymerase chain reaction (RT-PCR) in the E3.5 blastocyst before implantation and in F9 teratocarcinoma stem cells and parietal endoderm cells. IRS-1 mRNA is detected by Northern blot hybridization at high levels in stem cells and in differentiated progeny of F9 cells and C3H/NE trophectoderm cells. IRS-1 protein was detected in these cell lines and in an overexpressing CHO-IRS-1 fibroblast cell line by immunocytochemistry. Cultured blastocyst outgrowths are a model for implantation events of the trophoblast/placenta lineage and endoderm/yolk sac lineage. In the blastocyst outgrowth, IRS-1 protein is detected in inner cell mass cells (ICM cells), primitive endoderm, parietal endoderm, and trophectoderm cells. These data suggest that IRS-1 is expressed in all cell lineages of the peri-implantation mouse embryo and mediates some effects of insulin and IGFs at this stage.

Increased expression of c-jun, but not retinoic acid receptor β, is associated with F9 teratocarcinoma stem cell differentiation induced by polyamine depletion

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, and all-trans-retinoic acid (RA) are known to induce F9 teratocarcinoma stem cell differentiation. Both compounds induce the formation of the same cell type, i.e., parietal endoderm-like cells expressing tissue plasminogen activator and collagen type IV alpha-1. The present study shows that DFMO and RA induce terminal differentiation of F9 cells through different pathways. Thus, retinoic acid receptor (RAR) alpha mRNA is weakly expressed during DFMO treatment, but strongly induced during an early phase of RA treatment. RAR beta mRNA is not detectable in DFMO-treated cells, but very strongly induced by RA and maintained at a high level throughout the differentiative process. RAR gamma mRNA is relatively strongly expressed in untreated control cells and remains at approximately the same level during DFMO-induced differentiation. In RA-treated cells, however, RAR gamma mRNA is rapidly down-regulated and becomes nondetectable during the final course of differentiation. These experiments show that the differentiation of F9 cells into parietal endoderm-like cells does not necessarily involve changes in any of the RAR mRNA subtypes. Even though the steady-state levels of the RAR alpha and RAR gamma transcripts may be sufficient to support the differentiative process, our data clearly show that induction of RAR beta mRNA transcription is neither a prerequisite for F9 cell differentiation, nor an absolute consequence of the elevated c-jun mRNA expression that is consistently observed during the course of parietal endoderm differentiation.