Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase.

Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.     

Action potentials recorded intracellularly from differentiated mouse teratocarcinoma cells in culture

Under appropriate in vitro culture conditions, mouse embryonal carcinoma can give rise to a variety of differentiated cell types, including those having the morphology of nerve cells. We report here that it is possible to evoke from such cells potential changes resembling classical neuronal action potentials, thereby indicating functional differentiation of the malignant stem cell.     

The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.     

Regulation of the cellular p53 tumor antigen in teratocarcinoma cells and their differentiated progeny.

F9 embryonal carcinoma cells express high levels of a 53,000-molecular-weight cellular tumor antigen called p53. When F9 cell cultures are treated with retinoic acid and dibutyryl adenosine 3',5'-phosphate, they differentiate, predominantly into endoderm-like cells. This differentiation is accompanied by a marked decrease in the levels of p53. The mechanism(s) responsible for this decline in the level of p53 in differentiated cells was investigated. The results demonstrate that the high levels of p53 in F9 cells relative to their differentiated progeny were not due to alterations in the stability or turnover of this protein. Rather, the regulation during differentiation involved a marked decrease in the amount of in vitro translatable p53 mRNA detected in the differentiated cell cultures. This mechanism is unlike the one operating during the simian virus 40 infection or transformation, where the increased levels of p53 are largely due to the increased stability of the p53 protein.     

Expression of c-kit protooncogene is stimulated by cAMP in differentiated F9 mouse teratocarcinoma cells.

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.     

Genetic activity of X chromosomes in pluripotent female teratocarcinoma cells and their differentiated progeny

We have induced teratocarcinomas from female embryos heterozygous for electrophoretic variants of the X-linked gene coding for phosphoglycerate kinase (PGK). An embryonal carcinoma cell line, P10, has been isolated from such a teratocarcinoma. It has a normal female karyotype and cultures contain both PGK isoenzymic forms. Clonal populations derived from P10 also contain both PGK electrophoretic variants. In addition, both X chromosomes in these cells replicate in synchrony with the autosomes during early S phase of the cell cycle. These data indicate that the undifferentiated P10 embryonal carcinoma cells contain two active X chromosomes. When cultured under the appropriate conditions, the P10 cells differentiate to form a variety of tissue types. At least some of these differentiated cells contain an inactive X chromosome as determined by cytogenetic analysis. Apparently X chromosome inactivation accompanies the differentiation of these female embryonal carcinoma cells.     

High chemotactic response to platelet-derived growth factor of a teratocarcinoma differentiated mesodermal cell line

Summary Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/ml of PDGF, 2-fold at 2.5 ng/ml of PDGF. In contrast, PDGF induces the same increase in [³H]thymidine incorporation in both cell lines, made quiescent under reduced serum concentration. This high chemotactic response to PDGF seems specific for these mesodermal cells. Among the different teratocarcinoma cells tested, including stem cells (F9, PC 13, PCC4) and endodermal derivatives (PYS, F9 with retinoic acid, PSA 5E), only the visceral endodermlike cells (PSA5E) are slightly attracted by PDGF. This chemotactic response to PDGF is not related to the presence or characteristics of the type B PDGF receptors, which are less numerous in MES-1 cells (10⁵ receptors/cell, KDa 1,2 mM) compared to NIH 3T3 cells (64×10⁴ receptors per cell, KDa 1,8 nM). The MES-1 cell line might be of interest for studying the chemotactic effect of PDGF. These results also suggest a role for this soluble factor in cell migration during early embryogenesis. This investigation was supported by a grant of La Fondation pour la Recherche Médicale.     

Direct visualization, by beta-galactosidase histochemistry, of differentiated normal cells derived from malignant teratocarcinoma in allophenic mice.

Mice obtained from blastocysts injected with malignant teratocarcinoma stem cells may comprise tumor-derived cells in their tissues. Evidence for their presence has hitherto been indirect, i.e., through detection of tissue-specific products of the tumor genotype or of strain-specific enzyme variants in tissue homogenates from healthy mice. Direct visualization and identification of the tumor-derived cells would permit their normalcy and their state of differentiation to be assessed. For this purpose, a histochemical marker is required. The marker chosen was β-galactosidase (BGS), which allows high- vs low-activity cell strains to be distinguished in situ by their differences in staining intensity. BGS has previously been employed for such visualization only in brain [Dewey, M., Gervais, A., and Mintz, B. (1976). Develop. Biol.50, 68–81] and has here been shown to be applicable to other tissues, including kidney, pancreas, and salivary gland. Two unexpected results concerning the marker itself were obtained and affected its application to histochemical comparisons: BGS activity in some tissues of some inbred strains was not concordant with that of brain, on which the existing genotypic classification is based; and some cell types within a tissue varied independently in BGS levels among strains (e.g., exocrine vs endocrine pancreas). BGS visualization clearly disclosed the presence of large numbers of fully differentiated normal cells of the teratocarcinoma strain in tissues, including the Purkinje layer of the cerebellum, the kidney tubules, and the exocrine pancreas of experimental animals. In one individual, the relevant brain region was almost entirely derived from the teratocarcinoma. Yet all tissues were indistinguishable in structure and differentiation from adult controls, and showed no malignant growth. The pattern of cell-strain distribution, which was fine-grained in the brain and patchy in the other tissues named, also resembled that of ordinary allophenic mice produced from blastomere aggregates of two strains. Thus, teratocarcinoma stem cells are here seen to undergo normal histogenesis after they are successfully incorporated into a developing host embryo.     

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.     

The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics.

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.     

Gene expression of differentiated parent in teratocarcinoma cell hybrids. Repression or reprogramming?

The hybrid cell line H422 was constructed by fusing embryonal carcinoma (EC) cells of the PCC4AzaRCapR cell line with lymphocytes from a 129-tw32 mouse inbred strain female. An apparently complete extinction of stage-specific gene products of the lymphocyte parent was inferred from comparison of protein maps in two-dimensional PAGE of parental and hybrid cells, and from the reactivity patterns of monoclonal antibodies in radioimmunobinding assays. Furthermore, the hybrid cells display a true EC phenotype and EC functions, tumorigenicity and pluripotency. Taken together, the available evidence suggests either that the lymphocyte genome is a 'silent passenger' as far as the differentiated functions of hybrid cells are concerned, or that the developmentally restricted lymphocyte genome is reprogrammed in the hybrid cells back to a non-determined stage and both genomes thus act in a concerted manner.     

Interaction between endometrium epithelial cells (SNG-M) and teratocarcinoma cells (HT-H) differentiated into trophoblast

Many of the human teratocarcinoma cell lines resemble cells of preblastocyst to trophectoderm. The use of these cell lines is, however, limited since they differentiate poorly in vitro. HT-H cell line might be useful as it differentiates into syncytiotrophoblast. Because syncytiotrophoblast of the blastocyst are the cells which attach to the maternal endometrium epithelium during implantation, HT-H cells were examined for their adhesion to SNG-M cells, which feature exhibit cells of endometrium epithelium. Differentiated HT-H cells attached to SNG-M cells, and between these two cells desmosomes and adherent junctions were developed. Undifferentiated HT-H cells did not directly attach to SNG-M cells. Cell-to-cell adhesions are often mediated by the homophilic adhesion molecules present in both cells. As the first step in the investigation of adhesion between SNG-M and HT-H cells, we examined adhesion between SNG-M cells themselves. Calcium had no effect on aggregation of SNG-M cells, which may exclude the involvement of cadherin type molecule in this system. Development of monoclonal antibodies that bind to the cell surface and inhibit aggregation of SNG-M cells, and SNG-M to HT-H cells will allow us to identify the adhesion molecule(s) involved in ovum implantation in utero.     

Synthesis and retention of fibronectin (LETS protein) by mouse teratocarcinoma cells.

Mouse teratocarcinoma cells in culture were examined for both the synthesis (by metabolic labelling) and surface accumulation (by indirect immunofluorescence) of fibronectin, a glycoprotein with subunits of molecular weight 220000 D known to form part of the extracellular matrix of many cells in vivo. Although lines of both pluripotent and nullipotent embryonal carcinoma cells synthesize the protein and release it into the medium, they do not retain it on their surfaces. Monolayers of the endoderm line PSA5-E both synthesize fibronectin and lay it down in an extracellular network. A line of PYS parietal endoderm cells does not retain surface fibronectin, although it does accumulate other extracellular matrix material. When pluripotent embryonal carcinoma cells differentiate into cystic embryoid bodies, fibronectin accumulates in a basement membrane below the outer endoderm cells (both visceral and parietal-like) and may play a transient role in organizing the inside cells into an epithelial layer.     

Clonal growth of differentiated rabbit cartilage cells

Single cell suspensions have been prepared from rabbit cartilage by collagenase digestion in the presence of fetal bovine serum. When inoculated into medium F12 supplemented with 5% fetal bovine serum and 5% rabbit serum, the cartilage cell suspensions yield large healthy colonies of epithelial-like cells with an average plating efficiency of 47%. At the end of a 14 day incubation period, the centers of many of the colonies are thickened into multilayered aggregates. Such colonies exhibit serveral cartilage-like properties, including metachromatic staining with toluidine blue, dense brown staining with bismark brown, bright green staining with alcian green, and a refractile matrix which can be seen with the phase contrast microscope to surround the living cells. Similar properties have also been observed in second and third passage subcultures.     

Human teratocarcinoma cells express functional insulin-like growth factor I receptors

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.     

Multiangle light-scattering analysis of murine teratocarcinoma cells.

Stem cells of the mouse testicular teratocarcinoma are capable of giving rise in vivo and in vitro to a wide variety of cell and tissue types representative of each embryonic germ layer. Multiangle light-scattering measurements in a flow system have been made on these stem cells and on a variety of their differentiated derivatives. This technique is capable of distinguishing the stem cells from parietal yolk sac cells, visceral yolk sac cells, neuronal cells and squamous cells. However, multipotential stem cells cannot be distinguished from stem cells that are restricted in their development to a single pathway.