Intracellular Ca(2+) Regulation During Neuronal Differentiation of Murine Embryonal Carcinoma and Mesenchymal Stem Cells

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L-but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.

Studies on the embryology of the Sipunculidae. I. The embryonal envelope and its homologue. ... By John H. Gerould.

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The Hippo Transducer YAP1 Transforms Activated Satellite Cells and Is a Potent Effector of Embryonal Rhabdomyosarcoma Formation.

The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.

CCN2/CTGF silencing blocks cell aggregation in embryonal carcinoma P19 cell

Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y(2), P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-beta S-induced proliferation in P19 cells was mediated by P2Y, and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y, and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores. (C) 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference

Embryonic carcinoma cells are widely used models for studying the mechanisms of proliferation and differentiation occurring during early embryogenesis. We have now investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Wild-type P19 embryonal carcinoma cells or cells stably expressing shRNAs targeting P2X2 or P2X7 receptor expression were induced to differentiate into neurons and glial cells in the presence of retinoic acid. Silencing of P2X2 receptor expression along differentiation promoted cell proliferation and an increase in the percentage of cells expressing glial-specific GFAP, while the presence of beta-3 tubulin-positive cells diminished at the same time. Proliferation induction in the presence of stable anti-P2X2 receptor RNAi points at a mechanism where glial proliferation is favored over growth arrest of progenitor cells which would allow neuronal maturation. Differently from the P2X2 receptor, inhibition of P2X7 receptor expression during neural differentiation of P19 cells resulted in a decrease in cell proliferation and GFAP expression, suggesting the need of functional P2X7 receptors for the progress of gliogenesis. The results obtained in this study indicate the importance of purinergic signaling for cell fate determination during neural differentiation, with P2X2 and P2X7 receptors promoting neurogenesis and gliogenesis, respectively. The shRNAs down-regulating P2X2 or P2X7 receptor gene expression, developed during this work, present useful tools for studying mechanisms of neural differentiation in other stem cell models. (C) 2012 ISDN. Published by Elsevier Ltd. All rights reserved.

The quassinoid derivative NBT-272 targets both the AKT and ERK signaling pathways in embryonal tumors

The quassinoid analogue NBT-272 has been reported to inhibit MYC, thus warranting a further effort 7to better understand its preclinical properties in models of embryonal tumors (ET), a family of childhood malignancies sharing relevant biological and genetic features such as deregulated expression of MYC oncogenes. In our study, NBT-272 displayed a strong antiproliferative activity in vitro that resulted from the combination of diverse biological effects, ranging from G(1)/S arrest of the cell cycle to apoptosis and autophagy. The compound prevented the full activation of both eukaryotic translation initiation factor 4E (eIF4E) and its binding protein 4EBP-1, regulating cap-dependent protein translation. Interestingly, all responses induced by NBT-272 in ET could be attributed to interference with 2 main proproliferative signaling pathways, that is, the AKT and the MEK/extracellular signal-regulated kinase pathways. These findings also suggested that the depleting effect of NBT-272 on MYC protein expression occurred via indirect mechanisms, rather than selective inhibition. Finally, the ability of NBT-272 to arrest tumor growth in a xenograft model of neuroblastoma plays a role in the strong antitumor activity of this compound, both in vitro and in vivo, with its potential to target cell-survival pathways that are relevant for the development and progression of ET.

Karyotypic characterization of infant embryonal rhabdomyosarcoma

Despite embryonal rhabdomyosarcoma (eRMS) representing the most frequent form of RMS, the karyotypic characterization of this tumor subtype is still incomplete. We report the karyotypic analysis of two new cases of infant-onset eRMS. Both cases had a hyperdiploid karyotype, including gain of chromosomes 2 and 8. Only one of the cases showed a structural aberration, an unbalanced rearrangement involving 4p. These cases, together with a review of the literature, suggest that a karyotypic subgroup exists in infant eRMS that is defined by hyperdiploidy (<53 chromosomes) and includes gain of chromosomes 2, 8, 11, and 17, with few or no structural aberrations. Hence, this report illustrates that distinct karyotypic subgroups may be found in eRMS, which ultimately may be shown to have prognostic relevance.

Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^

Modulation of carbohydrates and carbohydrate binding proteins (lectins) during retinoic acid -induced differentiation of murine embryonal carcinoma cells

The current studies were undertaken to examine the effect of retinoic acid (RA)-induced differentiation of the murine embryonal carcinoma cell line, F-9, on the glycosylation of specific cellular glycoproteins and on the expression of two members of the family of endogenous lactoside-binding lectins. It was found that RA-induced differentiation of these cells into cells with the properties of primitive endoderm results in the increased fucosylation of 3 glycoproteins with molecular weights of 175 (gp175), 250 (gp250), and 400 (pg400) kDa. These three fucose-containing glycoproteins can be considered as new markers of differentiation in this system. The increased fucosylation of these glycoproteins preceded the 3-fold increase in fucosyltransferase (FT) activity that was seen upon RA-induced differentiation of these cells, indicating that an increase in fucosyltransferase activity alone cannot explain the increased fucosylation of these glycoproteins.^ The effect of RA and Ch55, a chalcone carboxylic acid with retinoid-like properties, induced differentiation of a variety of murine embryonal carcinoma cell lines on the activities of both FT and sialyltransferase (ST) was examined. The effect of differentiation on the activities of both glycosyltransferases was modulated and most probably is dependent upon the differentiation pathway that is triggered by the retinoids for each of the embryonal carcinoma cell lines.^ Two glycoproteins, Lysosomal Associated Membrane Glycoproteins 1 and 2 (LAMP-1 and LAMP-2) were examined in more detail during the course of RA-induced differentiation of F-9 cells. Both the levels and glycosylation of both glycoproteins are increased following differentiation of these cells. Differentiation results in the increased binding of $\sp{125}$l-labelled L-phytohemagglutinin to bind to LAMP-1 which indicates increased GlcNAc $\beta$1,6 branching of the oligosaccharide side chains.^ We found that RA-induced differentiation of F-9 cells results in the decreased expression of the 34 kDa lectin 24 h after addition of the retinoid to the medium. Additionally, 48 h of RA-treatment results in the increased expression of the 14.5 kDa lectin. By indirect immunofluorescence we were able to colocalize the 14.5 kDa lectin and laminin which suggests that laminin may be a ligand for the lectin in the F-9 cells. (Abstract shortened with permission of author.) ^

A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.