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1.
Lee JH Shin SY Kim S Choo J Lee YH 《Biochemical and biophysical research communications》2006,347(3):715-722
Apoptosis is thought to be involved in the maintenance of cellular homeostasis, as well as various pathological processes. However, little information is available about the regulation of apoptosis during the aggregation stage of P19 embryonal carcinoma (EC) cells. Here we report that aggregation-induced apoptosis is markedly attenuated by treatment with retinoic acid (RA). PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression was down-regulated during the aggregation phase of P19 EC cells in the presence, but not in the absence, of RA. Suppression of PTEN expression during the aggregation was accompanied by increased phosphorylation of serine/threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that RA attenuates the induction of apoptosis during the aggregation phase of P19 EC cells, probably by suppressing PTEN expression. 相似文献
2.
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 Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation. 相似文献
3.
Cornelia M. M. van der Kruijssen Tanja A. E. van Achterberg Alie Feijen Jean M. Hébert Peter de Waele Adriana J. M. van den Eijnden-van Raaij 《Development, growth & differentiation》1995,37(5):559-574
We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx-1 , goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose-dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA-responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10−9 mol/L RA or a factor produced by visceral endoderm-like cells (END-2 factor). The FGF effect is dose-independent. These results suggest an important function for RA and the END-2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development. 相似文献
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Tsukamoto K Arimitsu H Ochi S Nakamura K Tanaka Y Nuemket N Taniguchi K Kozaki S Tsuji T 《Microbiology and immunology》2012,56(10):664-672
Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs. 相似文献
6.
Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells 总被引:3,自引:0,他引:3
Tanaka T Satoh T Onozawa Y Kohroki J Itoh N Ishidate M Muto N Tanaka K 《Cell biology international》1999,23(8):541-550
We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation. 相似文献
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Shouyi Qiao 《Cell biology international》2012,36(12):1275-1279
ADAM23 (a disintegrin and metalloprotease 23), a member of brain MDC (macrophage‐derived chemokine) family, is important for the development of CNS (central nervous system). P19 mouse embryonal carcinoma cells can differentiate into neurons when cultured in aggregates and induced with RA (retinoic acid). We have found that under conditions without RA induction, knocking down ADAM23 with RNAi (RNA interference) promoted neuronal differentiation, and similarly recombinant GST (glutathione transferase)‐ADAM23‐DIS protein inhibited neuronal differentiation of P19/ADAM23KD (P19/ADAM23‐knockdown) cells. In P19/ADAM23KD, there were more cells arrested in G1 phase than normal P19 cells, due to the up‐regulation of P57KIP2 and P27KIP1 expression. P27KIP1 was up‐regulated during the differentiation process of both P19/ADAM23KD cells without RA induction, and P19 cells with RA induction. Transient overexpression of P27KIP1 in P19 cells also promoted neuronal differentiation of P19 cells. The findings indicate that ADAM23 suppresses neuronal differentiation through its disintegrin domain, and Adam23 KD up‐regulates P27KIP1 in P19/ADAM23KD cells, one reason that P19/ADAM23KD cells can differentiate into neurons without RA induction. 相似文献
9.
Summary The embryonal carcinoma cell line P19 is derived from mouse teratocarcinomas. These pluripotent cells can be induced to differentiate
into a variety of cell types by exposure to various drugs. We used retinoic acid to induce embryonal carcinoma cells to differentiate
into neuronlike cells. In this study, we show that changes occur in mitochondria during differentiation of embryonal carcinoma
cells to neuronlike cells. We found that various morphologic parameters such as mitochondrial fractional area and mitochondrial
size decrease as embryonal carcinoma cells differentiate into neuronlike cells. Similar changes were also observed in mitochondrial
DNA content. Stereologic analysis of cell preparations provided a measure of mitochondrial fractional area per cell and mtDNA
content was assessed by radiolabeled mtDNA probe. This study establishes that mitochondria are regulated as cells differentiate.
This study was financially supported by the Medical Research Council of Canada. 相似文献
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Jennifer Petsche Connell Emily Augustini Kenneth J. Moise Jr. Anthony Johnson Jeffrey G. Jacot 《Journal of cellular and molecular medicine》2013,17(6):774-781
Amniotic fluid‐derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte‐like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2‐week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co‐culture group. Protein expression of cardiac calsequestrin 2 was up‐regulated in direct transmembrane co‐cultures and media control cultures compared to the other experimental groups, but all groups were up‐regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape‐loading dye transfer assay, was significantly increased in direct transmembrane co‐cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co‐culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co‐culture does enhance connexin‐43‐mediated gap junction communication between AFSC. 相似文献
12.
Bing Huang ;Wei Li ;Botao Zhao ;Caihong Xia ;Ruqiang Liang ;Kangcheng Ruan ;Naihe Jing ;Youxin Jin 《Acta biochimica et biophysica Sinica》2009,(3):231-236
MicroRNAs (or miRNAs) are small non-coding RNAs (21-25 nucleotides) that are involved in a wide range of activities related to the development and differentiation of cells. Comparison of the miRNA expression profiles of mouse P19 embryonic carcinoma cells with those of differentiated neural stem cells showed that the expression level of 65 miRNAs changed (2-fold) after differentiation. MiR-124a was dramatically upregulated (more than 20-fold) while miRNAs of the miR-302 family and those in the miR-290-295 cluster were strongly down-regulated. Further analysis revealed that some important factors such as Oct4 and Sox2 appeared to be involved in the regulation of these miRNAs. These results may contribute to a better understanding of miRNA-regulated neural differentiation in early mouse embryos. 相似文献
13.
Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells
Yamada T Hashiguchi A Fukushima S Kakita Y Umezawa A Maruyama T Hata J 《In vitro cellular & developmental biology. Animal》2000,36(2):139-146
Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment.
Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation.
We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were
subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment,
at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein
level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by
heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order
to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression
of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited
by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β
alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological
critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation
into specific cell lineages. 相似文献
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Monique Cadrin Geoffrey O. Wasteneys Elizabeth M. V. Jones-Villeneuve David L. Brown Kenneth R. Reuhl 《Cell biology and toxicology》1988,4(1):61-80
Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 M resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronalAbbreviations -MEM
alpha minimal essential medium
- EC
embryonal carcinoma cells
- FCS
fetal calf serum
- MAP
microtubule-associated protein
- MeHg
methylmercury
- RA
retinoic acid 相似文献
16.
HSPs (heat shock proteins) have been recognized to maintain cellular homoeostasis during changes in microenvironment. The present study aimed to investigate the HSPs expression pattern in hierarchical neural differentiation stages from mouse embryonal carcinoma stem cells (P19) and its role in heat stressed exposed cells. For induction of HSPs, cells were heated at 42°C for 30 min and recovered at 37°C in different time points. For neural differentiation, EBs (embryoid bodies) were formed by plating P19 cells in bacterial dishes in the presence of 1 mM RA (retinoic acid) and 5% FBS (fetal bovine serum). Then, on the sixth day, EBs were trypsinized and plated in differentiation medium containing neurobasal medium, B27, N2 and 5% FBS and for an extra 4 days. The expression of HSPs and neural cell markers were evaluated by Western blot, flow cytometry and immunocytochemistry in different stages. Our results indicate that HSC (heat shock constant)70 and HSP60 expressions decreased following RA treatment, EB formation and in mature neural cells derived from heat-stressed single cells and not heat-treated EBs. While the level of HSP90 increased six times following maturation process, HSP25 was expressed constantly during neural differentiation; however, its level was enhanced with heat stress. Accordingly, heat shock 12 h before the initiation of differentiation did not affect the expression of neuroectodermal and neural markers, nestin and β-tubulin III, respectively. However, both markers increased when heat shock was induced after treatment and when EBs were formed. In conclusion, our results raise the possibility that HSPs could regulate cell differentiation and proliferation under both physiological and pathological conditions. 相似文献
17.
Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis. 相似文献
18.
P19 embryonal carcinoma cells were examined under different conditions of embryoid body (EB) formation to evaluate endothelial differentiation. A prominent capillary-like network including PECAM-1 positive cells and upregulated expression of endothelial markers Tie-1, Tie-2, Flk-1 and PECAM-1 were identified in the DMSO-treated group during EB formation as well as after VEGF treatment. About 4% of the P19 cells differentiated into PECAM-1 positive cells on day 14 of differentiation. These results suggest that the P19 cells have the potential for endothelial cell differentiation and provide a new model system for future research. 相似文献
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Kukharskyy V Sulimenko V Macůrek L Sulimenko T Dráberová E Dráber P 《Experimental cell research》2004,298(1):218-228
Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis. 相似文献