Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.     

Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.     

An increase in prolyl-4-hydroxylase activity occurs during the retinoic acid-induced differentiation of mouse teratocarcinoma stem cell lines F9 and P19

Monolayer cultures of F9 teratocarcinoma stem cells and P19 stem cells differentiate into endoderm, and fibroblast-like cells, respectively, when treated with retinoic acid. We demonstrate that this differentiation is associated with a large increase (greater than 40-fold) in the activity of an enzyme, prolyl-4-hydroxylase, involved in the posttranslational modification of collagens. This large increase in prolyl-4-hydroxylase activity occurs between 42 and 72 h after retinoic acid addition, and is associated with an increased amount of immunoprecipitable prolyl hydroxylase enzyme. This enzyme should be a useful marker for certain differentiated cell types produced during differentiation of teratocarcinoma stem cell lines.     

Modulation of protein biosynthesis during early stages of differentiation in retinoic acid treated F9 teratocarcinoma cells

Modulation of protein biosynthesis by retinoic acid during induction of differentiation of F9 teratocarcinoma stem cells was investigated by using computerized analysis of double label autoradiography of two-dimensional polyacrylamide gels. As early as 6 h after induction increased synthesis of 5 and decreased synthesis of 2 proteins occur. By 12 h after induction, synthesis of 13 proteins is elevated and by 24 h that of 17. At 24 h the range of stimulation is from two- to fourfold, as demonstrated by a 3H:14C ratio divided by the mode ratio. Examination of the Gaussian distributions of frequency of ratio indicates that many subtle changes in protein synthesis accompany the development of the new phenotype.     

The induction of differentiation in teratocarcinoma stem cells by retinoic acid.

Embryonal carcinoma cells, the stem cells of teratocarcinomas, usually undergo extensive differentiation in vivo and in vitro to a wide variety of cell types. There exist, however, several embryonal carcinoma cell lines that have almost completely lost the capacity to differentiate, so that the cells are propagated primarily as the stem cells. Using one such cell line, F9, we have found that retinoic acid at concentrations as low as 10(-9) M induces multiple phenotypic changes in the cultures in vitro. These changes include morphological alteration at the resolution of the light microscope, elevated levels of plasminogen activator production, sensitivity to cyclic AMP compounds and increased synthesis of collagen-like proteins. The nature of these changes, as well as their independence of the continued presence of retinoic acid, are consistent with the proposition that retinoic acid induces differentiation of embryonal carcinoma cells into endoderm.     

Changes of surface glycoproteins after retinoic acid-dibutyryl cAMP-induced differentiation of teratocarcinoma stem cells

Retinoic acid induces differentiation of embryonal carcinoma F9 cells into parietal endoderm. The surface proteins of F9 cells from induced and control cultures were labeled with the 125I-lactoperoxidase system and analyzed by two-dimensional gel electrophoresis. Their quantitative comparison has shown an 11-fold increase of protein p220 of apparent MW 220,000 and isoelectric point 5.6. Among other enhanced surface proteins, 3.5-fold increases of p50, p45, and p40 of MW 50,000-40,000 and isoelectric point 5.1-5.3 were observed. Simultaneously another surface protein, p70 of MW 70,000 and isoelectric point 6.1-6.3, disappeared. The quantitative changes of surface proteins produced after treatment with retinoic acid were enhanced in the presence of dibutyryl cAMP. Analysis of lectin-binding proteins demonstrated that increasing proteins p220, p50, p45, and p40 have an affinity for concanavalin A, whereas p70, which decreases, has an affinity for wheat germ agglutinin. Antibodies raised against p70 from undifferentiated cells have shown a specific immunoreaction with p220 from differentiated cells and also with the subunit B of purified laminin. The electrophoretic mobilities of p220 and of the B subunit of laminin are similar. It is suggested that p70, p220, and laminin B subunit share structural homology.     

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.     

Cyclic adenosine monophosphate-mediated induction of F9 teratocarcinoma differentiation in the absence of retinoic acid.

F9 teratocarcinoma stem cells differentiate into parietal endoderm-like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB-cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm-like cells in the absence of exogenous RA when treated with cholera toxin and 1-methyl,3-isobutyl xanthine (CT/MIX) or 8-bromo-cAMP/MIX (8B2-cAMP/MIX). Cells treated with CT/MIX or 8B2-cAMP/MIX were morphologically similar to parietal endoderm-like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage-specific embryonic antigen (SSEA-1) and an mRNA species of 6.8 kb (pST6-135) were markedly reduced in CT/MIX-treated cells. To prove that cAMP alone could induce differentiation Lipidex-1000, a hydrophobic gel, was used to remove 80-90% of the endogenous serum retinoids. F9 cells grown in this retinoid-depleted serum and treated with 8B2-cAMP/MIX differentiated to parietal endoderm-like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm-like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.     

The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.     

Integrin phosphorylation is modulated during the differentiation of F-9 teratocarcinoma stem cells

The retinoic acid-induced differentiation of F-9 teratocarcinoma cells in monolayer culture is accompanied by the accumulation of fibrillar fibronectin deposits, the appearance of a highly structured actin cytoskeleton, and the redistribution of integrin to apparent sites of substrate contact. We have studied the 140-kD fibronectin receptor during this process and report that although the integrin molecule is present in equivalent amounts before and after differentiation, the level of integrin phosphorylation decreases dramatically as the cells differentiate. This loss of phosphorylation coincides temporally with the observed changes in actin, fibronectin, and integrin organization. The phosphorylation state of integrin thus may mediate developmentally regulated cell-matrix interactions.     

Formation of vinculin plaques precedes other cytoskeletal changes during retinoic acid-induced teratocarcinoma cell differentiation

Immunofluorescence and immunoblotting techniques were used to study the presence and distribution of vimentin and keratin type intermediate filaments, actin, and vinculin (130 kD protein) during retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma (EC) cells. The undifferentiated F9 cells regularly expressed vimentin, usually concentrated close to the nucleus, but not keratin. Actin appeared as short intracellular filaments and as spikes at the edges of the colonies, together with some diffuse cytoplasmic staining. F9 cells also showed a weak, diffuse cytoplasmic vinculin-specific fluorescence in addition to occasional small focal vinculin patches at the edges of the cell colonies. RA treatment led into a series of changes in the cytoskeletal organization of F9 cells. These changes were initiated by the appearance of distinct vinculin plaques and followed by formation of actin stress fibers and by profound changes in the organization of vimentin in the flattening cells. RA treatment finally led to the appearance and co-expression of keratin fibrils in many of the vimentin-containing F9 cells. This sequence of changes suggests that the vinculin-containing adhesion plaques may be important in the mechanism of RA-induced differentiation of EC cells.     

F9 teratocarcinoma aggregates express villin upon differentiation into visceral endoderm-like cells

The visceral endoderm of the mouse embryo is a polarized epithelium which has recently been shown to express villin, a major actin binding component of absorptive epitheliums. We report here that villin is induced during differentiation of aggregates of the mouse embryonal carcinoma F9, an in vitro system widely used to study extraembryonic endoderm differentiation. Identical results were obtained with a variant of F9 which carries an immortalizing vector. Villin is coexpressed with F-actin and with alpha-foetoprotein, in most of the visceral endoderm-like cells lining the aggregates. This system is potentially useful to study (i) the induction of villin expression and (ii) the establishment of polarity in the visceral endoderm epithelium.     

Down-regulation of phospholipase D during differentiation of mouse F9 teratocarcinoma cells.

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.