Induction of CAT gene expression on a plasmid vector (L factor) by retinoic acid in mouse embryonal carcinoma (F9) cells

We reported previously that composite DNA constructed from a mammalian plasmid (L factor) and foreign gene can be reestablished as a plasmid in mouse embryonal carcinoma (F9) cells after transfection and the plasmid-bearing F9 cells undergo normal in vitro differentiation in response to retinoic acid, an inducer for F9 cell differentiation. We constructed F9 cells bearing plasmidal L factor DNA in which a reporter (chloramphenicol acetyltransferase; CAT) gene was placed under the control of a differentiation-responsive viral (Moloney murine leukemia virus or simian virus 40) enhancer-promoter. When such plasmid-bearing cells were treated with retinoic acid, the CAT gene was inducibly expressed. These results indicate that mammalian gene expression can be studied with the plasmidal expression vector which is structurally dissociated from complex chromosomes.     

Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.     

Amplification of plasmid (L factor) DNA and increased production of a plasmid gene product (gamma-interferon) in mouse L cells.

It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase (dhfr) gene, and gamma-interferon (gamma-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of gamma-IFN compared with the original clone, which was likely to be derived from the plasmid gamma-IFN gene amplified along with L factor and the dhfr gene.     

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.     

Neuronal differentiation in F9 embryonal carcinoma cells

F9 line embryonal carcinoma cells were induced to differentiate into neural direction by long-term treatment of monolayer cultures with retinoic acid and dibutyryl cyclic AMP. Bi- and multi-polar cells appeared, expressing acetylcholinesterase and neurofilament proteins but not markers of glial differentiation including GFA-protein. Nerve growth factor combined with both retinoic acid and dibutyryl cyclic AMP greatly enhanced the development of neuron-like morphology and induced expression of immunoreactivity to tyrosine hydroxylase as well as to Leu-encephalin-like peptides. Similarly, serotonin-like immunofluorescence but not substance P-like immunoreactivity was demonstrable in such cultures. In addition, synaptic-like vesicles were often found in the processes. Analysis of matrix expression in neuronally differentiated F9 cells revealed marked increase in laminin production, as judged by immunofluorescence and immuno-electron microscopy, but no demonstrable intracellular staining for fibronectin or type IV collagen. The results with neuronal cells contrast with the expression of all the three matrix components in endodermally differentiating F9 cells in the same cultures.     

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.     

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.     

Cellular HSP90 (HSP86) mRNA level and in vitro differentiation of mouse embryonal carcinoma (F9) cells

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.     

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.     

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.     

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.     

Growth conditions of F9 embryonal carcinoma cells affect the degree of DNA methylation

We have investigated differences in C^*pG methylation between F9 embryonal carcinoma cellsin vitro and as tumor cells grownin vivo usingMsp I andHpa II restriction isoschizomers. Southerns were hybridized with two low copy number probes, mouse major -globin (f7) and a class I, histocompatibility-2 cDNA clone (pH-2^d-4). In each case, the tumor-DNA was hypomethylated while the DNA from F9 cells grownin vitro was moderately methylated. We conclude that growth conditions or cell-cell interactions can greatly affect methylation of C^*pG sites.