Cell-surface changes during in vitro differentiation of pluripotent embryonal carcinoma cells

When aggregates of HM-1 embryonal carcinoma (EC) cells were exposed to 10(-6) M retinoic acid for 2 days and cultured in medium lacking retinoic acid, they differentiated to nerve cells, endoderm cells, and myoblasts. Cells 2 days after initial exposure to retinoic acid were not significantly different from the parental EC cells, as judged by cell-surface architecture and by reactivity to lectins. On the fourth day, the surface of the aggregates was covered with two kinds of cells distinguishable from the parental cells. The round cells with short villi seemed to be precursors to endoderm cells. Receptors for Dolichos biflorus agglutinin (DBA) newly appeared and receptors for peanut agglutinin (PNA) were still expressed on their surfaces. The other cells, which were round cells with a few processes, might be precursors to nerve cells. PNA receptors had disappeared from their surfaces, and DBA receptors were not expressed. On the sixth day of differentiation, possible precursors to myoblasts were detected; they were flat cells with smooth surfaces. These cells lacked cell-surface receptors for the two lectins, while the precursor cells and the myoblasts excreted intercellular fibers reacting with PNA. HM-1 cells synthesized much embryoglycan, the structure of which was similar to that of the glycan isolated from quasinullipotent F9 cells. The only difference was that the glycan from HM-1 cells lacked DBA binding sites. Synthesis of fucosylated embryoglycan mainly decreased between the second and fourth day of differentiation. As above, cell-surface changes occurred mainly between the second and fourth day. The period seems to be important in determining the fate of the cells, since endoderm cells were scarcely seen among differentiated cells which had been continuously exposed to 10(-6) M retinoic acid during the period.     

Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.     

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.     

Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells

Molecular clones complementary to the mRNA species for the A, B1 and B2 chains of murine laminin were identified by hybrid-selection and in vitro translation. Northern blot analysis demonstrated that the three clones, p59 (A), p2 (B1) and p16 (B2) hybridized to mRNA species 9.8, 6.0, and 8.0 kb in length, respectively. The three clones were used as probes to monitor the steady-state levels of laminin mRNA species during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid and dibutyryl cyclic AMP. The steady-state levels of the three mRNA species appeared to increase in a coordinate manner. Undetectable levels at the beginning of induction were followed by a dramatic increase in the levels of the three mRNA species between 48 and 72 h. The kinetics parallel the increase in laminin synthesis and the striking morphological changes previously reported.     

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.     

Induction of F9 embryonal carcinoma cell differentiation by inhibition of polyamine synthesis

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells

We have analyzed the importance of substrate methylation by S-adenosylmethionine-dependent methyltransferases for neuronal differentiation of P19 embryonal carcinoma cells. We show that treatment of cells with methyltransferase inhibitor adenosine dialdehyde (AdOx) interferes with neuronal differentiation. Retinoic acid (RA) and AdOx co-treated cells had a decreased number of neurites and a flattened morphology compared with cells differentiated by RA. Also, the amount of neuronal class III tubulin (Tuj1) decreased from 76% to 9.6% with AdOx-treatment. Gene expression levels of wnt-1, brn-2, neuroD, and mash-1 were also down-regulated by AdOx-treatment. But AdOx-treatment did not up-regulate BMP-4 and GFAP genes. Treatment of RA decreased E-cadherin expression during neuronal differentiation. However, in AdOx/RA co-treated cells, E-cadherin expression was restored to the control level. Also, mRNA expression of N-cadherin decreased with AdOx-treatment. Taken together, these data show that methylation reactions might influence the cell-fate decision and neuronal differentiation of P19 cells.     

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.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

Transient expression of the proto-oncogene int-1 during differentiation of P19 embryonal carcinoma cells.

In mouse embryos, the int-1 proto-oncogene is transiently expressed in areas of the developing neural system. Retinoic acid-treated P19 embryonal carcinoma cells have often been used as an in vitro model for the molecular basis of neural development. We shown here that int-1 is transiently expressed in differentiated P19 cells. The time course and retinoic acid dose dependence of int-1 expression suggest that the gene is specifically expressed during early neural differentiation. P19 cells may be a useful model to assist in the study, at the cellular level, of the role of int-1 in neural development.