Human Embryonal Carcinoma Research Articles

Establishment and functional analysis of a cell model expressing the C-type lectin receptor Langerin

Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro. Methods The cDNA of Langerin was obtained by PCR and cloned into a eukaryotic green fluorescent protein (EGFP) expression vector pEGFP-C1 with EGFP located in the N terminal region of the Langerin gene. Then, the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T. Subsequently, laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein, and flow cytometry to measure the expression of Langerin. Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen (nDer p 2) by Langerin. Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells. As flow cytometry revealed, the expression level of Langerin in transfected HEK293T cells was increased by 43% compared with untransfected cells. Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed, and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2. Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors, and could bind to and endocytose allergens. Key words: Dendritic cells; Receptors, mitogen; Recombinant fusion proteins; Pyroglyphidae; HEK293 cells; Receptors, pattern recognition; Langerin

Dimethyl sulfoxide induces chemotherapeutic resistance in the treatment of testicular embryonal carcinomas.

Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is used as a solvent in biological studies and as a vehicle for drug therapy. The present study was designed to evaluate the potential effects of DMSO as a solvent in the treatment of testicular embryonal carcinomas (ECs). DMSO was applied to two human EC cell lines (NEC8 and NEC14), with the treated cells evaluated in relation to cisplatin (CDDP) resistance, differentiation (using Vimentin, Fibronectin, TRA-1-60, and SSEA-1 and -3 as markers) and stemness (denoted by expression of SOX2 and OCT3/4). Furthermore, DNA methyltransferase (DNMT-1, -3A and -3L) expression and methylation status were analyzed. DMSO induced resistance to CDDP, aberrant differentiation and reduction of stemness-related markers in each of the EC cell lines. The expression levels of DNMT-3L and -3A were reduced in response to DMSO, while this treatment also affected DNA methylation. The data demonstrated that DMSO perturbed differentiation, reduced stemness and induced resistance to CDDP in human EC cells. Therefore, DMSO could reduce drug efficacy against EC cells and its use should be carefully managed in the clinical application of chemotherapy.

Curcumin induces apoptotic cell death via Oct4 inhibition and GSK-3β activation in NCCIT cells.

Octamer-binding transcription factor 4 (Oct4) is a key regulator of pluripotent embryonic stem cell maintenance. However, increasing evidence has suggested that Oct4 is also expressed in cancer stem cells (CSCs) and is associated with tumor progression and chemoresistance. Curcumin (CUR) is a widely used cancer chemopreventive agent, and it has been used to treat several diseases including cancers. Here, we investigated whether CUR-induced apoptotic cell death by inhibiting Oct4 levels and examining molecular mechanisms in NCCIT human embryonic carcinoma cells. CUR significantly inhibited Oct4 transcription levels in a dose-dependent manner by dual luciferase experiment, also decreased mRNA and protein levels in NCCIT human embryonic carcinoma cells, which express high levels of endogenous Oct4. Interestingly, we found that CUR treatment increased apoptotic cell death including subG0/G1 contents, cleavage caspases, and pro-apoptotic protein, as confirmed with a series of loss-of-function experiments using Oct4 siRNA. Furthermore, CUR induced marked total level of glycogen synthase kinase 3 beta (GSK-3β), resulting in an increase in apoptotic cell death, was evaluated using chemical inhibitor of GSK3-3β. These data suggest that CUR induces apoptotic cell death through Oct4 inhibition and GSK-3β activation. Thus, CUR may be a useful cancer chemopreventive agent to suppress tumor progression or to improve chemoresistance by eliminating CSCs.

Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure.

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.

Transcription factor NF-Y inhibits cell growth and decreases SOX2 expression in human embryonal carcinoma cell line NT2/D1.

Transcription factor NF-Y belongs to the embryonic stem cell transcription factor circuitry due to its role in the regulation of cell proliferation. We investigated the role of NF-Y in pluripotency maintenance using NT2/D1 cells as one of the best-characterized human embryonal carcinoma cell line. We investigated the efficiency of protein transduction and analyzed the effects of forced expression of short isoform of NF-Y A-subunit (NF-YAs) on NT2/D1 cell growth and expression of SOX2. We found that protein transduction is an efficient method for NF-Y overexpression in NT2/D1 cells. Next, we analyzed the effect of NF-YAs overexpression on NT2/D1 cell viability and detected significant reduction in cell growth. The negative effect of NF-YAs overexpression on NT2/D1 cell pluripotency maintenance was confirmed by the decrease in the level of the pluripotency marker SOX2. Finally, we checked the p53 status and determined that the NF-Y-induced inhibition of NT2/D1 cell growth is p53-independent.

PCDHB14- and GABRB1-like nervous system developmental genes are altered during early neuronal differentiation of NCCIT cells treated with ethanol.

Ethanol (EtOH) exposure during embryonic development causes dysfunction of the central nervous system (CNS). Here, we examined the effects of chronic EtOH on gene expression during early stages of neuronal differentiation. Human embryonic carcinoma (NCCIT) cells were differentiated into neuronal precursors/lineages in the presence or absence of EtOH and folic acid. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early brain development. EtOH exposure downregulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in CNS development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. GeneGo pathway analysis revealed that the deregulated genes mapped to disease pathways that were relevant to fetal alcohol spectrum disorders (FASD, such as neurotic disorders, epilepsy, and alcohol-related disorders). In conclusion, these findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from EtOH exposure could underlie the neurodevelopmental disorders in individuals with FASD.

Biomimetically Synthesized Aqueous Ferrofluids Having Antibacterial and Anticancer Properties

Synthesis of functional iron oxide nanoparticles, well dispersed in aqueous fluids still remains a challenge as its stability requires a delicate balance between electrostatic and magnetic interactions. Templated synthesis using biomolecules is useful because the biomolecules have their unique arrangement in aqueous systems that enhance stability, commonly called “biomimetic synthesis”. We have developed a one-pot in-situ, low energy process for the synthesis of highly monodispersed, Collagen Functionalized Ferrofluids (CFF) as a templating agent in an aqueous medium. The nanoparticles so obtained were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR). The antibacterial activity in terms of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and growth inhibition has been assessed against gram positive, Staphylococcus aureus, ATCC 13709 (native strain) and in Escherichia coli, DH5α gram negative bacteria. The cytotoxicity of the CFFs on cancer cell lines human embryonic kidney (HEK), breast adenocarcinoma (MCF-7) and Ehrlich ascitic carcinoma (EAC) have also been investigated. CFFs indicated variable MIC and MBC values against S. aureus and E. coli being minimum for 1.5% CFF (MIC:23.43 μg/ml and 93.75 μg/ml and MBC: 46.87 μg/ml and 187.5 μg/ml). The observed cytotoxicity in mammalian cells indicated the susceptibility of MCF-7 breast cancer cells when compared to HEK cells.

RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells.

Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.

Fluoxetine-induced regulation of heat shock protein 90 and 14-3-3ε in human embryonic carcinoma cells.

Fluoxetine, a serotonin-selective reuptake inhibitor, exerts antidepressant and antianxiety effects on major depressive and anxiety disorders. Previous studies suggest that treatment with fluoxetine influences the expression of various proteins that are involved in proliferation, differentiation, and apoptosis in the neuronal cells of the brain. However, many aspects of the molecular pathways that modulate antidepressant action are not well understood. Here, with the aim of identifying proteins involved in antidepressant action, we examined the protein expression profile of human embryonic carcinoma (NCCIT) cells in response to fluoxetine treatment using proteomic techniques such as two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We found several upregulated and downregulated proteins in fluoxetine-treated NCCIT cells, and then biochemically confirmed the increased expression of heat shock protein 90 and 14-3-3ε, which play an essential role in many cellular mechanisms including cell cycle control and other signaling pathways. Our data suggest that the regulated expression of heat shock protein 90, 14-3-3ε, and other identified proteins may be associated with the therapeutic action of fluoxetine.

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.

MiR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8) axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

Abstract 2319: Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells

Abstract The Ten-eleven translocation (TET) family of dioxygenases hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. 5mC provides important epigenetic instructions during development, and its aberrant control is a major contributor to cellular transformation; however TET functions in regulating the epigenome, particularly in cancer, remain largely unknown. We targeted TET1, TET2, and TET3 for siRNA-mediated depletion in pluripotent human embryonic carcinoma cells and examined the impact on 5mC and 5hmC genome-wide localization. TET1, TET2, and TET3 co-regulate 5hmC at many sites, and depletion of only one of the TETs is sufficient to reduce 5hmC at these co-regulated sites, suggesting a functional co-dependence for TETs. Depletion of TET1 and TET2 had the greatest impact on 5hmC levels at high and low CpG density promoters, respectively, indicating that TETs exhibit DNA sequence-based functional specificity. All TETs prevent hypermethylation throughout the genome, especially in CpG island shores, where TET depletion resulted in prolific hypermethylation. Promoter hypermethylation resulting from TET depletion was associated with histone H2AK119 monoubiquitination, DNMT1, and DNMT3B occupancy. Surprisingly, TETs also promote cytosine methylation, as many loci became hypomethylated following TET depletion. Induction of differentiation generally caused 5hmC reduction, except at transcriptionally activated genes, which become enriched for 5hmC. Importantly, genes prone to promoter hypermethylation in cancer become depleted of intragenic 5hmC and 5mC with TET deficiency. This study highlights the multi-dimensional functions of TETs in mediating DNA methylation, hydroxymethylation, and gene expression patterns, and the results reveal that chromatin landscape and DNA sequence composition are regulators of TET function. Citation Format: Emily L. Putiri, Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2014-2319

Abstract 1934: Role of CDK1 in human stem cell biology

Abstract Self-renewal and pluripotency of stem cells (SC) is maintained by well-characterized transcriptional factors that assemble a core regulatory network, although less defined auto- and cross-regulatory signaling pathways are also critical. Accumulating evidence indicates that cell cycle regulators play an important role in control of pluripotency and differentiation that goes beyond their roles in cell cycle regulation. Cyclin-dependent kinase 1 (CDK1) is essential for cell cycle progression and embryonic development, and has recently been implicated in a signaling network with pluripotency proteins. In this study, we investigated the potential role of CDK1 in regulation of pluripotency and differentiation. CDK1 expression is enriched in the pluripotent state of human embryonic stem cells (hESCs) and down regulated during differentiation. shRNA knockdown of CDK1 or a selective CDK1 inhibitor (RO-3306) promoted hESC differentiation, shown by the reduced expression of the pluripotency markers Oct4, Nanog, and Sox2, and the up-regulation of the mesendoderm transcripts Brachyury, Eomes, Goosecoid, and MixL1. Interestingly, down-regulation of CDK1 by both shRNA and RO-3306 could not block ES cells at G2/M or other phases of the cell cycle, suggesting that relatively milder knockdown of CDK1 did not induce hESC cell cycle arrest or impair overall proliferation but was enough to promote differentiation. Knockdown of CDK1 also decreased phosphorylation of Akt at Thr308 and Ser473, one of the key regulators of pluripotency signaling. Further, JNJ-770621, a dual inhibitor of CDK1/CDK2 and Aurora kinases but not Akt or Erk, induced human embryonal carcinoma cell (NCCIT) differentiation accompanied by reduced phosphorylation of Akt. Addition of UO126 and SB431542, which inhibit MEK/ERK and TGFβ/Smad2/ respectively (downstream of PI3K/Akt signaling), reversed the differentiation induced by shCDK1 or RO-3306 treatment. Moreover, phosphorylation of PDK1 (upstream of Akt) was notably reduced in CDK1 knockdown cells but not in serum-induced differentiation, indicating that the inhibition of activation of PDK1 or Akt could be the primary effect of CDK1 down-regulation. The results suggest that threshold levels of CDK1 might direct- or cross-regulate key signaling pathways, such as that operating through PI3K-PDK1-Akt. In summary, CDK1 is required for maintenance of stem cell pluripotency, in a manner that is distinct from its role in regulation of cell cycle progression, potentially through direct or indirect cross-regulation of fundamental pluripotency signaling of PI3K/PDK1/Akt-ERK-GSK3β and TGFβ/Smad. Citation Format: Xiao Qi Wang, Lin Chen, Chris Norbury. Role of CDK1 in human stem cell biology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1934. doi:10.1158/1538-7445.AM2014-1934

Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2)

Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model.

Beclin-1-p53 interaction is crucial for cell fate determination in embryonal carcinoma cells.

Emerging interest on the interrelationship between the apoptotic and autophagy pathways in the context of cancer chemotherapy is providing exciting discoveries. Complexes formed between molecules from both pathways present potential targets for chemotherapeutics design as disruption of such complexes could alter cell survival. This study demonstrates an important role of Beclin-1 and p53 interaction in cell fate decision of human embryonal carcinoma cells. The findings provide evidence for p53 interaction with Beclin-1 through the BH3 domain of the latter. This interaction facilitated Beclin-1 ubiquitination through lysine 48 linkage, resulting in proteasome-mediated degradation, consequently maintaining a certain constitutive level of Beclin-1. Disruption of Beclin-1–p53 interaction through shRNA-mediated down-regulation of p53 reduced Beclin-1 ubiquitination suggesting requirement of p53 for the process. Reduction of ubiquitination consequently resulted in an increase in Beclin-1 levels with cells showing high autophagic activity. Enforced overexpression of p53 in the p53 down-regulated cells restored ubiquitination of Beclin-1 reducing its level and lowering autophagic activity. The Beclin-1–p53 interaction was also disrupted by exposure to cisplatin-induced stress resulting in higher level of Beclin-1 because of lesser ubiquitination. This higher concentration of Beclin-1 increased autophagy and offered protection to the cells from cisplatin-induced death. Inhibition of autophagy by either pharmacological or genetic means during cisplatin exposure increased apoptotic death in vitro as well as in xenograft tumours grown in vivo confirming the protective nature of autophagy. Therefore, Beclin-1–p53 interaction defines one additional molecular subroutine crucial for cell fate decisions in embryonal carcinoma cells.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells.

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Induced pluripotency enables differentiation of human nullipotent embryonal carcinoma cells N2102Ep

Embryonal carcinoma (EC) cells, which are considered to be malignant counterparts of embryonic stem cells, comprise the pluripotent stem cell component of teratocarcinomas, a form of testicular germ cell tumors (GCTs). Nevertheless, many established human EC cell lines are nullipotent with limited or no capacity to differentiate under normal circumstances. In this study, we tested whether an over-expression of Yamanaka's reprogramming factors OCT4, SOX2, c-MYC and KLF4 might enable differentiation of the human nullipotent EC cells N2102Ep. Using OCT4 knockdown differentiated N2102Ep cells, we are able to derive reprogrammed N2102Ep cell lines. The induced pluripotency of N2102Ep allows the cells to differentiate toward neural lineage by retinoic acid; the expression of SSEA3 and SSEA4 is down-regulated, whereas that of neural surface markers is up-regulated. Consistent with the up-regulation of neural surface markers, the expression of the master neuroectodermal transcription factor PAX6 is also induced in reprogrammed N2102Ep. We next investigated whether PAX6 might induce spontaneous differentiation of nullipotent stem cells N2102Ep. However, while an ectopic expression of PAX6 promotes differentiation of NTERA2, it induces cell death in N2102Ep. We nevertheless find that upon induction of retinoic acid, the reprogrammed N2102Ep cells form mature neuronal morphology similar to differentiated pluripotent stem cells NTERA2 as determined by TUJ1 expression, which is absent in N2102Ep parental cells. Altogether, we conclude that the nullipotent state of human EC cells can be reprogrammed to acquire a more relaxed state of differentiation potential by Yamanaka's factors.

Impact of human MLL/COMPASS and polycomb complexes on the DNA methylome.

The correlation between DNA methylation and a subset of histone post-translational modifications (positive and negative) has hinted at an underlying regulatory crosstalk between histone marks and DNA methylation in patterning the human DNA methylome, an idea further supported by corresponding alterations to both histone marks and DNA methylation during malignant transformation. This study investigated the framework by which histone marks influence DNA methylation at a genome-wide level. Using RNAi in a pluripotent human embryonic carcinoma cell line we depleted essential components of the MLL/COMPASS, polycomb repressive complex 2 (PRC2), and PRC1 histone modifying complexes that establish, respectively, the post-translational modifications H3K4me3, H3K27me3, and H2AK119ub, and assayed the impact of the subsequent depletion of these marks on the DNA methylome. Absence of H2AK119ub resulted predominantly in hypomethylation across the genome. Depletion of H3K4me3 and, surprisingly, H3K27me3 caused CpG island hypermethylation at a subset of loci. Intriguingly, many promoters were co-regulated by all three histone marks, becoming hypermethylated with loss of H3K4me3 or H3K27me3 and hypomethylated with depletion of H2AK119ub, and many of these co-regulated loci were among those commonly targeted for aberrant hypermethylation in cancer. Taken together, our results elucidate novel roles for polycomb and MLL/COMPASS in regulating DNA methylation and define targets of this regulation.

Transcriptional activation of OCT4 by the ETS transcription factor PEA3 in NCCIT human embryonic carcinoma cells

We examined the molecular mechanism of OCT4 gene regulation by polyomavirus enhancer activator 3 (PEA3) in NCCIT cells. Endogenous PEA3 and OCT4 were significantly elevated in undifferentiated cells and reduced upon differentiation. PEA3 knockdown led to a reduction in OCT4 levels. OCT4 promoter activity was significantly up-regulated by dose-dependent PEA3 overexpression. Deletion and site-directed mutagenesis of the OCT4 promoter revealed a putative binding site within the conserved region 2 (CR2). PEA3 interacted with the binding element within CR2 in NCCIT cells. This study reveals the molecular details of the mechanism by which the oncogenic factor PEA3 regulates OCT4 gene expression as a transcriptional activator.