Human Embryonal Carcinoma Cell Line Research Articles

Proteomic Analysis of the Copper Ion-Induced Stress Response in a Human Embryonic Carcinoma Cell Line

Excessive exposure to copper, a redox-active metal, generates free radicals, which can cause cellular damage. In this study, we aim to identify the proteins that are up- or downregulated by copper exposure in human embryonic carcinoma (NCCIT) cells and to understand the mechanisms that play a role in the copper-induced stress response. After exposure to copper ions, the cells showed upregulated levels of 78 kDa glucose-regulated protein, fibrillin 1, CWC22 spliceosome-associated protein (KIAA1604), heat shock protein (HSP) 60, and HSP70, while the tumor necrosis factor receptor-associated factor 6, vimentin, 14-3-3 protein zeta, and RAC-beta (AKT2) serine/threonine protein kinase were downregulated. The GeneGo Process Networks of the proteins upregulated by copper ions were analyzed, and the 3 highest-scoring networks from the proteins upregulated by copper ions are presented here. In particular, the increased level of HSP70 in response to copper ions occurred in a dose-dependent manner, indicating that HSP70 could be a potential biomarker for copper toxicity in mammalian cells.

Cytoplasmic NANOG-Positive Stromal Cells Promote Human Cervical Cancer Progression

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, IHC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer-derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal microenvironment.

P2.19 Aaptamine, Demethyloxyaaptamine, and Isoaaptamine: A Proteomic-Based Screening of Protein Targets in Cisplatin-Resistant Tumor Cells

ABSTRACT Marine sponges are a rich source of natural compounds that possess potent anticancer activities. In continuation of our research of aaptamines - bioactive marine sponge metabolites - we investigated the alkaloid aaptamine (1) and two natural derivatives, demethyloxyaaptamine (2), and isoaaptamine (3), previously isolated from the marine sponge Aaptos sp., and shown to have promising anticancer properties via a largely undefined mode of action ( Fig. 1 ). All three alkaloids showed equal levels of cytotoxicity in vitro against a cisplatin-sensitive human embryonal carcinoma cell line, NTERA2, and NTERA2-R, a cisplatin-resistant subline derived from this cell line. We investigated the effect of the alkaloids on NTERA2-R cells at IC50 (concentration causing 50 % reduction of trypan blue excluding cells after 48 h, compared to untreated control). Aaptamine caused significant inhibition of cell proliferation and induction of a G2/M cell cycle arrest in NTERA2-R cells without influencing cell viability, whereas demethyloxyaaptamine and isoaaptamine potently induced apoptosis. Proteomic screening was applied to assess proteins altered by treatment with 1, 2 and 3. Treatment at the IC50 for 48 h resulted in an alteration of 16-22 proteins (Fig. 2). Selected targets were subjected to analysis with Western blotting. Changes in the 2D map frequently resulted from post-transcriptional modifications rather then from changes in total protein expression. For example, aaptamine treatment caused hypusination of eIF5A at lysin50 (activation of eIF5A), phosphorylation of cofilin-1, and dephosphorylation of vimentin. Demethyloxyaaptamine treatment resulted in up-regulation of Hsp70 and differential phosphorylation of isoforms of alpha-enolase. Isoaaptamine caused dissociation of the nuclear matrix-associated nucleolin complex. Interestingly, 2 and 3 did not induce the eIF5A hypusination. A hypothetical pathway analysis, performed by IPA network analysis, put the following factors in a central position of the network: myc and p53 for aaptamine; TNF for demethyloxyaaptamine; and all three for isoaaptamine, thus identifying potential targets or effectors of the substances. Download : Download full-size image Fig. 1 . Aaptamine (1), demethyl-oxyaaptamine (2) and isoaaptamine (3).

Proteomic Profiling of Germ Cell Cancer Cells Treated with Aaptamine, a Marine Alkaloid with Antiproliferative Activity

Aaptamine is a marine compound isolated from the sponge Aaptos aaptos showing antiproliferative properties via an undefined mode of action. We analyzed the effects of aaptamine treatment on the proliferation and protein expression of the pluripotent human embryonal carcinoma cell line NT2. Effects on proliferation, cell cycle distribution, and induction of apoptosis were analyzed. At lower concentrations, including the IC50 of 50 μM, aaptamine treatment resulted in a G2/M phase cell cycle arrest, whereas at higher concentrations, induction of apoptosis was seen. Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of the most significantly up- and down-regulated proteins. Aaptamine treatment at the IC50 for 48 h resulted in alteration of 10 proteins, of which five each showed up- and down-regulation. Changes in the 2D map were frequently noticed as a result of post-transcriptional modifications, e.g., of the hypusine modification of the eukaryotic initiation factor 5A (eIF5A). Observed alterations such as increased expression of CRABP2 and hypusination of eIF5A have previously been identified during differentiation of pluripotent cells. For the first time, we describe changes in protein expression caused by aaptamine, providing valuable information regarding the mode of action of this compound.

A specific increase in inositol 1,4,5-trisphosphate 3-kinase B expression upon differentiation of human embryonic stem cells

Human embryonic stem cells (hESCs) are of great hope for regenerative medicine due to their dual pluripotency and self-renewal properties. We report a comparison of inositol phosphate (InsPs) production in undifferentiated, differentiated hESCs and in two cancer cell lines, Ntera2 cells, a human embryonal carcinoma cell (hECC) line and HeLa cells. To evaluate the potential impact of InsPs in differentiation, hESCs were spontaneously differentiated in culture for two weeks. The distribution of the different InsPs was affected upon differentiation: the level of highly phosphorylated InsPs was decreased. In contrast, the total level of phosphoinositides (PI) was increased. Using real time quantitative PCR (qPCR), the mRNA expression of several enzymes of the metabolism of InsPs was determined: a specific increase in inositol 1,4,5-trisphosphate 3-kinase A and B (ITPKA and ITPKB) was observed upon hESCs spontaneous differentiation. Ins(1,4,5)P3 3-kinase activity, undetectable in undifferentiated hESCs, increased upon differentiation. The same observation was made by Western blotting using an antibody directed against human ITPKB. This is the first report showing the potential implication of soluble InsPs in hESCs and possible function of isoenzymes of the inositol trisphosphate 3-kinase family in differentiation.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Comparative epigenetic analysis of Oct4 regulatory region in RA-induced differentiated NT2 cells under adherent and non-adherent culture conditions

Oct4 is a POU domain homeobox gene, expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down-regulated upon induction of differentiation. Transcriptional repression of Oct4 is followed by pronounced epigenetic changes on the regulatory region of the gene. Oct4 has a long upstream regulatory region of about 2,600 bp, consisting of proximal enhancer (PE), distal enhancer (DE), and proximal promoter (PP). In this study, we induced differentiation of a human embryonic carcinoma cell line, NT2, under two different adherent and non-adherent culture conditions, and compared histone modifications as the epigenetic marks on the regulatory region of Oct4 gene after 3 days of differentiation. Using chromatin immunoprecipitation coupled with real-time PCR technique, it was shown that the after induction of differentiation the repressive epigenetic marks of hypoacetylation and methylation on lysine-9 of histone H3 occurred very effectively on the upstream of Oct4, especially in PP region. Also, comparing the two culturing systems it was shown that methylation of lysine-9 of H3 histone was more drastic in PE region of adherent cells rather than suspension cells. This epigenetic profile was in agreement with the difference observed in the expression level of Oct4 in these two culturing systems. The current study clearly shows the effective role of cell culture condition on the epigenetic regulation of gene expression.

Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells

Serious concerns have been expressed about potential risks of engineered nanoparticles. Regulatory health risk assessment of such particles has become mandatory for the safe use of nanomaterials in consumer products and medicines; including the potential effects on reproduction and fertility, are relevant for this risk evaluation. In this study, we examined effects of silver particles of nano- (20nm) and submicron- (200nm) size, and titanium dioxide nanoparticles (TiO2-NPs; 21nm), with emphasis on reproductive cellular- and genotoxicity. Ntera2 (NT2, human testicular embryonic carcinoma cell line), and primary testicular cells from C57BL6 mice of wild type (WT) and 8-oxoguanine DNA glycosylase knock-out (KO, mOgg1−/−) genotype were exposed to the particles. The latter mimics the repair status of human testicular cells vs oxidative damage and is thus a suitable model for human male reproductive toxicity studies. The results suggest that silver nano- and submicron-particles (AgNPs) are more cytotoxic and cytostatic compared to TiO2-NPs, causing apoptosis, necrosis and decreased proliferation in a concentration- and time-dependent manner. The 200nm AgNPs in particular appeared to cause a concentration-dependent increase in DNA-strand breaks in NT2 cells, whereas the latter response did not seem to occur with respect to oxidative purine base damage analysed with any of the particles tested.

Sodium arsenite dependent protein expression analysis on human embryonic carcinoma (NCCIT) cell line

Due to their pluripotent nature, embryonic carcinoma (EC) cell lines are useful for the study of basic and applied aspects of medical therapeutics, disease management and environmental mutagenesis. We evaluated the teratogenic like effects of inorganic arsenic during the early stages of cellular development in NCCIT cells, a type of human EC cells. Using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and MetaCore pathway analysis software (GeneGo), the proteomic expression profiles of NCCIT cells after either short- or long-term sodium arsenite (NaAsO2) treatment and of NCCIT cell-derived embryoid bodies (EBs) after short-term NaAsO2 treatment were studied to determine the toxic effects on the process of normal growth and differentiation. The protein expression profiles of the NaAsO2-treated NCCIT cells and EBs were compared with that of untreated NCCIT cells. Short-term NaAsO2 treatment resulted in the down-regulation of most proteins, with heat shock 90-kDa protein (HSP90) and valosin-containing protein (VCP) being significantly downregulated. Long-term NaAsO2 treatment caused marked up-regulation of B23/nucleophosmin, glucose regulated protein 78-kDa (GRP78), unc-51-like kinase 3 (ULK3), toll-like receptor 6 precursor (TLR6) and mitogen-activated protein kinase 7 isoform A (MAP3K7). NaAsO2 treatment of the NCCIT cell-derived EBs resulted in the down-regulation of several tumor-suppressor proteins. Taken together, these data suggest that a wide spectrum of cellular responses is induced to cope with chronic non-lethal toxic stresses in NCCIT cells.

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

BackgroundIn human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.Methodology/Principal FindingsWe show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.Conclusions/SignificanceWe demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription.

Distinct Roles for Wnt-4 and Wnt-11 During Retinoic Acid-Induced Neuronal Differentiation

Retinoic acid and Wnt/β-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited β-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of β-catenin but did not involve competition between retinoic acid receptors and β-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit β-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.

P60. Functional analysis of stem cell-specific long non-coding RNAs in human embryonic carcinoma cell line NCCIT

Human stem cells are characterized by the ability for self-renewal and differentiation potential. These characteristics are likely governed by tight regulation of a molecular network that includes POU5F1, SOX2, and NANOG. In addition to known genes, transcriptome profiling studies have also identified a large number of novel transcripts that appear to be significantly expressed in stem cells. Several novel targets previously identified by reverseSAGE were investigated and these transcripts appear to be long non-coding RNAs. Two of these, HEST097 and HEST147, were further studied to determine possible connections to the stem cell fate. Real-time PCR showed specific expression of these targets in human embryonic stem cells (hESCs) and human embryonic carcinomas (hECCs), with little expression in other cell types, and was confirmed by SAGE database analysis. RNAi knockdown of HEST097 and HEST147 in hECC cell line NCCIT produced minor morphological changes and no overt signs of differentiation despite downregulation of NANOG. Cell proliferation appeared to be reduced and cell cycle markers were also affected. Intriguingly, siRNA knockdown of HEST147 resulted in downregulation of FGF4 expression, a gene that is located on the chromosome region downstream to HEST147. This may possibly point towards a role in which the stability of a long non-coding RNA may affect expression of a neighbouring transcript on its chromosome location. The discovery of these stem cell-specific transcripts raises interesting questions where further studies would be required in order to determine the possible mechanisms in which long non-coding RNAs may affect the stem cell fate.

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

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

Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting

BackgroundTesticular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.MethodsHuman embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-trans retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.ResultsCanonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.ConclusionDuring induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.

Evaluation of four cell lines for assay of infectious adenoviruses in water samples

Human viral contamination in drinking and recreational waters poses health risks. The application of PCR-based molecular technology has advanced our knowledge of the occurrence and prevalence of human viruses in water; however, it has provided no information on viral viability and infectivity. Four human cell lines were compared for their sensitivity to different serotypes of human adenoviruses using the TCID50 test. The sensitivity of each cell line varied with different serotypes of adenovirus. Human embryonic kidney cell line 293A and human lung carcinoma cell line A549 were the most sensitive, especially to enteric adenovirus 40 and 41. Plaque assay of primary sewage samples showed 293A can detect viral plaques in 7 of 13 primary sewage samples tested. Adenoviruses were also isolated using 293A from environmental water concentrates. Cloning and sequencing of environmental adenoviral isolates indentified them to be aligned with adenoviruses serotype 40 and serotype 5. The result of this study suggests that plaque assay with 293A cell line is suitable for detection of adenovirus in the aquatic environment. Combining this cell culture with molecular methods for viral assay in the aquatic environment will provide critical information for risk assessment.

Caveolin-1 Up-regulation during Epithelial to Mesenchymal Transition Is Mediated by Focal Adhesion Kinase

Emerging evidence has shown that caveolin-1 is up-regulated in a number of metastatic cancers and can influence various aspects of cell migration. However, in general, the role of caveolin-1 in cancer progression is poorly understood. In the present study, we examined alterations in caveolin-1 expression during epithelial-to-mesenchymal transition (EMT) and the ability of caveolin-1 to alter cancer cell adhesion, an aspect of cell motility. We employed two EMT cell models, the human embryonic carcinoma cell line NT2/D1, and TGF-beta1-treated NMuMG cells, which are derived from normal mouse mammary epithelia. Caveolin-1 expression was substantially up-regulated in both cell lines following the induction of EMT and was preceded by increased activation of focal adhesion kinase (FAK) and Src, two known tyrosine kinases involved in EMT. We hypothesized that caveolin-1 expression could be influenced by increased FAK phosphorylation, to which Src is a known contributor. Examination of FAK+/+ and FAK-/- mouse embryonic fibroblasts revealed that in cells devoid of FAK, caveolin-1 expression is strikingly diminished. Using FAK and superFAK constructs and the novel FAK inhibitor PF-228, we were able to demonstrate that indeed, FAK can regulate caveolin-1 expression. We also found that Src can contribute to increases in caveolin-1 expression, however, only in the presence of FAK. From the culmination of this data and our functional analyses, we conclude that caveolin-1 expression can be up-regulated during EMT, and further, once expressed, caveolin-1 can greatly influence cancer cell adhesion.

Cell-Cell Signaling Through NOTCH Regulates Human Embryonic Stem Cell Proliferation

Unlike pluripotent mouse embryonic stem (ES) cells, human ES cells and their malignant equivalents, embryonal carcinoma (EC) cells, require close cell-cell contact for efficient growth. Signaling through the NOTCH receptor, initiated by interaction with ligands of the DELTA/JAGGED family expressed on neighboring cells, plays a role in regulating the self-renewal of several stem cell systems. Members of the NOTCH and DELTA/JAGGED families are expressed by human EC and ES cells, and we have therefore investigated the possible role of NOTCH in the maintenance of these cells. Cleavage of both NOTCH1 and NOTCH2 to yield the intracellular domain responsible for the canonical signaling pathway of NOTCH was detected in several human EC and ES cell lines, suggesting that NOTCH signaling is active. Furthermore, the proliferation of human EC cells, as well as the expression of several downstream NOTCH target genes, was markedly reduced after small interfering RNA knockdown of NOTCH1, NOTCH2, and the canonical effector CBF-1 or after blocking NOTCH signaling with the gamma-secretase inhibitor L-685,458. The inhibitor also caused a reduction in the growth of human ES cells, although without evidence of differentiation. The results indicate that cell-cell signaling through the NOTCH system provides a critical cue for the proliferation of human EC and ES cell in vitro.

Testicular Maturation Arrest to Testis Cancer: Spectrum of Expression of the Vitamin D Receptor and Vitamin D Treatment In Vitro

We investigated the qualitative distribution of vitamin D receptor in human testis pathologies and performed an in vitro study of vitamin D receptor expression in a human testis cancer cell line model. Qualitative immunohistochemical analysis of vitamin D receptor in testis tumors, normal testis and specimens from infertile patients was performed. The human embryonal carcinoma cell line NT2/D1 (American Type Culture Collection, Manassas, Virginia) was cultured. Vitamin D receptor expression was examined by Western immunoblot analysis after incubating the cells with 250 to 800 nM vitamin D, 10 to 70 nM testosterone, 2 nM calcium or a combination of the 3 products. Negative controls, synctiotrophoblasts and interstitial stroma did not stain positive for vitamin D receptor. Spermatogenic, Sertoli's, Leydig and tumor cells stained positive in all specimens. Embryonal carcinoma demonstrated more nuclear and cytoplasmic staining than other tumors. Vitamin D receptor expression was seen at 50 kDa in the cell line. Sequential concentrations of vitamin D increased vitamin D receptor expression intensity. Simultaneous addition of vitamin D and testosterone decreased the vitamin D receptor signal, as did testosterone alone. Delayed administration of vitamin D 5 hours after testosterone showed the return of vitamin D receptor expression. A combination of calcium, testosterone and vitamin D showed decreased or no vitamin D receptor expression. Calcium alone increased vitamin D receptor expression at later passages. To our knowledge this is the first description of vitamin D receptor in different primary testis pathologies and in an embryonal carcinoma cell line. The in vitro model showed that vitamin D receptor is an active receptor and it is inducible with the addition of vitamin D. Testosterone may be important for vitamin D receptor down-regulation. Calcium may be an important co-factor in vitamin D receptor expression.

Characterisation of Urogenital Ridge Gene Expression in the Human Embryonal Carcinoma Cell Line NT2/D1

The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sex-determining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY, SOX9, SF-1, DHH and FGF9. Genes not expressed included WT1, DAX1 and the ovary-specific genes FOXL2 and WNT4. Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.

Transient and Stable Transgene Expression in Human Embryonic Stem Cells

Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells. Disclosure of potential conflicts of interest is found at the end of this article.