Spontaneous high expression of heat-shock proteins in mouse embryonal carcinoma cells and ectoderm from day 8 mouse embryo

When submitted to a heat-shock, mouse embryonal carcinoma (EC) and fibroblast cells show very different behavior. All the EC cells so far analyzed express very high levels of several heat-shock proteins (HSP) in the absence of stress and independent of their origin and culture conditions. In such cells, the 89-kd, 70-kd and 59-kd HSP are the most prominent proteins after actin. In addition, the 89-kd and 59-kd HSP are not stimulated by an arsenite shock in contrast to what is observed with fibroblasts or cells of the parietal yolk sac type. Arsenite induces the synthesis of a 105-kd polypeptide in fibroblasts but not in EC cells. In vitro differentiation of F9 cells induced by retinoic acid and dibutyryl cAMP is accompanied by a decrease in the spontaneous relative abundance of HSP and restores the arsenite-induced synthesis of the 105-kd polypeptide. EC cells are usually believed to be similar to inner cell mass cells of mouse blastocyst. Furthermore, data in the literature together with our own results suggest that the same three HSP are also spontaneously expressed in high amounts in the early mouse embryo.     

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.     

Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain of the trophoblast cells. These are believed to be involved in facilitating the accumulation of fluid into the blastocyst cavity. We have investigated the ability of mouse embryos to regulate AQP gene expression in response to different treatments expected to affect the passage of water across the trophoblast cells using real-time PCR. In the first experiment 8-cell embryos were allowed to develop to blastocysts in media from 300 to 400 mOsm. Blastocyst formation was unaffected by media made hyperosmolar by glycerol, whereas blastocyst formation was significantly reduced in sucrose-based 350 and 400 mOsm media. AQP 8 mRNA levels were reduced when embryos were cultured in glycerol-based hyperosmolar media. The mRNA levels of AQP 3, 7, 9, and 11 were not significantly affected by hyperosmolar media. In the second experiment blastocysts were punctured (0 hr) and allowed to re-expand. AQP mRNA levels were examined after 2, 6, and 10 hr. Compared to control embryos, the expression of AQP 3, 7, and 9 were upregulated after 2 hr. Upregulation was sustained only for AQP 9 and this was sustained up to 6 and 10 hr after puncture. In the third experiment we compared expression of AQPs between in vitro cultured and in vivo developed blastocysts. We found that in vitro culture resulted in lower levels of AQP 8, 9, and 11 compared to in vivo development. These experiments show that mouse embryos are capable of regulating AQP mRNA abundances in response to environmental alterations.     

Effects of tunicamycin on the differentiation of F9 cells induced by either retinoic acid or retinoic acid and dibutyryl cyclic AMP

Tunicamycin (0.5 micrograms/ml) inhibited differentiation of F9 cells treated either with retinoic acid or with retinoic acid and dibutyryl cyclic AMP, as monitored by the activity of alkaline phosphatase and expression of cytokeratins. On the other hand, the pattern of the polysaccharide chain synthesis changed drastically with the treatment irrespective of the presence of tunicamycin. Therefore, phenotypes induced with retinoic acid are dissociated into two categories, one that is directly induced by the drug and the other that is induced indirectly by a mechanism in which glycoproteins play a role.     

Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.     

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)     

Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.     

Effects of tunicamycin on preimplantation mouse embryos: prevention of molecular differentiation during blastocyst formation

To examine the role of carbohydrate-containing molecules of preimplantation mouse embryos in early development, effects of tunicamycin were analyzed at the molecular level. Embryos cultured in the presence of tunicamycin (0.1 micrograms/ml) from earlier than the 16-cell stage did not develop into blastocysts, though cell divisions continued normally. Tunicamycin inhibited not only the synthesis of carbohydrate chains of usual N-glycosidic glycoproteins but also that of characteristic large polysaccharides (Mr greater than 9K) of early embryos. Expression of several polypeptides which are characteristic of the blastocyst stage (blastocyst-characteristic proteins: BCPs) was strongly inhibited in the embryos treated by tunicamycin from earlier than the 16-cell stage, while the expression was not inhibited in the embryos treated by the drug after that stage as analyzed by two-dimensional polyacrylamide gel electrophoresis. The expression of BCPs appeared to be dependent on de novo mRNA synthesis, since it was also inhibited by alpha-amanitin treatment. Since tunicamycin was shown not to inhibit expression of most other proteins and the bulk of mRNA, the inhibitory effects of tunicamycin appeared to be specific for the induction of BCPs. These observations suggest that the glycoprotein(s) and/or the characteristic large polysaccharides on the morula stage embryos play an essential role not only for morphological development but also for triggering differentiation at the molecular level.     

Identification of murine extra-embryonic endodermal cells by reaction with teratocarcinoma basement membrane antiserum

Embryonal carcinoma cells from the PSA1 cell line will differentiate in vitro to form structures called embryoid bodies composed of an inner core of embryonal carcinoma cells surrounded by a basement membrane matrix and an outer layer of extra-embryonic endodermal cells. Immunization of rabbits with basement membranes isolated from embryoid bodies resulted in an antiserum, which binds to fixed extra-embryonic endodermal cells of either embryonic or teratocarcinoma origin but does not bind substantially to mouse embryonal carcinoma cells, fibroblasts, myoblasts or erythroleukemic cells. The F9-22 embryonal carcinoma cell line normally differentiates only to a very limited extent in vitro or in vivo. However, incubation of these cells in medium containing retinoic acid results in the appearance of cells resembling extra-embryonic endoderm. The embryoid body basement membrane antibodies were used to measure, by flow microfluorometry, the appearance of reactive cells in F9-22 cultures treated with retinoic acid. The kinetics of appearance of cells reactive with the basement membrane antibodies are similar to the kinetics of appearance of cells secreting plasminogen activator, a known marker of extraembryonic endoderm.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Induction of neural-like cells and acetylcholinesterase activity in cultures of F9 teratocarcinoma treated with retinoic acid and dibutyryl cyclic adenosine monophosphate

Cultures of F9 embryonal carcinoma cells treated with retinoic acid showed partial differentiation to endoderm cells as previously reported [Strickland, S., and Mahdavi, V. (1978).Cell15, 393–403]. Addition of dibutyryl cAMP to cultures pretreated with retinoic acid led to a second distinctive change in the cell population, with the formation of many neural-like cells. The appearance of these cells coincided with large increases in specific acetylcholinesterase activity of the cultures. Provided the cultures had been exposed to retinoic acid for at least 48 hr beforehand, the morphological and enzymatic changes became apparent between 24 and 48 hr after the addition of dibutyryl cAMP. The changes proceeded more abruptly and extensively when cells were grown in nongelatinized culture dishes. On gelatin-coated surfaces, the differentiated cells occasionally showed local areas of ordered arrangements. It is suggested that this system may be useful in analyzing early events in neural differentiation.     

Cell-cycle characteristics of undifferentiated and differentiating embryonal carcinoma cells

The cell-cycle parameters of undifferentiated and differentiating embryonal carcinoma cells were determined. Undifferentiated cultures of an F9 subclone, OTF9-63, had a 12-hr generation time, 10-hr S, and 2-hr G2 + M. G1 was less than 0.5 hr. In contrast, OTF9-63 cells induced to differentiate by treatment with retinoic acid had a 16.8-hr generation time, 12.5-hr S, 2-hr G2 + M, and 2.3-hr G1. Similar results were obtained with undifferentiated cultures and aggregation-induced differentiating cultures of PSA-1 cells. These data demonstrate that the undifferentiated stem cells have little or no G1, and that both G1 and S lengthen during 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.     

Differential expression of mouse Disabled 2 gene in retinoic acid-treated F9 embryonal carcinoma cells and early mouse embryos.

Using a differential display PCR, we identified a differentially expressed cDNA fragment which was detectable in retinoic acid (RA) treated F9 embryonal carcinoma (EC) cells but not in untreated F9 cells. A homology search of the Gene Bank indicated that the cDNA fragment is part of the mouse homolog of the Drosophila Disabled (mDab2) gene. Aggregate cultures of F9 EC cells grown in the presence of the RA differentiated into nonmalignant cells resembling the visceral endoderm of the mouse embryo. Upon induction of endodermal differentiation with 10(-7) M RA, the gene expression of mDab2 was increased gradually during the first 96 h. Neither undifferentiated F9 cells, nor the undifferentiated aggregate cells without RA expressed mDab2. Whole-mount in situ hybridization and quantitative RT-PCR also showed that the temporal expression pattern of the mDab2 gene coincides with the initiation pattern of RA synthesis that occurs during mouse embryogenesis. Also, two alternative splicing messages of mDab2 were detected in a tissue specific manner. All the data indicate that mDab2 may play an important role in RA-induced signal transduction during mouse development.