Transglutaminase activity and embryonal carcinoma cell differentiation

Murine embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) modulate transglutaminase (TGase) activity shortly after exposure to the inducer. Compounds that inhibit TGase enzyme activity in vitro can successfully block RA induced EC cell differentiation in culture. These observations suggest that TGase may play a role in mediating RA induced EC cell differentiation.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.     

Comparison of the effects of transforming growth factor beta, N,N-dimethylformamide, and retinoic acid on transformed and nontransformed fibroblasts

In order to compare the effects of transforming growth factor (TGF beta) with those of the differentiation promoters N,N-dimethylformamide (DMF) and retinoic acid (RA), the antiproliferative and fibronectin-inducing activities of the three agents were examined. AKR-2B mouse embryo fibroblasts and their chemically transformed counterpart AKR-MCA cells were used as the model system. Growth in monolayer culture of both cell lines was inhibited by TGF beta (EC50 approximately 1 ng/ml), DMF (EC50 approximately 0.5%), and RA (EC50 approximately 1 microM) in a concentration-dependent manner. Time-dependent elevation in fibronectin expression was also observed with all three agents. The EC50 for growth inhibition of both cell lines by TGF beta agreed well with that obtained for stimulation of fibronectin synthesis. A 3-h exposure to TGF beta is sufficient to obtain the maximal fibronectin level observed at 48 h in AKR-2 B cells but not in AKR-MCA cells. Our results indicate that in this system the effects of TGF beta are similar to those of the chemical differentiation inducers DMF and RA. Furthermore, our data also suggest that the TGF beta signal may be processed differently by nontransformed and transformed fibroblasts.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Modulation of lysosomal-associated membrane glycoproteins during retinoic acid-induced embryonal carcinoma cell differentiation

Differentiation of the murine embryonal carcinoma (EC) cell lines F-9 and PC-13, induced by beta-all transretinoic acid (RA) resulted in an increased level of two lysosomal-associated membrane glycoproteins (LAMP-1 and LAMP-2). After differentiation, the levels of both LAMPs in the EC cells were comparable to those found in visceral and parietal endoderm cell lines (PSA-5E and PYS-2, respectively). RA treatment of the EC cells also resulted in an increase in the apparent Mr of both LAMPs apparently due to increased glycosylation because the deglycosylated LAMP-1 from undifferentiated and from differentiated cells had a similar electrophoretic migration. Indeed, the binding of 125I-labeled L-phytohemagglutinin (L-PHA) to glycoproteins with Mr or 90,000-130,000 increased after differentiation and about 24 times more 125I-labeled L-PHA bound to LAMP-1 isolated by immunoprecipitation from extracts of RA-treated F-9 cells than to LAMP-1 from undifferentiated cells. The increased level of the LAMPs was detected in F-9 cells treated with greater than 10(-7) M RA and required greater than 48 h of treatment as did the increased expression of the B1 chain of laminin, an established marker for differentiation in this system. LAMP-1- and L-PHA-reactive glycoproteins were localized by fluorescence techniques to intracellular vesicles, presumably lysosomes, and to the cell surface and both increased after RA treatment. LAMP-2 was barely detectable intracellularly in undifferentiated cells but could be detected clearly after differentiation. In contrast, no LAMP-2 could be detected on the cell surface either before or after differentiation of F-9 cells. The increased level and glycosylation of both LAMP-1 and LAMP-2 was observed also in cells treated with a synthetic chalcone carboxylic acid analog of RA and by combination of either retinoid with dibutyryl cyclic AMP. These results demonstrate that differentiation of EC cells is accompanied by changes in the synthesis and glycosylation of LAMP glycoproteins and that these changes are specific for the cell type that results after differentiation.     

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.     

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.     

Metabolism of retinoids by embryonal carcinoma cells

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Cell cycle analysis during retinoic acid induced differentiation of a human embryonal carcinoma-derived cell line

Several subclones of the human embryonal carcinoma (EC) cell line Tera-2 can be induced to differentiate in monolayer culture by retinoic acid (RA) to a flattened cell type with reduced growth rate. Using a method based on the transition probability model, we have analysed changes in cell cycle kinetics of Tera-2 cells during the differentiation process. Growth inhibition was shown to occur without a lag period and to be partly due to an increase in the duration of the S-phase, but with a relatively greater contribution from an increase in the duration of G1-phase. Since the fraction of the cell population in the G1-phase then doubled, cells accumulated in this part of the cycle. In contrast, the reduced proliferation rate of two murine EC cell lines, PC13 and P19, treated with RA occurs after a lag period of about two cell cycles and is mainly attributable to an increase in the duration of the S-phase. The results illustrate a differential response of human and murine EC cells to growth regulation by RA and again emphasize that although the stem cells of murine teratocarcinomas may provide a useful model, they are not identical to their human counterparts.     

Commitment in a murine embryonal carcinoma cell line during differentiation induced by retinoic acid

Murine embryonal carcinoma (EC) cells are induced to differentiate when cultured in the presence of retinoic acid (RA). Whereas the EC cells have a high plating efficiency, the differentiated cells have little or no colony-forming ability under the same conditions. We have assumed that the loss of colony-forming ability following exposure of EC cells to RA corresponds to the irreversible commitment of EC cells to differentiate. We found that uncommitted EC cells persist in RA-treated aggregates of EC cells and that the proportion of EC cells stabilizes at a level inversely related to the RA concentration. Both experimental evidence and mathematical modelling results are consistent with the interpretation that there is a dynamic equilibrium achieved by a balance between the processes of EC cell proliferation and differentiation. Since different cell types are induced by different RA concentrations, our results suggest that the commitment to differentiate is not related in any simple way to the developmental program which ensues.     

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( Bvcycll ).     

Early cycling-independent changes to p27, cyclin D2, and cyclin D3 in differentiating mouse embryonal carcinoma cells.

Changes to cell cycle-regulating machinery that occur during differentiation of cells are thought to be responsible mostly for withdrawal from cycling. Here, embryonal carcinoma (EC) cell lines were found that differ in their basal levels of p27 inhibitor of cyclin-dependent kinases but not in their growth rates, distribution of cells in phases of cell cycle, and their ability to differentiate. High basal levels of p27 did not substitute for up-regulation of p27 that in EC cells normally occurs early after entering a differentiation pathway. Under both standard and differentiation-supporting culture conditions, variances in the levels of p27 were strictly followed by variances in the levels of cyclins D2 and D3. In EC cells genetically manipulated to overexpress p27 protein, cyclin D3 became up-regulated and vice versa. Supposedly, titration of p27 by D-type cyclins, which prevents its inhibitory action toward cyclin-dependent kinase 2, allows for the maintenance of elevated p27 in proliferating EC cells. Increased levels of p27 in early embryonal cells thus may, at least in certain phases of embryo development, serve a differentiation-associated, rather than proliferation-associated, function.     

Polyamine metabolism and gene regulation during the transition of autonomous sugar beet cells in suspension culture from quiescence to division

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S -adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S -adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S -adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence ( BvcycII ).     

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.