Embryonal Carcinoma Cells Research Articles

Identification of a candidate enhancer for DMRT3 involved in spastic cerebral palsy pathogenesis

Cerebral palsy (CP) is a major neuronal disease and the most common movement disorder in children. Although environmental factors leading to CP have been greatly investigated, the genetic mechanism underlying CP is not well understood. Here we focused on two clinical reports that characterized a deletion involving the KANK1 gene locus in the 9p24.3 region. One report shows spastic CP and the other shows no spastic CP phenotype. Based on the epigenetic status and evolutionary conservation, we first found a functional genomic element at the noncoding region that was deleted only in patients with spastic CP. This element contains the retinoic acid receptor/retinoid X receptor (RAR/RXR) complex-binding motif that is widely conserved among placental mammals. RAR/RXR ChIP-seq data from mouse F9 embryonal carcinoma cells that were treated with trans-retinoic acids showed that the element has a binding ability. In addition, data regarding chromosome conformation capture from mouse neural progenitor and ES cells suggested that the element spatially interacts with the Doublesex and mab-3 related transcription factor 3 (Dmrt3) gene promoter that is located approximately 120 kb downstream of the RAR/RXR-binding site. Dmrt3 is detected in the developing mouse forebrain and in some interneurons in the spinal cord, and it works as a locomotion coordinator in horses and mice. Thus, the deletion of the cis-regulatory element for DMRT3 in humans may cause impaired development of the forebrain and gait abnormalities, resulting in spastic CP. In conclusion, this study provides new mechanistic insights into the genetic basis of CP.

Clinicopathological Significance and Antitumor Effect of MPHOSPH1 in Testicular Germ Cell Tumor

MPHOSPH1, which is one of the kinesin superfamily proteins, has been reported to play an essential role in the carcinogenesis and progression of several kinds of cancers. MPHOSPH1 has also been suggested to be involved in STAT3 phosphorylation in hepatocellular carcinoma. However, the biological behavior of MPHOSPH1 in testicular germ cell tumors (TGCTs) is unclear at present. The purposes of this study were to investigate the correlation between the expression of MPHOSPH1 and clinicopathological factors and to examine the efficacy of MPHOSPH1 target therapy in TGCTs. We investigated 75 formalin-fixed paraffin-embedded TGCT samples, containing a total of 86 germ cell tumor components, by immunohistochemistry and 12 frozen samples by Western blotting. Moreover, we carried out in vitro studies to clarify the antitumor effect of MPHOSPH1 knockdown in embryonal carcinoma cell lines, NEC8 and NEC14, using small interference RNA (siRNA). A significantly high expression of MPHOSPH1 was recognized in embryonal carcinoma and yolk sac tumor components compared to the seminoma component (p<0.001, respectively). Clinically, non-seminoma cases are known to have worse prognosis than pure-seminoma cases. Interestingly, high MPHOSPH1 expression was associated with distant metastasis (p=0.001), and thus with advanced-stage disease in this study. High expression of MPHOSPH1 interacted with high expression of phosphorylated STAT3 (p=0.01). The in vitro experiments demonstrated that MPHOSPH1 interruption by siRNA resulted in a significant reduction of cell migration, invasion, proliferation and colony formation in both embryonal carcinoma cell lines (p<0.001, respectively). In conclusion, MPHOSPH1 may be a potential treatment option for TGCTs, and its expression may be a novel biomarker of poor prognosis.

66 Unexpected Protein Dynamics During the Oocyte-to-Embryo Transition in Mice: a Mass Spectrometry and RNA Sequencing Tandem Study

The development of a zygote from fertilization through implantation is orchestrated by a series of changes in gene expression at the RNA and protein levels. In mice, the hexagonal-shaped free ribosomes that translate mRNAs into proteins are rare during cleavage and only become abundant at the morula-blastocyst stage. Thus, how well can we understand development by an analysis of transcripts as proxy for the proteins? This question led us to combine mass spectrometry with RNA sequencing, with a long-term aim to illuminate the functional interconnection between transcriptome, proteome, and morphogenetic transitions in mouse embryos. To obtain cellular material in sufficient numbers for a proteomic approach, we recovered B6C3F1 × CD1 fertilized oocytes in vivo after ovarian stimulation, and cultured them in KSOM(aa) medium under 5% CO2 in air. We collected unfertilized oocytes and embryos from 6 pre-implantation developmental stages (pronuclear oocyte, 2-cell embryo, 4-cell embryo, 8-cell embryo, advanced morula, and blastocyst) in triplicates (600 oocytes or embryos per replicate). The protein lysates of these samples were added with equimolar amounts of isotopically labelled F9 embryonal carcinoma cells, resulting in light/heavy (L/H) protein mixtures. These were analysed using LTQ Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) and Q-Exactive (Thermo Fisher Scientific) instruments to yield L/H ratios for each measured protein, which were compared to transcript levels measured by RNA sequencing using an Illumina HiSEqn 2500 platform (100 oocytes or embryos per replicate; Illumina Inc., San Diego, CA, USA). All statistical analyses were performed in R (https://www.r-project.org/). Collectively, 6976 proteins were detected in at least the F9 cells (precondition for quantifying the L/H ratios in oocytes or embryos). In particular, 4991 proteins were detected in all developmental stages, and 1893 proteins were detected in all replicates. Spearman correlation analysis of each stage relative to unfertilized oocyte revealed distinct proteome and transcriptome developmental profiles. Furthermore, hierarchical clustering of the proteomes identified 2 main clusters (cluster 1: oocyte to 16-cell embryo; cluster 2: blastocyst), which are different from the well-known clusters of the transcriptomes (cluster 1: oocyte to 2-cell embryo; cluster 2: 4-cell embryo to blastocyst). Functional analysis of the genes that are differently expressed across adjacent stages highlighted the developmental roles of DNA damage response and Wnt signalling among the proteins and metabolism and translation among the RNAs. Our data provide new insight into the regulation of the transition from the differentiated oocyte into the embryo, highlighting the different gene identities and different biological processes featured by the proteins that change quantitatively across consecutive developmental stages, compared with mRNAs. The status of the mouse as model system in developmental biology is enriched with a protein dimension, and caution is called for in the use of transcript dynamics as proxy for protein dynamics.

Arsenic Induces Members of the mmu-miR-466-669 Cluster Which Reduces NeuroD1 Expression.

Chronic arsenic exposure can result in adverse development effects including decreased intellectual function, reduced birth weight, and altered locomotor activity. Previous in vitro studies have shown that arsenic inhibits stem cell differentiation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate multiple cellular processes including embryonic development and cell differentiation. The purpose of this study was to examine whether altered miRNA expression was a mechanism by which arsenic inhibited cellular differentiation. The pluripotent P19 mouse embryonal carcinoma cells were exposed to 0 or 0.5 μM sodium arsenite for 9 days during cell differentiation, and changes in miRNA expression was analyzed using microarrays. We found that the expression of several miRNAs important in cellular differentiation, such as miR-9 and miR-199 were decreased by 1.9- and 1.6-fold, respectively, following arsenic exposure, while miR-92a, miR-291a, and miR-709 were increased by 3-, 3.7-, and 1.6-fold, respectively. The members of the miR-466-669 cluster and its host gene, Scm-like with 4 Mbt domains 2 (Sfmbt2), were significantly induced by arsenic from 1.5- to 4-fold in a time-dependent manner. Multiple miRNA target prediction programs revealed that several neurogenic transcription factors appear to be targets of the cluster. When consensus anti-miRNAs targeting the miR-466-669 cluster were transfected into P19 cells, arsenic-exposed cells were able to more effectively differentiate. The consensus anti-miRNAs appeared to rescue the inhibitory effects of arsenic on cell differentiation due to an increased expression of NeuroD1. Taken together, we conclude that arsenic induces the miR-466-669 cluster, and that this induction acts to inhibit cellular differentiation in part due to a repression of NeuroD1.

Extract of mouse embryonic stem cells induces the expression of pluripotency genes in human adipose tissue-derived stem cells.

Objective(s):In some previous studies, the extract of embryonic carcinoma cells (ECCs) and embryonic stem cells (ESCs) have been used to reprogram somatic cells to more dedifferentiated state. The aim of this study was to investigate the effect of mouse ESCs extract on the expression of some pluripotency markers in human adipose tissue-derived stem cells (ADSCs).Materials and Methods:Human ADSCs were isolated from subcutaneous abdominal adipose tissue and characterized by flow cytometric analysis for the expression of some mesenchymal stem cell markers and adipogenic and osteogenic differentiation. Frequent freeze-thaw technique was used to prepare cytoplasmic extract of ESCs. Plasma membranes of the ADSCs were reversibly permeabilized by streptolysin-O (SLO). Then the permeabilized ADSCs were incubated with the ESC extract and cultured in resealing medium. After reprogramming, the expression of some pluripotency genes was evaluated by RT-PCR and quantitative real-time PCR (qPCR) analyses.Results:Third-passaged ADSCs showed a fibroblast-like morphology and expressed mesenchymal stem cell markers. They also showed adipogenic and osteogenic differentiation potential. QPCR analysis revealed a significant upregulation in the expression of some pluripotency genes including OCT4, SOX2, NANOG, REX1 and ESG1 in the reprogrammed ADSCs compared to the control group.Conclusion:These findings showed that mouse ESC extract can be used to induce reprogramming of human ADSCs. In fact, this method is applicable for reprogramming of human adult stem cells to a more pluripotent sate and may have a potential in regenerative medicine.

Transplantation of iPS-Derived Tumor Cells with a Homozygous MHC Haplotype Induces GRP94 Antibody Production in MHC-Matched Macaques.

Immune surveillance is a critical component of the antitumor response in vivo, yet the specific components of the immune system involved in this regulatory response remain unclear. In this study, we demonstrate that autoantibodies can mitigate tumor growth in vitro and in vivo We generated two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from monkey-induced pluripotent stem cells (iPSC) carrying a homozygous haplotype of major histocompatibility complex (MHC, Mafa in Macaca fascicularis). To establish a monkey cancer model, we transplanted these cells into monkeys carrying the matched Mafa haplotype in one of the chromosomes. Neither Mafa-homozygous cancer cell line grew in monkeys carrying the matched Mafa haplotype heterozygously. We detected in the plasma of these monkeys an IgG autoantibody against GRP94, a heat shock protein. Injection of the plasma prevented growth of the tumor cells in immunodeficient mice, whereas plasma IgG depleted of GRP94 IgG exhibited reduced killing activity against cancer cells in vitro These results indicate that humoral immunity, including autoantibodies against GRP94, plays a role in cancer immune surveillance. Cancer Res; 77(21); 6001-10. ©2017 AACR.

Distinct retinoic acid receptor (RAR) isotypes control differentiation of embryonal carcinoma cells to dopaminergic or striatopallidal medium spiny neurons

Embryonal carcinoma (EC) cells are pluripotent stem cells extensively used for studies of cell differentiation. Although retinoic acid (RA) is a powerful inducer of neurogenesis in EC cells, it is not clear what specific neuronal subtypes are generated and whether different RAR isotypes may contribute to such neuronal diversification. Here we show that RA treatment during EC embryoid body formation is a highly robust protocol for generation of striatal-like GABAergic neurons which display molecular characteristics of striatopallidal medium spiny neurons (MSNs), including expression of functional dopamine D2 receptor. By using RARα, β and γ selective agonists we show that RARγ is the functionally dominant RAR in mediating RA control of early molecular determinants of MSNs leading to formation of striatopallidal-like neurons. In contrast, activation of RARα is less efficient in generation of this class of neurons, but is essential for differentiation of functional dopaminergic neurons, which may correspond to a subpopulation of inhibitory dopaminergic neurons expressing glutamic acid decarboxylase in vivo.

OCT4B2, a novel alternative spliced variant of OCT4, is significantly upregulated under heat-stress condition and downregulated in differentiated cells.

OCT4 is a crucial transcription factor that maintains self-renewal and pluripotency of embryonic stem and embryonic carcinoma cells. The human OCT4 gene can generate at least three variants (OCT4A, OCT4B, and OCTB1) via alternative splicing and alternative promoters. It has been previously reported that OCT4A is the main isoform, retaining stemness state in embryonic stem and embryonic carcinoma cells. There are several reports on the expression of OCT4A, OCT4B, and OCT4B1 in some cancers and tumor cells. The expression of OCT4 in cancer tissues and cell lines appeared to be highly controversial since it was believed that OCT4 is exclusively expressed in embryonic stem/embryonic carcinoma cells. Here, we are reporting the detection of a novel alternatively spliced variant of OCT4, OCT4B2, in several pluripotent and tumor cell lines. Moreover, the expression pattern of OCT4B2 in the course of neural differentiation of NT2 and NCCIT, embryonic carcinoma cells, was similar to that of OCT4A. OCT4B2 was highly expressed in undifferentiated cells; however, its expression was sharply downregulated upon induction of differentiation. Overexpression of OCT4B2 did not affect the distribution of cells in different cell-cycle phases of transfected cells, compared to the mock transfected cells. Interestingly, the expression of OCT4B2 transcript was elevated under the heat-shock induction. In conclusion, we are reporting a new variant of OCT4, which is expressed under different physiological conditions. The finding shed more light on complexity of OCT4 expression and functions.

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci.

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.

Brentuximab vedotin exerts profound antiproliferative and pro-apoptotic efficacy in CD30-positive as well as cocultured CD30-negative germ cell tumour cell lines.

Prognosis in patients suffering from high‐risk, refractory and relapsed germ cell tumours (GCT) often comprising of CD30‐positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody‐drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30‐expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30‐negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS‐ and flow cytometry‐based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT‐PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30‐positive GCT27 EC line exerting marked time‐dependent antiproliferative and pro‐apoptotic activity. CD30‐negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose‐dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30‐negative JAR cocultured with CD30‐positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30‐negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro‐apoptotic activity against both CD30‐positive as well as CD30‐negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high‐risk GCT of heterogeneous CD30 positivity.

Retinoic Acid Signaling in P19 Stem Cell Differentiation.

Retinoic acid (RA), especially all-trans retinoic acid is the most potent natural metabolite of vitamin A. RA is involved in a variety of biological functions including embryogenesis, cell differentiation and apoptosis. RA acts through its nuclear receptors to induce transcription of specific target genes. Mouse P19 embryonic carcinoma (EC) stem cells (ES) are one of the most studied in vitro systems for RA-induced differentiation. P19 ES cells can differentiate to endodermal-like, mesodermal-like, and neuronal-like phenotypes in response to specific morphogens including RA and dimethyl sulfoxide (DMSO). At low concentrations, RA directs P19 ES cells to differentiate into cells displaying an endodermal phenotype, whereas at higher concentrations it induces differentiation to neuroectoderm. In the past, many RA---regulated genes have been discovered in EC and ES cells and efforts are ongoing to elucidate the exact mechanisms of RA-induced ES cell differentiation and apoptosis. In the RA-triggered differentiation process of the P19 ES cells, several proteins belonging to different families participate, some being obligatory while others, dispensable. Revealing the mechanisms behind RA-induced effects on ES cells has a bearing on understanding how cells proliferate, differentiate and undergo apoptosis that can provide greater insight into cancer biology and therapy. In addition to summarizing the reports on gene/protein targets of RA in stem cells, the signaling pathways driven by some of the specific class of proteins in the presence or absence of RA in P19 ES cell differentiation, especially to an endodermal phenotype, are the focus of this review.

MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

Asymmetric DNA methylation between sister chromatids of metaphase chromosomes in mouse embryos upon bisphenol A action

Earlier we showed that asymmetric methylation of sister chromatids (AMSC) was a specific characteristic of differentiation potency, and supposed that AMSC could be a useful marker of environmental impact connected with differentiation and/or dedifferentiation. Here we investigated the level of AMSC in chromosomes and the nuclei methylation in mouse preimplantation and postimplantation embryos, in comparison with the undifferentiated cells of mouse embryonal carcinoma cell line F9, and human differentiated HEK293 cells upon BPA influence. We found that exposure of mouse preimplantation embryos to BPA caused a significant decrease in the level of AMSC in chromosomes and the nuclei methylation. The BPA exposure of potentially differentiating F9 cells had no any influence on DNA methylation in nuclei but significantly decreased the number of AMSC. The level of DNA methylation and AMSC in HEK293 cells were not also changed. These data indicate that BPA exerts significant influence on differentiating and potentially differentiable cells. The most sensitive BPA targets are preimplantation embryos and stem cells.

Abstract 3505: The expression of two key long non coding RNAs involved in reprogramming, linc-ROR and lin-P21, in gastric cancer

Abstract Introduction: Gastric cancer (GC) is the second leading cause of cancer-related death in the world and the most common cancer in southeast of Caspian Sea (Golestan province) and northwest of Iran (Ardabil). Based on cancer stem cell (CSC) hypothesis, there are similarities between process of somatic cell reprogramming, embryogenesis and tumorigenesis. Malfunction of signaling pathways that are essential for normal development also involved in the tumor initiation and CSC formation. In recent years long noncoding RNAs (lncRNAs), more than 200 nt in length, have been highly regarded as promising biomarkers for early diagnosis and prognosis of cancers. Reprogramming-Related Long Intergenic Noncoding RNA (lincRNA-ROR) plays as modulator in the reprogramming of human induced pluripotent stem cells (iPSCs) and also maintenance of embryonic stem cells (ESCs). On the other hand, lincRNA-P21 induced by P53 and prevents the somatic cell reprogramming by silencing of pluripotency gene promoters. In this project, motivate to CSCs, we evaluated the expression level of lincRNA-ROR and linc-P21 in samples of patients with gastric cancer and also in human gastric cancer cell lines (AGS and MKN45) and human embryonal carcinoma cell line (NT2). Methods: Thirty pairs of gastric samples, including gastric adenocarcinoma and their matched non-tumor tissue samples, were collected from the Iran National Tumor Bank (INTB). Cell lines were cultured in the RPMI1640. Total RNA of samples and cell lines was extracted using TRIZOL reagent (invitrogene). cDNA synthesis was performed by PrimeScript™ 1st strand cDNA Synthesis Kit (TAKARA) and real time PCR was performed by using TaqMan master mix (TAKARA) on Step one Plus™ instrument (ABI). Gene expression analysis performed by GenEx software program. Results: Despite of reports indicating the high expression of linc-ROR in some cancers, our results showed no expression for linc-ROR in gastric samples (both tumor and non tumor) and also gastric cancer cell lines. But NT2 embryonal carcinoma cell line revealed high expression of linc-ROR compared to AGS and MKN45cell lines. According to our expectation, linc-P21 represents a significant decrease in tumor versus non tumor samples (p&amp;lt;0.01) in different grads of malignancy and also in AGS and MKN45 versus to NT2 cell line. Conclusion: Dysfunction of linc-ROR and lincRNA-P21 has reported in some cancers. According to our results, it seems that the expression of linc-ROR is limited to embryonic stem cells and hasn’t remarkable expression in gastric samples and cell lines. Based on the low expression of linc-P21 in tumor samples, it seems that linc-P21 functions as a tumor suppressor. Citation Format: Mitra Khalili, Pourandokht Farhangiyan, Somayeh Jahandoost, Fatemeh Kamali. The expression of two key long non coding RNAs involved in reprogramming, linc-ROR and lin-P21, in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2017-3505

Abstract 152: Dual mTORC1/2 inhibition sensitizes testicular cancer cell lines toward cisplatin treatment

Abstract Testicular cancer (TC) patients with metastatic disease and poor prognosis have a 50% 5-year survival. It has been reported that resistant TC cell lines show hyperactivation of the PI3K/Akt/mTOR pathway. In this study we investigated the potential benefit from PI3K, Akt and mTOR inhibition in combination with cisplatin in TC. Our panel of embryonal carcinoma cell lines include the cisplatin-sensitive cells: Tera and 833KE and the cisplatin-resistant cells: Scha, TeraCP and NCCIT. Western blotting showed that the resistant TC cell line TeraCP expresses higher levels of p-Akt compared to the sensitive cell line Tera. To evaluate sensitization towards cisplatin, TC cell lines were treated with cisplatin and/or PI3K inhibitor GDC0941, Akt inhibitor MK2206, mTORC1 inhibitor everolimus and mTORC1/2 inhibitors AZD8055 and AZD2014 for 24 hours and DilC5/PI staining was performed to estimate apoptosis with flow cytometry. All TC cell lines were strongly sensitized by mTORC1/2 inhibition by increasing cisplatin induced apoptosis to 60-80% when cisplatin alone induced only 15-30%. TC cells were also sensitized by mTORC1 and PI3K inhibition but to a lesser extent. Akt inhibition did not sensitize Scha or Tera cells to cisplatin, while TeraCP was marginally sensitized. We then evaluated clonogenic capacity in cells pretreated with AZD8055 for 24 hours and seeded in the presence of different concentrations of cisplatin for 6 days. Clonogenic capacity was reduced in Tera and TeraCP after mTORC1/2 inhibition with AZD8055 compared with control cells treated with cisplatin only. Western blot done with Scha, Tera and TeraCP lysates treated for 24 hours with AZD8055, everolimus, GDC0941 and MK2206 showed that only mTOR inhibition was able to block S6 phosphorylation. In addition, we performed immunohistochemistry of p-S6 and Ki-67 in paraffin embedded tissue from TC patients. IHC showed that patient derived xenografts showed high expression of these markers compared to non-cancerous tissue, meaning that the mTOR pathway is very active in this tumor type. These data indicate that TC relies on the PI3K/Akt/mTOR pathway for survival and mTORC1/2 inhibition showed the stronger sensitizing effect towards cisplatin treatment. We consider adding mTORC1C/2 inhibition to cisplatin based treatment a potential therapeutic option for chemoresistant TC patients that warrants further in vivo investigations. Supported by CONACYT grant 381543 and Dutch Cancer Society grant RUG 2014-6691 Citation Format: Fernanda Ximena Rosas Plaza, Gerda de Vries, Albert J. Suurmeijer, Jourik A. Gietema, Marcel A.T.M. van Vugt, Steven de Jong. Dual mTORC1/2 inhibition sensitizes testicular cancer cell lines toward cisplatin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 152. doi:10.1158/1538-7445.AM2017-152

LRWD1 Regulates Microtubule Nucleation and Proper Cell Cycle Progression in the Human Testicular Embryonic Carcinoma Cells.

Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. J. Cell. Biochem. 119: 314-326, 2018. © 2017 Wiley Periodicals, Inc.

Novel variant of OCT4B4 is differentially expressed in human embryonic stem and embryonic carcinoma cells

POU domain proteins are an important family of transcription factors that regulates cell type-specific gene expression. One of the most crucial members of this family that maintains pluripotency and self-renewal of embryonic stem cells is POU5F1/OCT4. The OCT4 gene can generate several variants under different situations/cell types includes OCT4A that is the major factor sustains pluripotency in embryonic stem and embryonic carcinoma cells, and also OCT4B and OCT4B1, which are transcribed from a different potential promoter located in intron1 and are expressed in various tissues and cell types.In present study, during expression check of OCT4B1 in embryonic carcinoma cells (NT2), we discovered a novel OCT4 transcript for the first time and designated it as OCT4B4. This variant is expressed in various human pluripotent cells and its expression is down-regulated upon induction of differentiation. Moreover, knocking down of OCT4B4 by shRNA resulted in increased accumulation of transfected cells in G0/G1 phase compared to the mock-transfected control cells.

Do Cancer Cell Lines Have Fixed or Fluctuating Stem Cell Phenotypes? - Studies with the NTera2 Cell Line.

One of the important questions when studying established cancer cell lines is whether such cells contain a subpopulation of primitive cancer stem cells that maintains the expansion of the cell line. To address this issue, we performed studies on the established human embryonal carcinoma cell line NTera2 by evaluating the potential stemness of cells sorted according to their expression of the cell surface stem cell markers CD133 and SSEA4. By performing in vitro and in vivo assays, we observed different properties of cells expressing both, one, or neither of these antigens. While sorted SSEA4+ subpopulations exhibited the greatest propensity for migration toward normal serum and the highest seeding efficiency in the lungs of immunodeficient mice, CD133-SSEA4- cells displayed high seeding efficiency to the bone marrow after injection in vivo. It is worth noting that these properties did not depend on the size of the evaluated cells. To address the question of whether cancer stem cell phenotypes in cell lines are fixed or fluctuating, we sorted single cells according to their expression of CD133 and SSEA4 antigens and observed that cells which did not express these cancer stem cell markers gave rise to cells that express these markers after expansion in vitro. Therefore, our results support the idea that within established cancer cell lines, the phenotype of the cell subpopulation expressing cancer stem cell markers is not fixed but fluctuates during cell line expansion, and cells negative for these markers may acquire their expression.

The estimation of morphological state of the lungs and cytokine cascade in experimental acute necrotizing pancreatitis (ANP)

Low oxygen availability (hypoxia) is a hallmark of rapidly proliferating tumors and has been suggested to be a characteristic of the embryonic and adult stem cell niche. The idea of relating cancer to stem cells is increasingly popular due to the identification of specific cancer stem cells sharing the typical plasticity and motility of pluripotent stem cells. Hypoxia plays a critical role in early embryonic development and in tumor progression, participating in processes such as angiogenesis, apoptosis, cell migration, invasion and metastasis. Some of the molecular pathways that have been shown to mediate these hypoxia-induced responses, such as the hypoxia inducible factor (HIF)-1alpha and Notch signaling, appear to be active in both embryonic and neoplastic pluripotent stem cells. Nevertheless, the mechanisms underlying these regulatory processes are not yet fully understood. In this review, we attempt to shed some light on the mechanisms involved in hypoxia-dependent processes related to stem cell features and tumor progression, such as the maintenance of the undifferentiated state, cell proliferation, tumor neovascularization, extra-cellular matrix degradation and motility factor up-regulation. With this purpose in mind, we summarize recent observations in embryonic, adult and cancer stem cells that demonstrate the parallelism existing in their hypoxia responses. Finally, based on the observations of our own laboratory and others, we suggest that the comparative analysis of the response to low oxygen levels of embryonic stem cells and cancer stem cells (such as embryonal carcinoma cells), may throw fresh light on our understanding of the mechanisms underlying hypoxia-induced invasiveness and the resistance to anticancer treatments, thereby stimulating the development of novel therapeutic strategies.

2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells, it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line).We examined three parameters related to cellular bioenergetics: phosphotransfer system, aerobic glycolysis, and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells.2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells, while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass, increased proton leak, and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference, and highlight the importance of further research concerning energy metabolism of stem cells.