Differentiation Of F9 Embryonal Carcinoma Cells Research Articles

Antiproliferation and Redifferentiation in Thyroid Cancer Cell Lines by Polyphenol Phytochemicals

Thyroid carcinogenesis is accompanied by loss of thyroid-specific functions and refractory to radioiodine and thyroid stimulating hormone (TSH) suppression therapy. Redifferentiating agents have been shown to inhibit tumor growth and improve the response to conventional therapy. Polyphenol phytochemicals (PPs) in fruits and vegetables have been reported to inhibit cancer initiation, promotion, progression and induce redifferentiation in selected types. In this study we examined PPs induce redifferentiation in thyroid cancer cell lines. We investigated the effects of genistein, resveratrol, quercetin, kaempferol, and resorcinol on the F9 embryonal carcinoma cell differentiation model. The thyroid cancer cell lines, TPC-1, FTC-133, NPA, FRO, and ARO, displayed growth inhibition in response to genistein, resveratrol, quercetin. We further demonstrated that genistein decreased the dedifferention marker CD97 in NPA cells and resveratrol decreased CD97 in FTC-133, NPA, FRO cells and quercetin decreased CD97 in all cell lines. We observed increased expression of differentiation marker NIS in FTC-133 cells in response to genistein, and resveratrol but no change in NPA, FRO, ARO cells. Quercetin increased or induced NIS in FTC-133, NPA, FRO cells. These findings suggest that PPs may provide a useful therapeutic intervention in thyroid cancer redifferentiation therapy.

Stimulation of the murine type II transforming growth factor-beta receptor promoter by the transcription factor Egr-1.

Previous studies have demonstrated that differentiation of murine embryonal carcinoma (EC) cells leads to the appearance of high affinity receptors for transforming growth factor-beta (TGF-beta). Subsequently, it was demonstrated that differentiation of F9 EC cells leads to increases in the transcription of the type II TGF-beta-receptor gene (TbetaR-II) and leads to significant increases in the steady-state levels of TbetaR-II mRNA. Analysis of the human TbetaR-II promoter in F9-differentiated cells identified several cis-regulatory elements that influence the activity of the promoter, including a CRE/ATF site and a CCAAT box motif. In the work described in this report, we focused on the effect of the transcription factor Egr-1 on the murine TbetaR-II promoter. We have identified an Egr-1 response-element approximately 150 bp upstream of the major transcription start site of the murine TbetaR-II gene. We demonstrate by electrophoretic mobility shift analysis (EMSA) that this cis-regulatory element binds Egr-1, and we demonstrate that disruption of this site eliminates the response to Egr-1. As part of this analysis, we also examined the effect of Egr-1 on human TbetaR-II promoter. In contrast to a previous report, which reported that Egr-1 inhibits expression of human TbetaR-II promoter/reporter gene constructs, we did not observe an inhibitory effect of Egr-1 that was specific for the human TbetaR-II promoter. Taken together, the findings described in this report identify important differences between the human and the murine TbetaR-II promoter, and our findings identify an Egr-1 cis-regulatory element that is capable of stimulating the activity of the murine TbetaR-II promoter.

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

An analysis of F9 embryonal carcinoma cell differentiation into parietal endoderm using DNA microarrays

An analysis of F9 embryonal carcinoma cell differentiation into parietal endoderm using DNA microarrays

Transcriptional Regulation of the Transforming Growth Factor-β2 Promoter by cAMP-responsive Element-binding Protein (CREB) and Activating Transcription Factor-1 (ATF-1) Is Modulated by Protein Kinases and the Coactivators p300 and CREB-binding Protein

Transcription of the transforming growth factor-beta2 (TGF-beta2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-beta2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-beta2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-beta2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-beta2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-beta2 gene.

Interdependent action of RalGEF and Erk in Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells.

Previous work by us and others has implicated a role for Ral guanine exchange factors (RalGEFs) in Ras-induced cell growth and oncogenic transformation. Here we show for the first time that RalGEFs are involved in Ras-induced differentiation as well. Expression of oncogenic Ras in F9 embryonal carcinoma (EC) cells is known to induce differentiation to a primitive endoderm (PrE)-like phenotype, but the downstream signal transduction mechanisms involved are unclear. We found that PrE differentiation is induced by the Ras effector domain mutants, RasV12G37 and RasV12E38, but not by RasV12C40. Accordingly, expression of constitutively active forms of RalGEF (Rlf-CAAX) or Rafl (Raf-CAAX) is sufficient to induce differentiation. Inhibition of RalGEF activity by expression of dominant negative Ral completely abolishes Rlf-CAAX- and RasV12G37-induced differentiation, while it reduces differentiation by RasV12 and Raf-CAAX. Finally, while Rlf-CAAX does not increase Erk activity, inhibition of MEK blocks both Ras- as well as Rlf-CAAX-induced differentiation, suggesting that RalGEFs induce PrE differentiation in a manner depending on basal MEK or Erk activity. Based on these results we conclude that Ras induces PrE differentiation of F9 EC cells via an interplay of Erk-and RalGEF-mediated pathways.

The Ras/Erk Pathway Induces Primitive Endoderm but Prevents Parietal Endoderm Differentiation of F9 Embryonal Carcinoma Cells

The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear. We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK. In conclusion, our data suggest that activation of the Ras/Erk is sufficient to induce differentiation to PrE and to prevent subsequent differentiation toward PE. Activation of PKA down-regulates Ras activity, resulting in disappearance of this blockade and transmission of signal(s) triggering PE differentiation.

RXRalpha-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.

Gene expression in visceral endoderm: a comparison of mutant and wild-type F9 embryonal carcinoma cell differentiation.

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.

Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation

B2 genes are rodent-specific middle repetitive elements transcribed by RNA polymerase III. They are expressed in the ectoderm and mesoderm but not in the embryonic or extraembryonic endoderm of early mouse embryos. This tissue specificity is mimicked in vitro by embryonal carcinoma and embryonic stem cell lines. Nuclear run-on experiments show that the down-regulation of B2 genes during F9 embryonal carcinoma cell differentiation into endoderm occurs at the transcriptional level and that other class III genes, including those encoding tRNA, show a similar response. We have used cell-free extracts to investigate the molecular mechanisms responsible. The specific down-regulation of transcription by RNA polymerase III during F9 cell differentiation is due to a reduction in the activity of the general class III transcription factor TFIIIB.

Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells.

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.

Biological activity of retinoids correlates with affinity for nuclear receptors but not for cytosolic binding protein.

In order to better understand the respective roles of the nuclear retinoic acid receptors (RARs) and the cytosolic retinoic acid binding protein (CRABP) in the mode of action of retinoic acid (RA), several types of RA analogs have been synthesized. Representative compounds have been radiolabeled to a high specific activity and their binding (direct and competition) to RARs and CRABP was determined. Their biological activity on F9 embryonal carcinoma cell differentiation has been determined by a quantitative assay of plasminogen activator (PA). All biologically active analogs studied in this work bound to RARs. A good correlation was found between PA induction and affinity for the RARs, with the exception of RA itself which was a good ligand but a moderate inducer of F9 differentiation. Two biologically active analogs (compounds II and III) did not bind to the CRABP. One biologically inactive analog (compound VIII) bound to CRABP. These results strongly suggest that retinoids must bind to RARs but not necessarily to CRABP in order to induce cell differentiation in F9 cells.

DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells

Restriction fragments encompassing the DNA sequence changes, which allow polyoma mutants to productively infect embryonal carcinoma (EC) cells, have been coupled to the Herpes simplex virus thymidine kinase (HSV TK) gene and to the bacterial chloramphenicol acetyl transferase (CAT) gene. F9 TK(−) EC cells have been transfected with the TK constructions and transformation to TK(+) colonies has been determined. F9 EC cells, retinoic-acid-induced differentiating F9 cultures, mouse fibroblasts, and mouse myoblasts have been transfected with the CAT constructions and CAT enzyme activity has been measured from the transfected cells. The results suggest that the mutant changes function at the level of gene-expression enhancement, the mutant constructions increasing the TK transformation frequency and also yielding a higher level of CAT enzyme activity when compared with constructions having no polyoma fragment or the analogous wildtype polyoma restriction fragment. The sequence specificity for enhancement changes upon differentiation of F9 EC cells, with the CAT enzyme levels from the wild-type construction approaching the values obtained from a PyEC mutant construction having a single base-pair difference.