Differentiation of F9 embryonal carcinoma cells

We found that monolayer cultures of F9 cells induced to differentiate with trans-retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step.     

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells

The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 μM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [³⁵S]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.     

Pluripotent differentiation of single F9 embryonal carcinoma cells

F9 embryonal carcinoma cells were induced to form a variety of differentiated cell types in monolayer culture. Cells with the morphological, histochemical and immunocytochemical properties of parietal and visceral endoderm, neurones and adipocytes were identified. Cells expressing Thy-1 antigen and large, multinucleated cells expressing cytoplasmic fibronectin were also observed. Various cell types were found together in colonies derived from individual F9 cells, allowing us to conclude that F9 cells are pluripotent in vitro.     

Retroviral vector gene expression in F9 embryonal carcinoma cells.

When F9 embryonal carcinoma (EC) cells are infected with retroviral vectors, the efficiency of expression of selectable genes is considerably lower than that in mouse fibroblasts infected with the same retroviral vectors. In this study, several retroviral vectors with regulatory sequences placed immediately 5' to a selectable gene were constructed, packaged, and used to infect mouse fibroblasts and F9 EC cells. With selection as an assay, there was a hierarchy of relative expression in F9 cells compared with that in mouse fibroblasts. These internally placed regulatory sequences are the source of the mRNAs detected in F9 EC cells, while both retroviral long-terminal-repeat promoters and internal promoters are the source of steady-state mRNAs in mouse fibroblasts. This effect was observable with both the internally placed herpes simplex virus thymidine kinase promoter and the Moloney murine leukemia virus promoter.     

Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.     

New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells.

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.     

Undifferentiated F9 embryonal carcinoma cells produce a short-chain collagen molecule.

The undifferentiated F9 embryonal carcinoma cells produce a unique collagen that decreases in amount during retinoic acid-induced differentiation of F9 cells into basement-membrane parietal endoderm. A bacterial-collagenase-sensitive protein of approx. 60,000 Da was resolved on polyacrylamide-gel electrophoresis. After pepsin digestion, two pepsin-resistant fragments containing hydroxyproline were demonstrated, suggesting that a portion of the molecule has a stable triple helix. The mRNA from the undifferentiated F9 cells translates a collagenase-sensitive protein with a molecular mass consistent with the 60,000 Da collagenous protein produced by undifferentiated F8 cells.     

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells.

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.     

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

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