Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

FOXC1 Downregulates Nanog Expression by Recruiting HDAC2 to Its Promoter in F9 Cells Treated by Retinoic Acid.

FOXC1, a transcription factor involved in cell differentiation and embryogenesis, is demonstrated to be a negative regulator of Nanog in this study. FOXC1 is up-regulated in retinoic acid-induced differentiation of F9 Embryonal Carcinoma (EC) cells; furthermore, FOXC1 specifically inhibits the core pluripotency factor Nanog by binding to the proximal promoter. Overexpression of FOXC1 in F9 or knockdown in 3T3 results in the down-regulation or up-regulation of Nanog mRNA and proteins, respectively. In order to explain the mechanism by which FOXC1 inhibits Nanog expression, we identified the co-repressor HDAC2 from the FOXC1 interactome. FOXC1 recruits HDAC2 to Nanog promoter to decrease H3K27ac enrichment, resulting in transcription inhibition of Nanog. To the best of our knowledge, this is the first report that FOXC1 is involved in the epigenetic regulation of gene expression.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells.

Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 µM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 µM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

Selective ROS-dependent p53-associated anticancer effects of the hypoxoside derivative rooperol on human teratocarcinomal cancer stem-like cells.

Cancer stem cells (CSCs) are potential targets for innovative anticancer therapies that involve natural products with potential chemopreventive effects. We therefore analyzed the antineoplastic activity of rooperol, the aglycone of the plant-derived compound hypoxoside, on a model of Oct4-expressing cancer stem-like cell, i.e. the human embryonal carcinoma (EC) cell NT2/D1. Rooperol selectively inhibited the proliferation of NT2/D1 cells in a concentration-dependent manner and had no effect on either normal embryonic fibroblasts which are more restrictive pluripotent stem cells or on NCCIT p53-mutant EC cells. Accordingly, rooperol only eliminates colon carcinoma cells expressing p53. Rooperol treatment triggered cell death on NT2/D1 cells through the alteration of mitochondrial membrane potential and production of reactive oxygen species (ROS). Rooperol-induced apoptosis was associated with activation of p53 and concentration-dependent changes of the expression levels of both caspase 3 and poly ADP ribose polymerase type 1 cleaved subunits. These modifications were accompanied by a downregulation of Oct4 and its two partners involved in the maintenance of cell pluripotency and self-renewal, Nanog and Sox2.Treatment with intracellular membrane permeant O2 (-) scavengers prevented rooperol-induced apoptosis and upregulation of the expression of p53 and active caspase-3. Our findings indicate that rooperol mediates its growth inhibitory effects on CSCs via a mitochondrial redox-sensitive mechanism. We propose that abrogating the expression of the stemness regulators is a prerequisite for rooperol to fully exert its pro-apoptotic properties on wild-type p53-bearing CSCs.

Vitamin C enhances Nanog expression via activation of the JAK/STAT signaling pathway.

Vitamin C (Vc), also known as ascorbic acid, is involved in many important metabolic and physiological reactions in the body. Here, we report that Vc enhances the expression of Nanog and inhibits retinoic acid-induced differentiation of embryonic stem cells. We investigated Vc regulation of Nanog through Janus kinase/signal transducer and activator of transcription pathway using cell signaling pathway profiling systems, and further confirmed by specific pathway inhibition. Using overexpression and knockdown strategies, we demonstrated that STAT2 is a new positive regulator of Nanog and is activated by phosphorylation following Vc treatment. In addition, site mutation analysis identified that STAT2 physically occupies the Nanog promoter, which was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, our data suggest a role for Vc in Nanog regulation networks and reveal a novel role for STAT2 in regulating Nanog expression.

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells.

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced immediately after retinoic acid (RA)-initiated differentiation of pluripotent P19 embryonal carcinoma cells, correlated with the downregulated expression of pluripotency-related genes such as Nanog. To study whether Foxa1 participates in the repression of pluripotency factors, we expressed Foxa1 ectopically in P19 cells and identified that Nanog was repressed directly by Foxa1. We confirmed that Foxa1 was able to interact with Grg3, which is a transcriptional corepressor that expresses in P19 cells as well as during RA-induced P19 cell differentiation. Knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during RA-induced P19 cell differentiation. Furthermore, we found that Foxa1 recruited Grg3 to the Nanog promoter -2kb upstream region and switched the promoter to an inactive chromatin status represented by typical modifications in histone H3. Together, our results suggested a critical involvement of Foxa1 in the negative regulation of Nanog expression during the differentiation of pluripotent stem cells.

Human embryonal carcinoma cells in serum-free conditions as an in vitro model system of neural differentiation.

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.

Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives.

BACKGROUND: Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2. METHODS: We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC. RESULTS: Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells. CONCLUSIONS: Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells.

Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/ß-catenin pathway.

Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/ß-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3ß phosphorylation, further induced the nuclear accumulation of ß-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/ß-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/ß-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

Genomic and phenotypic alterations of the neuronal-like cells derived from human embryonal carcinoma stem cells (NT2) caused by exposure to organophosphorus compounds paraoxon and mipafox.

Historically, only few chemicals have been identified as neurodevelopmental toxicants, however, concern remains, and has recently increased, based upon the association between chemical exposures and increased developmental disorders. Diminution in motor speed and latency has been reported in preschool children from agricultural communities. Organophosphorus compounds (OPs) are pesticides due to their acute insecticidal effects mediated by the inhibition of acetylcholinesterase, although other esterases as neuropathy target esterase (NTE) can also be inhibited. Other neurological and neurodevelopmental toxic effects with unknown targets have been reported after chronic exposure to OPs in vivo. We studied the initial stages of retinoic acid acid-triggered differentiation of pluripotent cells towards neural progenitors derived from human embryonal carcinoma stem cells to determine if neuropathic OP, mipafox, and non-neuropathic OP, paraoxon, are able to alter differentiation of neural precursor cells in vitro. Exposure to 1 µM paraoxon (non-cytotoxic concentrations) altered the expression of different genes involved in signaling pathways related to chromatin assembly and nucleosome integrity. Conversely, exposure to 5 µM mipafox, a known inhibitor of NTE activity, showed no significant changes on gene expression. We conclude that 1 µM paraoxon could affect the initial stage of in vitro neurodifferentiation possibly due to a teratogenic effect, while the absence of transcriptional alterations by mipafox exposure did not allow us to conclude a possible effect on neurodifferentiation pathways at the tested concentration.

RING finger protein 10 regulates retinoic acid-induced neuronal differentiation and the cell cycle exit of P19 embryonic carcinoma cells.

Rnf10 is a member of the RING finger protein family. Recently, a number of RING finger proteins were reported to be involved in neuronal differentiation, development, and proliferation. In this study, we observed that the mRNA levels and protein expression of Rnf10 increase significantly upon the retinoic acid-induced neuronal differentiation of P19 cells. Knockdown of Rnf10 by RNA interference significantly impaired neuronal differentiation of P19 cells by attenuating the expression of neuronal markers. Cell cycle profiling revealed that Rnf10-depleted cells were unable to establish cell cycle arrest after RA treatment. In agreement with flow cytometry analysis, increased cell proliferation was observed after RA induction in Rnf10 knockdown cells as determined by a BrdU incorporation assay. Moreover, like Rnf10, the mRNA levels and protein expression of p21 and p27 also increased upon RA induction. Rnf10 knockdown only resulted in a reduction of p21 expression, while p27 and p57 expression remained unchanged, indicating that Rnf10 may regulate cell cycle exit through the p21 pathway. Ectopic p21 expression partially rescued the effect of Rnf10 depletion on the neuronal differentiation of P19 cells. Collectively, these results showed that increase in Rnf10 expression upon RA induction is necessary for the positive regulation of cyclin kinase inhibitor p21 expression, which leads to cell cycle arrest and is critical for neuronal differentiation.

α-Lipoic acid ameliorates mitochondrial impairment and reverses apoptosis in FABP3-overexpressing embryonic cancer cells.

Fatty acid-binding protein 3 (FABP3) is a low molecular weight protein with distinct tissue distribution, which may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. We have previously shown FABP3 was more highly expressed in myocardium with ventricular septal defects than in normal myocardium and furthermore, that overexpression of FABP3 causes mitochondrial dysfunction and induces apoptosis in the P19 mouse teratocarcinoma cell line (P19), which is a suitable model for the investigation of cardiac differentiation at the molecular and functional levels. α-Lipoic acid (α-LA), a natural dithiol compound with antioxidant properties, has been reported to protect mitochondrial function in cells. In this study, we established an FABP3-overexpressing P19 cell line for the investigation of the impact of α-LA on mitochondrial impairment and apoptosis in these cells. Mitochondrial morphology was evaluated by transmission electron microscopy, while the effects of α-LA on reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), intracellular ATP content and the amount of mitochondrial DNA were analyzed by flow cytometry, a commercially available assay and quantitative real-time PCR, respectively. The results revealed that α-LA ameliorated mitochondrial deformation and decreased intracellular ROS production. Furthermore, the MMP, intracellular ATP synthesis and the amount of mitochondrial DNA were also increased. Most significantly, α-LA was shown to reverse apoptosis. Collectively, our results indicate that abnormalities in FABP3 expression contribute to mitochondrial dysfunction and apoptosis, and that α-LA represents a suitable candidate for development as a treatment for apoptosis-related congenital cardiac malformations.

Comparative SRY incorporation on the regulatory regions of pluripotency/differentiation genes in human embryonic carcinoma cells after retinoic acid induction.

Members of the SOX (SRY box) family proteins play critical roles in multiple aspects of development. SRY, as a founder member of SOX family, has been long believed to be involved in the development of sexual gonads by triggering signaling cascades which lead to the formation of testis or ovary from bipotential gonads. However, less is known about other potential regulatory roles of SRY in the development and differentiation. In order to gain further insight into the possible roles of SRY during development, we looked into possible SRY-regulated genes and their levels of expression in a human embryonic carcinoma cell line, named NTera2, before and after induction of differentiation. For this respect, SRY incorporation on the regulatory regions of two groups of genes including OCT4, NANOG, and SOX2 as pluripotency marker genes, and NESTIN and PAX6 as differentiation marker genes were evaluated quantitatively. Chromatin immunoprecipitation using SRY antibody was performed on chromatin extract of a human embryonic carcinoma cell line, NT2/NTERA-2, before and after onset of differentiation. The results showed that incorporation of SRY in both groups of genes was increased after induction of differentiation. Besides, lower expression of OCT4, SOX2, and NANOG and higher expression of PAX6 and NESTIN genes in differentiated cells suggest that SRY may act as a transcription repressor for pluripotency-associated genes and as a transcription activator for differentiation-related genes.

Knockdown of nucleosome assembly protein 1-like 1 promotes dimethyl sulfoxide-induced differentiation of P19CL6 cells into cardiomyocytes.

Transplantation of cardiomyocytes derived from stem cells is a promising option for cardiac repair. However, how to obtain efficient cardiomyocytes from stem cells is still a great challenge. Understanding of the mechanism that regulates the cardiac differentiation of stem cells is necessary for the effective induction of cardiomyocytes. A clonal derivative named P19CL6 cells can easily differentiate into cardiomyocytes with 1% dimethyl sulfoxide (DMSO) treatment, which offers a valuable model to study cardiomyocytes differentiation in vitro. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ) proteomics were performed to identify proteins associated with cardiomyocytes differentiation of P19CL6 cells induced by DMSO. Out of 543 non-redundant proteins identified, 207 proteins showed significant changes during differentiation with ≥1.2-fold or ≤0.83-fold changes cut-offs. Nine proteins were confirmed by the quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis respectively. Notably, broad consistency was well showed between mRNA and protein expression for down-regulation of nucleosome assembly protein 1-like 1 (Nap1l1). Further study revealed that knockdown of Nap1l1 by stable transfection of shRNA vector significantly accelerated DMSO-induced cardiomyocytes differentiation of P19CL6 cells characterized by increases in expression of cardiac specific transcription factors, genes, and proteins (GATA4, MEF-2C, ANP, BNP, cTNT, and ß-MHC). Therefore, Nap1l1 is a novel protein that regulates cardiomyocytes differentiation of P19CL6 cells induced by DMSO.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and ßIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Mono-(2-ethylhexyl) phthalate (MEHP) promotes invasion and migration of human testicular embryonal carcinoma cells.

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins.

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.

The expression of peroxisomal protein transcripts increased by retinoic acid during neural differentiation.

Peroxisomal matrix protein is mainly expressed in heart, skeletal muscle, and brain tissues. To study the expression of peroxisomal protein (PEP) during neurogenesis, we employed mouse embryonic carcinoma cells (P19) and embryonic stem cells (mESCs) as an in vitro model for neural differentiation by retinoic acid (RA) induction. Expression pattern of PEP was investigated under distinct steps of differentiation by RT-PCR and real-time PCR. The results revealed that expression of PEP transcripts was markedly increased after the RA treatment at embryoid body and neural stages. Therefore, we concluded that PEP might be involved in the early process of neurogenesis, which needs further verification.