Expression of EGF receptor and transferrin by F9 and PC13 teratocarcinoma cells

We document the time of appearance and the levels of two markers of differentiation during the formation of embryoid bodies by two embryonal carcinoma (EC) cell lines. Neither of these markers has been described before for EC cells differentiating in aggregate culture, and they further extend the identification and characterization of new cell types. Both F9 and PC13 EC cell lines form embryoid bodies (so-called because they resemble early mouse embryos) with an outer epithelial layer of visceral endoderm cells, after suspension culture in the presence of retinoic acid. However, the two cell lines differ in the procedures needed to initiate the differentiation process. Once floating aggregate cultures have been formed, the time course of the appearance of epidermal growth factor (EGF) receptors and of the secretion of transferrin are similar in both cell lines, although the levels differ. EGF receptors and transferrin are quantified by 125I-EGF binding assays and enzyme-linked immunosorbent assays (ELISA) using specific antibodies, respectively. The expression of EGF receptors increases about two fold while that of transferrin increases up to 40 fold after treating F9 aggregates with retinoic acid. The EGF receptors reach a maximum 4 days after adding retinoic acid and then decline, while transferrin only increases later from a low but detectable level. For PC13 cells, EGF receptors increase tenfold, and transferrin synthetic rate increases 40 fold during the time-course. Interestingly, unstimulated F9 cells in monolayer cultures also express low levels of these markers, while the levels in PC13 EC cells are barely detectable above background.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: Modulation by cyclic AMP

The addition of dibutyryl cyclic AMP (dbcAMP) to aggregate cultures of F9 cells in medium containing retinoic acid (RA) directs the pathway of differentiation into parietal endoderm instead of visceral endoderm. We examined the levels of some of the markers that characterize the two pathways and studied the time of commitment of cells to either direction of differentiation by using immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). For either pathway, the levels and patterns of laminin, type IV collagen, and fibronectin are the same on the first day of differentiation, characterized by slightly decreased levels of laminin and type IV collagen synthesis and an increased level of fibronectin synthesis. These levels reverse on the second day of culture when the pathways diverge markedly. The differentiation pathway, however, can be redirected into the alternate one; parietal endoderm cells become committed after 3 days, whereas visceral endoderm cells are able to change into parietal endoderm cells at any time. Thus, α-fetoprotein (AFP)-producing F9 embryoid bodies switched to dbcAMP-containing medium lose the capacity to synthesize AFP and start to express genes characteristic of parietal endoderm. Our results indicate that at least some visceral endoderm cells may redifferentiate into parietal endoderm cells. These phenomena thus mimic features of endoderm differentiation in the mouse embryo.     

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.     

Induction of the expression of retinol-binding protein and transthyretin in F9 embryonal carcinoma cells differentiated to embryoid bodies

Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.     

The outgrowth of parietal endoderm from mouse teratocarcinoma stem-cell embryoid bodies

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.     

In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

In vitro Differentiation of Mouse Teratocarcinoma Cells Monitored by Intermediate Filament Expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

Changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases with retinoic-acid-induced differentiation of embryonal carcinoma cells

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variant embryonal carcinoma cells lacking SSEA-1 and Forsmann antigens remain developmentally pluripotent

Six embryonal carcinoma (EC) cell lines that are resistant to the cytotoxic, galactose-specific lectin abrin were isolated from mutagenized populations of either PSA-1 or F9 cells. The LD10 for each of the variant lines was at least 150-fold greater than that for parental cells. Indirect cytotoxicity tests demonstrated that all of the variant cell lines lacked both Stage Specific Embryonic Antigen-1 (SSEA-1, less than 1% of wild-type levels) and Forsmann antigen (less than 5% of wild-type levels). When abrin-resistant cells were fused to previously isolated SSEA-1-negative cells (M. J. Rosenstraus (1983), Dev. Biol. 99, 318-323) that express Forsmann antigen, the resulting hybrids expressed SSEA-1. This implies the mutation conferring abrin resistance is in a different gene than that defined by the previously isolated mutation. Thus, we have identified two genes that are required for SSEA-1 expression, one of which also appears to be required for Forsmann antigen expression. The F9-derived variants differentiated into visceral-like or parietal-like endoderm when treated with retinoic acid in the absence or presence of 8-bromo-cAMP, respectively. PSA-1-derived variants formed differentiated teratocarcinomas containing derivatives of all three germ layers. Thus the SSEA-1 and Forsmann haptenic determinants are not required for EC cells to differentiate into a broad spectrum of cell types; nor do they appear to be involved in the cell-cell interactions that are postulated to regulate visceral versus parietal endoderm differentiation.     

RXRalpha-null F9 embryonal carcinoma cells are resistant to the differentiation, anti-proliferative and apoptotic effects of retinoids.

The F9 murine embryonal carcinoma (EC) cell line, a well established model system for the study of retinoic acid (RA)-induced differentiation, differentiates into cells resembling three types of extra-embryonic endoderm (primitive, parietal and visceral), depending on the culture conditions and RA concentration used. A number of previously identified genes are differentially expressed during this process and serve as markers for the different endodermal cell types. Differentiation is also accompanied by a decreased rate of proliferation and an apoptotic response. Using homologous recombination, we have disrupted both alleles of the retinoid X receptor (RXR) alpha gene in F9 cells to investigate its role in mediating these responses. The loss of RXRalpha expression impaired the morphological differentiation of F9 EC cells into primitive and parietal endoderm, but has little effect on visceral endodermal differentiation. Concomitantly the inducibility of most primitive and parietal endoderm differentiation-specific genes was impaired, while several genes upregulated during visceral endodermal differentiation were induced normally. We also demonstrate that RXRalpha is required for both the anti-proliferative and apoptotic responses in RA-treated F9 cells. Additionally, we provide further evidence that retinoic acid receptor (RAR)-RXR heterodimers are the functional units transducing the effects of retinoids in F9 cells.     

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.     

Up-regulation of embryonic NCAM in an EC cell line by retinoic acid

The impact of retinoic acid (RA) on the expression of the neural cell adhesion molecules (NCAMs) and their developmentally regulated polysialic acid (PSA) moiety was studied in embryonal carcinoma (EC) cell lines. These cell lines are known to be capable of RA-induced differentiation into neurons (murine P19 cells) or parietal endoderm (murine F9 cells), respectively. Monoclonal antibodies were employed to monitor expression of NCAM and PSA. F9 and P19 cells were both found to express NCAM but only P19 cells carried the highly polysialylated "embryonic form" of NCAM (E-NCAM). The amount of NCAM in aggregated P19 cells but not in F9 cells was dramatically increased upon treatment with RA. Since NCAMs play an important role in cell interactions during embryogenesis it is tempting to speculate that the regulative impact of RA on NCAMs is related to its morphogenic property.     

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.     

A teratocarcinoma-derived endoderm stem cell line (1H5) that can differentiate into extra-embryonic endoderm cell types

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into "embryoid bodies" or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.     

Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.     

Plasminogen activator expression in F9 teratocarcinoma embryoid bodies and their endoderm derivatives

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.     

Epithelial layer formation in differentiating aggregates of F9 embryonal carcinoma cells

F9 embryonal carcinoma (EC) cells, cultured in suspension in medium containing 5 X 10(-8) M retinoic acid, aggregate and differentiate into embryoid bodies with an outer layer of visceral endoderm cells that synthesize and secrete alphafetoprotein (AFP) (Hogan, B. L. M., A. Taylor, and E. Adamson, 1981, Nature (Lond.). 291:235-237). Here we analyze the formation of the outer layer of cells as a model for epithelial differentiation. Three morphological phases are described, but analyses of cell numbers and the synthetic rates of some proteins, as well as the appearance of markers of visceral endoderm and basement membrane, show that the formation of the outer layer occurs as an orderly progression of multiple events. The markers used to follow the ontogeny of epithelial layer formation include SSEA-1, l, and i blood group antigens, laminin, fibronectin, type IV collagen, cytoskeletal intermediate filament proteins (vimentin, Endo A, and B), and AFP. The onset of epithelium formation occurs between the third and fourth day of culture, but its function is maximally expressed only when it is well organized. We found the rate of AFP secretion to be a measure of the proper alignment and maturity of the epithelium which occurs at the seventh or eighth day. This model of epithelium formation may help to explain how similar processes occur during embryogenesis.