scholarly journals Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation.

1989 ◽  
Vol 108 (6) ◽  
pp. 2467-2476 ◽  
Author(s):  
S J Braunhut ◽  
L J Gudas ◽  
T Kurokawa ◽  
J Sasse ◽  
P A D'Amore
Keyword(s):  
Stem Cells ◽  
Cdna Probe ◽  
Biologically Active ◽  
Endothelial Cell Proliferation ◽  
Fibroblast Growth ◽  
Basic Fgf ◽  
F9 Cells ◽  
Parietal Endoderm ◽  
F9 Teratocarcinoma ◽  
Proliferation Assays

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.

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

2017 ◽  
Vol 95 (2) ◽  
pp. 251-262 ◽  
Author(s):  
Gregory Golenia ◽  
Mohamed I. Gatie ◽  
Gregory M. Kelly
Keyword(s):  
Feedback Loop ◽  
Negative Regulation ◽  
Negative Feedback Loop ◽  
Primitive Endoderm ◽  
F9 Cells ◽  
Knock Down ◽  
Teratocarcinoma Cells ◽  
Parietal Endoderm ◽  
F9 Teratocarcinoma ◽  
Canonical Wnt

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.

c-myc regulation during retinoic acid-induced differentiation of F9 cells is posttranscriptional and associated with growth arrest

1986 ◽  
Vol 6 (2) ◽  
pp. 518-524
Author(s):  
M Dean ◽  
R A Levine ◽  
J Campisi
Keyword(s):  
Retinoic Acid ◽  
Cyclic Amp ◽  
Posttranscriptional Regulation ◽  
Growth Arrest ◽  
Mrna Levels ◽  
F9 Cells ◽  
Induction Of Differentiation ◽  
Parietal Endoderm ◽  
Arrest Growth ◽  
F9 Teratocarcinoma

We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.

Oxygen affects human endothelial cell proliferation by inactivation of fibroblast growth factors

1992 ◽  
Vol 263 (3) ◽  
pp. L370-L375
Author(s):  
M. M. Grant ◽  
H. C. Koo ◽  
W. Rosenfeld
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Fibroblast Growth Factors ◽  
Endothelial Cell Proliferation ◽  
Complete Medium ◽  
Fibroblast Growth ◽  
Basic Fgf ◽  
Endothelial Cell Growth

The fibroblast growth factors (FGF), including endothelial cell growth factor (ECGF)/acidic FGF and basic FGF, are important modulators of endothelial cell replication in vitro and in vivo. Premature infants and adults with lung injuries are often treated with high levels of inspired O2, which can be necessary for survival but potentially injurious to developing lungs and in tissue repair following injury. Human umbilical artery and vein endothelial cells were grown in ECGF- or FGF-supplemented Medium 199 and exposed to ambient levels of O2 from 10 to 95%. Endothelial cell growth, measured by [3H]thymidine incorporation, was inhibited by increasing levels of O2 and ceased above 50% O2. Vein endothelial cells could recover from up to 24 h of hyperoxic exposure when given fresh medium, but not after 48 h. Artery-derived cells were more sensitive to O2 than were vein-derived cells. Complete medium without endothelial cells, preincubated 24 h in 95% O2, lost its ability to support cell growth under normoxic conditions. Exposing individual medium components to high O2 demonstrated that purified natural ECGF and recombinant acidic or basic FGF were all inactivated by O2. Human recombinant superoxide dismutase prevented FGF inactivation. O2 inactivation of essential growth factors could thus have major consequences for lung development or repair of injured capillaries in infants or adults inspiring high levels of O2

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture

2015 ◽  
Vol 25 (6) ◽  
pp. 372-380 ◽  
Author(s):  
Sumeth Imsoonthornruksa ◽  
Kamthorn Pruksananonda ◽  
Rangsun Parnpai ◽  
Ruttachuk Rungsiwiwut ◽  
Mariena Ketudat-Cairns
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Fusion Proteins ◽  
Embryonic Stem ◽  
Biologically Active ◽  
Fibroblast Growth ◽  
Simple Method

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in <i>Escherichia coli</i> was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells

1993 ◽  
Vol 13 (5) ◽  
pp. 2919-2928
Author(s):  
B A Hosler ◽  
M B Rogers ◽  
C A Kozak ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Zinc Finger ◽  
Chromosome 8 ◽  
Lacz Gene ◽  
F9 Cells ◽  
Zinc Finger Gene ◽  
Teratocarcinoma Cells ◽  
Octamer Motif ◽  
F9 Teratocarcinoma

The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage.

scholarly journals An octamer motif contributes to the expression of the retinoic acid-regulated zinc finger gene Rex-1 (Zfp-42) in F9 teratocarcinoma cells.

1993 ◽  
Vol 13 (5) ◽  
pp. 2919-2928 ◽  
Author(s):  
B A Hosler ◽  
M B Rogers ◽  
C A Kozak ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Zinc Finger ◽  
Chromosome 8 ◽  
Lacz Gene ◽  
F9 Cells ◽  
Zinc Finger Gene ◽  
Teratocarcinoma Cells ◽  
Octamer Motif ◽  
F9 Teratocarcinoma

The message for the zinc finger gene Rex-1 (Zfp-42) is expressed in undifferentiated murine F9 teratocarcinoma cells and embryonic stem cells. Expression of Rex-1 is reduced at the transcriptional level when F9 cells are induced by the addition of retinoic acid (RA) to differentiate. We have isolated genomic DNA for the Rex-1 gene (Zfp-42), characterized the gene's structure, and mapped the gene to mouse chromosome 8. Promoter elements contributing to the regulation of the Rex-1 promoter in F9 cells have been identified. A region required for Rex-1 promoter activity in F9 stem cells contains an octamer motif (ATTTGCAT) which is a binding site for octamer transcription factor members of the POU domain family of DNA-binding proteins. Rex-1 reporter plasmids including this octamer site also exhibited reduced expression in F9 cells treated with RA. Thus, the octamer motif is a regulatory element required for the activity of the Rex-1 promoter in F9 stem cells, and this motif contributes to the negative regulation by RA of the transcription of the Rex-1 gene. As an initial confirmation of the in vivo relevance of the isolated fragment, a larger Rex-1 promoter fragment, also containing the octamer site, was able to promote expression of the bacterial lacZ gene in mouse embryos at the morula stage.

Targeting angiogenesis driven by fibroblast growth factor using RPT835, an FGFR2 inhibitor.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22097-e22097
Author(s):  
Evgenia Stepanova ◽  
Eliso Solomko ◽  
Oxana Ryabaya ◽  
Nina Peretolchina ◽  
Ilya Tsimafeyeu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Umbilical Vein ◽  
Endothelial Cell Proliferation ◽  
Control Group ◽  
Fibroblast Growth ◽  
Basic Fgf ◽  
Signaling Axis

e22097 Background: The fibroblast growth factor (FGF)/FGF receptors (FGFR) signaling axis plays a key role in driving tumor angiogenesis. There is little data on the targeting of FGF-induced angiogenesis. We describe here the targeting angiogenesis driven by FGF using RPT835, novel inhibitor of the FGFR2 extracellular domain (received from RusPharmTech, LLC). Methods: To assess the efficacy of RPT835 on FGF-mediated endothelial cells proliferation, the human umbilical vein endothelial FGFR-expressing cells (HUVEC) were incubated in a 96-well microculture plate and were treated with serially diluted RPT835 or Brivanib as a control. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega’s Cell Titer-Glo assay. In vivo angiogenesis was measured with subcutaneously implanted Matrigel plugs containing bFGF (100 ng/ml) or bFGF (100 ng/ml) + bevacizumab (10 mg/kg) or bFGF (100 ng/ml) + RPT835 (15 mg/kg). Control group was without stimulation and treatment. Each group included 3 mice. Number of endothelial cells/vessels was calculated. Results: Basic FGF significantly increased proliferation of the HUVEC cells (P=0.001) in untreated control group. RPT835 significantly inhibited FGF-triggered endothelial cell proliferation when compared with control (P<0.001) or brivanib (P<0.001, IC50=289 nmol/L) with IC50 of 11 nmol/L. In vivo, bFGF induced proliferation of endotheliocytes and mature vessels formation (P<0.001). There were no vessels in FGFR2 inhibitor group. Bevacizumab did not decrease number of vessels in comparison with FGF-stimulated angiogenesis (P=0.9). Conclusions: Inhibition of bFGF/FGFR2 pathway resulted in effects on endothelial cells proliferation andmature vessels formation. Bevacizumab had no activity in FGF-induced angiogenesis.

scholarly journals c-myc regulation during retinoic acid-induced differentiation of F9 cells is posttranscriptional and associated with growth arrest.

1986 ◽  
Vol 6 (2) ◽  
pp. 518-524 ◽  
Author(s):  
M Dean ◽  
R A Levine ◽  
J Campisi
Keyword(s):  
Retinoic Acid ◽  
Cyclic Amp ◽  
Posttranscriptional Regulation ◽  
Growth Arrest ◽  
Mrna Levels ◽  
F9 Cells ◽  
Induction Of Differentiation ◽  
Parietal Endoderm ◽  
Arrest Growth ◽  
F9 Teratocarcinoma

We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.

Use of F9 teratocarcinoma STEM cells to study cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 602-603
Author(s):  
Marc Lenburg ◽  
Rulang Jiang ◽  
Lengya Cheng ◽  
Laura Grabel
Keyword(s):  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Embryoid Bodies ◽  
F9 Cells ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Cell Matrix Interactions ◽  
F9 Teratocarcinoma

We are interested in defining the cell-cell and cell-matrix interactions that help direct the differentiation of extraembryonic endoderm in the peri-implantation mouse embryo. At the blastocyst stage the mouse embryo consists of an outer layer of trophectoderm surrounding the fluid-filled blastocoel cavity and an eccentrically located inner cell mass. On the free surface of the inner cell mass, facing the blastocoel cavity, a layer of primitive endoderm forms. Primitive endoderm then generates two distinct cell types; parietal endoderm (PE) which migrates along the inner surface of the trophectoderm and secretes large amounts of basement membrane components as well as tissue-type plasminogen activator (tPA), and visceral endoderm (VE), a columnar epithelial layer characterized by tight junctions, microvilli, and the synthesis and secretion of α-fetoprotein. As these events occur after implantation, we have turned to the F9 teratocarcinoma system as an in vitro model for examining the differentiation of these cell types. When F9 cells are treated in monolayer with retinoic acid plus cyclic-AMP, they differentiate into PE. In contrast, when F9 cells are treated in suspension with retinoic acid, they form embryoid bodies (EBs) which consist of an outer layer of VE and an inner core of undifferentiated stem cells. In addition, we have established that when VE containing embryoid bodies are plated on a fibronectin coated substrate, PE migrates onto the matrix and this interaction is inhibited by RGDS as well as antibodies directed against the β1 integrin subunit. This transition is accompanied by a significant increase in the level of tPA in the PE cells. Thus, the outgrowth system provides a spatially appropriate model for studying the differentiation and migration of PE from a VE precursor.

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