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Abundant Nucleostemin Expression Supports the Undifferentiated Properties of Germ Cell Tumors

2013; Elsevier BV; Volume: 183; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2013.04.018

1525-2191

Noriyuki Uema, Takako Ooshio, Kenichi Harada, Masako Naito, Kazuhito Naka, Takayuki Hoshii, Yuko Tadokoro, Kumiko Ohta, Mohamed Ashraf Ali, Miyuki Katano, Tomoyoshi Soga, Yasuni Nakanuma, Akihiko Okuda, Atsushi Hirao,

Pluripotent Stem Cells Research

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors. Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors. Testicular germ cell tumors (TGCTs) are the most common solid tumor in young adult males.1Gilbert D. Rapley E. Shipley J. Testicular germ cell tumours: predisposition genes and the male germ cell niche.Nat Rev Cancer. 2011; 11: 278-288Crossref PubMed Scopus (74) Google Scholar, 2Oosterhuis J.W. Looijenga L.H. Testicular germ-cell tumours in a broader perspective.Nat Rev Cancer. 2005; 5: 210-222Crossref PubMed Scopus (726) Google Scholar Histologically, TGCTs consist of two groups: seminomas and nonseminomas. The nonseminomas include embryonal carcinoma (undifferentiated), teratoma (differentiated), and yolk sac tumor and choriocarcinoma (extra-embryonic). TGCTs are thought to be initiated in utero as intratubular germ cell neoplasias unclassified, which originate from developing germ cells, primordial germ cells (PGCs), or gonocytes. PGCs are derived from epiblasts during early embryogenesis.3Ginsburg M. Snow M.H. McLaren A. Primordial germ cells in the mouse embryo during gastrulation.Development. 1990; 110: 521-528PubMed Google Scholar Epiblasts are pluripotent cells that differentiate to form all three layers of the trilaminar germ disk in a process called gastrulation. Consistently, pluripotent stem cell lines, termed epiblast stem cells, can be established from epiblasts in vitro.4Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D. New cell lines from mouse epiblast share defining features with human embryonic stem cells.Nature. 2007; 448: 196-199Crossref PubMed Scopus (1677) Google Scholar, 5Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Derivation of pluripotent epiblast stem cells from mammalian embryos.Nature. 2007; 448: 191-195Crossref PubMed Scopus (1549) Google Scholar In mice, PGCs become identifiable as a cluster of cells at the base of the allantois at 7.25 days after coitus (dpc).3Ginsburg M. Snow M.H. McLaren A. Primordial germ cells in the mouse embryo during gastrulation.Development. 1990; 110: 521-528PubMed Google Scholar The PGCs express OCT3/4, which support pluripotency. By 11.5 dpc, PGCs migrate from the allantois through the hindgut and settle in the gonadal ridge. By 13.5 dpc, PGCs start to display sexually dimorphic patterns of development. In males, PGCs become surrounded by differentiating Sertoli cells and grow continuously until 16.5 dpc, when they undergo mitotic arrest and become gonocytes.6Hasthorpe S. Barbic S. Farmer P.J. Hutson J.M. Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method.J Reprod Fertil. 1999; 116: 335-344Crossref PubMed Scopus (44) Google Scholar The differentiation of a PGC into a gonocyte restricts its subsequent developmental potential to the generation of male germ cells and is associated with down-regulation of markers of pluripotency.7McLaren A. Primordial germ cells in the mouse.Dev Biol. 2003; 262: 1-15Crossref PubMed Scopus (536) Google Scholar, 8Matsui Y. The molecular mechanisms regulating germ cell development and potential.J Androl. 2010; 31: 61-65Crossref PubMed Scopus (21) Google Scholar TGCTs and PGCs or gonocytes have similar gene expression profiles.9Almstrup K. Hoei-Hansen C.E. Nielsen J.E. Wirkner U. Ansorge W. Skakkebaek N.E. Rajpert-De Meyts E. Leffers H. Genome-wide gene expression profiling of testicular carcinoma in situ progression into overt tumours.Br J Cancer. 2005; 92: 1934-1941Crossref PubMed Scopus (79) Google Scholar Embryonal carcinomas highly express genes that support pluripotency of embryonic stem (ES) cells, including genes for OCT3/4, SOX-2, and NANOG.10Santagata S. Ligon K.L. Hornick J.L. Embryonic stem cell transcription factor signatures in the diagnosis of primary and metastatic germ cell tumors.Am J Surg Pathol. 2007; 31: 836-845Crossref PubMed Scopus (146) Google Scholar, 11Gopalan A. Dhall D. Olgac S. Fine S.W. Korkola J.E. Houldsworth J. Chaganti R.S. Bosl G.J. Reuter V.E. Tickoo S.K. Testicular mixed germ cell tumors: a morphological and immunohistochemical study using stem cell markers, OCT3/4, SOX2 and GDF3, with emphasis on morphologically difficult-to-classify areas.Mod Pathol. 2009; 22: 1066-1074Crossref PubMed Scopus (61) Google Scholar OCT3/4 and NANOG are sensitive and specific markers for primary seminoma and embryonal carcinoma, whereas SOX-2 is not expressed in seminomas.10Santagata S. Ligon K.L. Hornick J.L. Embryonic stem cell transcription factor signatures in the diagnosis of primary and metastatic germ cell tumors.Am J Surg Pathol. 2007; 31: 836-845Crossref PubMed Scopus (146) Google Scholar These data suggest that the differences among the subtypes of TGCTs may depend on the differentiation status of the tumor. Nucleostemin (NS), alias guanine nucleotide-binding protein-like 3 (GNL3), is a nucleolar GTP-binding protein that is expressed preferentially in the nucleolus12Tsai R.Y. McKay R.D. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells.Genes Dev. 2002; 16: 2991-3003Crossref PubMed Scopus (396) Google Scholar but it can also shuttle to and from the nucleoplasm.13Meng L. Yasumoto H. Tsai R.Y. Multiple controls regulate nucleostemin partitioning between nucleolus and nucleoplasm.J Cell Sci. 2006; 119: 5124-5136Crossref PubMed Scopus (57) Google Scholar NS belongs to the class of nucleolar GTPases that includes yeast Nug1, which exports pre-60S ribosomal subunits out of the nucleolus.14Bassler J. Kallas M. Hurt E. The NUG1 GTPase reveals and N-terminal RNA-binding domain that is essential for association with 60 S pre-ribosomal particles.J Biol Chem. 2006; 281: 24737-24744Crossref PubMed Scopus (44) Google Scholar In Caenorhabditis elegans, nst-1 mutants exhibit reduced rRNA levels, suggesting a critical role for NS in ribosome biogenesis.15Kudron M.M. Reinke V. C. elegans nucleostemin is required for larval growth and germline stem cell division.PLoS Genet. 2008; 4: e1000181Crossref PubMed Scopus (31) Google Scholar NS knockdown apparently delays processing of 32S pre-rRNA into 28S rRNA and is accompanied by a substantial decrease both in protein synthesis and in the levels of rRNAs and some mRNAs.16Romanova L. Grand A. Zhang L. Rayner S. Katoku-Kikyo N. Kellner S. Kikyo N. Critical role of nucleostemin in pre-rRNA processing.J Biol Chem. 2009; 284: 4968-4977Crossref PubMed Scopus (89) Google Scholar NS is also involved in regulating telomere integrity. NS and another protein in the same family, GNL3-like (GNL3L), interact with one of the telomeric proteins, telomeric repeat-binding factor 1 (TRF1), preventing telomere damage.17Zhu Q. Yasumoto H. Tsai R.Y. Nucleostemin delays cellular senescence and negatively regulates TRF1 protein stability.Mol Cell Biol. 2006; 26: 9279-9290Crossref PubMed Scopus (90) Google Scholar, 18Meng L. Hsu J.K. Zhu Q. Lin T. Tsai R.Y. Nucleostemin inhibits TRF1 dimerization and shortens its dynamic association with the telomere.J Cell Sci. 2011; 124: 3706-3714Crossref PubMed Scopus (22) Google Scholar, 19Hsu J.K. Lin T. Tsai R.Y. Nucleostemin prevents telomere damage by promoting PML-IV recruitment to SUMOylated TRF1.J Cell Biol. 2012; 197: 613-624Crossref PubMed Scopus (41) Google Scholar NS and GNL3L form a complex with the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT).20Okamoto N. Yasukawa M. Nguyen C. Kasim V. Maida Y. Possemato R. Shibata T. Ligon K.L. Fukami K. Hahn W.C. Masutomi K. Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.Proc Natl Acad Sci USA. 2011; 108: 20388-20393Crossref PubMed Scopus (88) Google Scholar NS deficiency in mice is early embryonic lethal,17Zhu Q. Yasumoto H. Tsai R.Y. Nucleostemin delays cellular senescence and negatively regulates TRF1 protein stability.Mol Cell Biol. 2006; 26: 9279-9290Crossref PubMed Scopus (90) Google Scholar, 21Beekman C. Nichane M. De Clercq S. Maetens M. Floss T. Wurst W. Bellefroid E. Marine J.C. Evolutionarily conserved role of nucleostemin: controlling proliferation of stem/progenitor cells during early vertebrate development.Mol Cell Biol. 2006; 26: 9291-9301Crossref PubMed Scopus (97) Google Scholar and NS thus plays a critical role in fetal development in vivo. NS was originally reported to be highly expressed in stem cells from several different tissues, including ES cells, immature hematopoietic cells, and neural stem/progenitor cells.12Tsai R.Y. McKay R.D. A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells.Genes Dev. 2002; 16: 2991-3003Crossref PubMed Scopus (396) Google Scholar A study using tetracycline (Tet)–inducible NS-deficient ES cells showed that NS is essential for the survival of ES cells.22Nomura J. Maruyama M. Katano M. Kato H. Zhang J. Masui S. Mizuno Y. Okazaki Y. Nishimoto M. Okuda A. Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.Stem Cells. 2009; 27: 1066-1076Crossref PubMed Scopus (25) Google Scholar Our research group has previously characterized NS expression in murine male germ cells.23Ohmura M. Naka K. Hoshii T. Muraguchi T. Shugo H. Tamase A. Uema N. Ooshio T. Arai F. Takubo K. Nagamatsu G. Hamaguchi I. Takagi M. Ishihara M. Sakurada K. Miyaji H. Suda T. Hirao A. Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.Stem Cells. 2008; 26: 3237-3246Crossref PubMed Scopus (31) Google Scholar In that study, NS protein was highly expressed in the nucleoli of PGCs. In addition, we successfully identified NS-expressing neonatal germ cells as spermatogonial stem cells with long-term repopulating capacity. In the present study, we investigated the expression pattern of NS in human germ cell tumors and its function in a mouse teratoma model and found that NS is essential for maintaining the undifferentiated status of germ cell tumors. The NS-GFP transgenic mice were generated as described previously.23Ohmura M. Naka K. Hoshii T. Muraguchi T. Shugo H. Tamase A. Uema N. Ooshio T. Arai F. Takubo K. Nagamatsu G. Hamaguchi I. Takagi M. Ishihara M. Sakurada K. Miyaji H. Suda T. Hirao A. Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.Stem Cells. 2008; 26: 3237-3246Crossref PubMed Scopus (31) Google Scholar BALB/c nu/nu mice (6 to 8 week old males) used as xenograft recipients were purchased from Sankyo Labo Service (Tokyo, Japan). All procedures were performed in accordance with the animal care guidelines of Kanazawa University, Kanazawa, Japan. For analysis of timed pregnant mice, the date the vaginal plug was observed was defined as 0.5 dpc of gestation. Tissue microarray sections consisting of human samples from patients with germ cell tumors (catalog no. TE2081) and of human fetal tissue (catalog no. BE01015) were purchased from US Biomax (Rockville, MD). For detailed immunohistochemical analysis, formalin-fixed, paraffin-embedded sections of surgically resected specimens from 13 germ cell tumors (4 seminomas, 4 mature teratomas, and 5 mixed germ cell tumors composed of teratoma and embryonal carcinoma) were obtained from surgical specimens in the Department of Human Pathology, Kanazawa University School of Medicine. All human materials and protocols were approved by the ethics committees of Kanazawa University. Informed consent was obtained from all patients at the time of their surgery. Surgical human specimens were immediately fixed in 10% neutral-buffered formalin and were embedded in paraffin. Serial sections (4 μm thick) were prepared from each formalin-fixed, paraffin-embedded block. Several of the sections were routinely stained for histological evaluation, and the remainder were processed for immunohistochemistry. Mouse ES cells (E14K) were maintained on a layer of mitomycin C–treated mouse embryonic fibroblasts in KnockOut Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 1% nonessential amino acids, 2 mmol/L l-glutamine, 50 units/mL penicillin plus 50 μg/mL streptomycin, 55 μmol/L 2-mercaptoethanol (all from Life Technologies–Invitrogen, Carlsbad, CA), and 1000 units/mL leukemia inhibitory factor (ESGRO; Millipore–Chemicon International, Temecula, CA). The NS-GFP mouse ES cells were established by transfection of an NS-GFP fragment23Ohmura M. Naka K. Hoshii T. Muraguchi T. Shugo H. Tamase A. Uema N. Ooshio T. Arai F. Takubo K. Nagamatsu G. Hamaguchi I. Takagi M. Ishihara M. Sakurada K. Miyaji H. Suda T. Hirao A. Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.Stem Cells. 2008; 26: 3237-3246Crossref PubMed Scopus (31) Google Scholar into parental ES cells by electroporation followed by subcloning. For labeling of ES cells with orange fluorescence, we transfected the NS-GFP ES cells with a Kusabira Orange expression vector (CAG-KO), in which Kusabira Orange cDNA (Medical & Biological Laboratories, Nagoya, Japan) was cloned into pCAGGS vector plasmid. ES cells with Tet-inducible OCT3/4 deficiency (ZHBTcH4),24Niwa H. Miyazaki J. Smith A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.Nat Genet. 2000; 24: 372-376Crossref PubMed Scopus (2896) Google Scholar ES cells with Tet-inducible NS deficiency,22Nomura J. Maruyama M. Katano M. Kato H. Zhang J. Masui S. Mizuno Y. Okazaki Y. Nishimoto M. Okuda A. Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.Stem Cells. 2009; 27: 1066-1076Crossref PubMed Scopus (25) Google Scholar and control ES cells (ERBTcH3)22Nomura J. Maruyama M. Katano M. Kato H. Zhang J. Masui S. Mizuno Y. Okazaki Y. Nishimoto M. Okuda A. Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.Stem Cells. 2009; 27: 1066-1076Crossref PubMed Scopus (25) Google Scholar were maintained without feeder cells, as described previously.22Nomura J. Maruyama M. Katano M. Kato H. Zhang J. Masui S. Mizuno Y. Okazaki Y. Nishimoto M. Okuda A. Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.Stem Cells. 2009; 27: 1066-1076Crossref PubMed Scopus (25) Google Scholar, 24Niwa H. Miyazaki J. Smith A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.Nat Genet. 2000; 24: 372-376Crossref PubMed Scopus (2896) Google Scholar These ES cells were cultured with Glasgow minimum essential medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mmol/L l-glutamine, 50 U/mL penicillin plus 50 mg/mL streptomycin, 1 mmol/L sodium pyruvate (Gibco), 55 mmol/L 2-mercaptoethanol, and 1000 U/mL leukemia inhibitory factor. The differentiation of ES cells was prevented by passaging the cells every 2 or 3 days. For Western blotting analysis, ES cells were incubated in Glasgow minimum essential medium containing 10 μg/mL Doxycycline (DOX) (Sigma-Aldrich). All ES cells were trypsinized and resuspended in 1:1 PBS/Matrigel (BD Biosciences, San Jose, CA). Viable ES cells (1 × 106 to 5 × 106 cells in 100 μL) were injected subcutaneously into the left and right dorsal flanks of BALB/c nu/nu mice. The mice were monitored for 5 weeks for teratoma formation. Any teratoma observed was measured with calipers, and size was calculated as length (mm) × width (mm). When appropriate, mice were given 0.2 mg/mL Dox (Sigma-Aldrich) in drinking water. For immunohistochemistry using human specimens, including those on tissue array, the deparaffinized and rehydrated sections were microwaved in citrate buffer (pH 6.0) for OCT3/4 or EDTA buffer (pH 9.0) for NS in a microwave oven for 20 minutes. After endogenous peroxidase was blocked, these sections were incubated at 4°C overnight with antibodies against OCT3/4 or NS (both from Abcam, Cambridge, UK) and then at room temperature for 1 hour with anti-rabbit IgG conjugated to a peroxidase-labeled dextran polymer (N-Histofine Simple Stain MAX PO kit; Nichirei Bioscience, Tokyo, Japan). After a benzidine reaction, the sections were lightly counterstained with hematoxylin. Mouse teratomas were fixed with 4% paraformaldehyde at 4°C overnight and embedded in paraffin. For some experiments, testes and teratomas were fixed with periodate-lysine paraformaldehyde for 1 hour and dehydrated with 15% sucrose for 3 hours. Sections fixed with periodate-lysine paraformaldehyde were embedded in optimal cutting temperature frozen-embedding medium (OCT Tissue-Tek; Sakura Finetek, Tokyo, Japan). Sections were immunostained with the following primary antibodies: anti-NS (Abcam and Novus Biologicals, Littleton, CO), anti-OCT3/4 (Santa Cruz Biotechnology, Santa Cruz, CA; BD Pharmingen, San Diego, CA; and Abcam), anti–Ki-67 (BD Biosciences), anti-bromodeoxyuridine (anti-BrdU) (Calbiochem; Merck Millipore, Darmstadt, Germany), and anti-GFP (Medical & Biological Laboratories and Aves Labs, Tigard, OR). The staining signals in paraffin-embedded sections were visualized with a horseradish peroxidase–conjugated secondary antibody (GE Healthcare, Chalfont St Giles, UK, and Dako, Carpinteria, CA) and a coloring reaction using a 3,3′-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Burlingame, CA), counterstained with hematoxylin, and viewed using a microscope (Axio Imager A1; Carl Zeiss, Jena, Germany). The staining signals in frozen sections were visualized with Alexa Fluor dye–conjugated secondary antibodies: anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG (Life Technologies–Invitrogen). For visualization of nuclei, specimens were stained with TOTO-3 dye (Life Technologies–Invitrogen). Immunostaining was visualized under a confocal microscope (FV1000; Olympus, Tokyo, Japan). To investigate the expression of GFP, dissociated cells were analyzed by flow cytometry. In brief, teratomas were dissected and incubated with 1% collagenase type B (Roche Diagnostics, Meylan, France), 2.5 mmol/L CaCl2, and 1 unit/mL Dispase II (Roche Diagnostics) in PBS and were dissociated with a pipetting procedure. Dissociated teratoma cells were stained with phycoerythrin-conjugated anti-CD45 and anti-Ter119 antibodies (BD Pharmingen). CD45+ and Ter119+ cells were negatively selected on an LD MACS column using anti-phycoerythrin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, the flow-through fraction was incubated with 1 μg/mL propidium iodide to allow the removal of dead cells. Teratoma cell sorting and analysis were performed using a FACSAria fluorescence-activated cell sorting system (BD Biosciences) and a JSAN cell sorter (Bay Bioscience, Kobe, Japan). Sorted cells were resuspended in PBS containing 10% fetal bovine serum or KnockOut Dulbecco’s modified Eagle’s medium, and were prepared for further analysis. Cells fractionated by flow cytometry were plated at 3000 cells per well in 24-well dishes. Colonies were visualized and counted after 1 week. The colonies were rinsed three times with PBS and fixed with 4% paraformaldehyde for 10 minutes at 4°C. The fixed colonies were washed with Tris-buffered saline and were measured with alkaline phosphatase (ALP) staining. ALP activity was detected with a fuchsin+ substrate-chromogen system (Dako, Glostrup, Denmark). RNA samples were purified from fractionated teratoma cells using an RNeasy kit (Qiagen, Valencia, CA) and reverse-transcribed using an Advantage RT-for-PCR kit (Clontech Laboratories, Mountain View, CA). Real-time RT-PCR was performed with an Mx3000P quantitative PCR system (Stratagene, La Jolla, CA) and SYBR premix Ex Taq (Takara, Shiga, Japan), according to the manufacturer’s recommended protocol. The following primers were used: GAPDH forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′), NS forward 5′-GAGTGGACAGGTGCCTCATTAGGTTACT-3′ and reverse 5′-GATGGCTTACCTGCTGTTGATTGCTC-3′, and OCT3/4 forward 5′-GGCGTTCTCTTTGGAAAGGTGTTC-3′ and reverse 5′-CTCGAACCACATCCTTCTCT-3′. For capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) analysis, 8 × 105 NS-GFPneg, NS-GFPlow, or NS-GFPhigh cells from teratomas were lysed to extract metabolites. Metabolomic profiling and data analysis were performed twice, as described previously.25Soga T. Ohashi Y. Ueno Y. Naraoka H. Tomita M. Nishioka T. Quantitative metabolome analysis using capillary electrophoresis mass spectrometry.J Proteome Res. 2003; 2: 488-494Crossref PubMed Scopus (803) Google Scholar, 26Soga T. Baran R. Suematsu M. Ueno Y. Ikeda S. Sakurakawa T. Kakazu Y. Ishikawa T. Robert M. Nishioka T. Tomita M. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption.J Biol Chem. 2006; 281: 16768-16776Crossref PubMed Scopus (554) Google Scholar For BrdU labeling experiments, mice were injected intraperitoneally with 100 mg/kg body weight BrdU (Sigma-Aldrich), and the tissues were removed 2 hours later. Detection of BrdU-labeled cells was achieved with a BrdU labeling kit (Calbiochem; Merck Millipore), according to the manufacturer’s instructions. ES cells were lysed with cell lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-HCl pH 8.0, 1% NP-40 detergent, and Roche Diagnostics protease inhibitor cocktail); these were then used as total cell lysates. Protein concentrations were measured with a Pierce BCA protein assay (Thermo Fisher Scientific, Rockford, IL), and equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk in PBS containing Tween 20 for 1 hour at room temperature. Membranes were then incubated with a goat anti-NS antibody (1:1000; Neuromics, Edina, MN) or an anti-OCT3/4 antibody (1:2000; BD Pharmingen) for 16 hours at 4°C, or a mouse anti–β-actin antibody (1:1000; Sigma-Aldrich) for 1 hour at room temperature. Immune complexes were detected using peroxidase-conjugated secondary antibodies (1:1000; 30 minutes incubation at room temperature; GE Healthcare and Dako) and an ECL Prime Western blotting detection system (GE Healthcare). To detect apoptosis in vivo, freshly isolated teratomas were immediately fixed with 4% paraformaldehyde, and tissue sections were prepared. The sections were stained by using a DeadEnd colorimetric TUNEL system (Promega, Madison, WI), according to the manufacturer’s instructions. Statistical significance was determined using the unpaired Student’s t-test. To analyze expression of NS in germ cell tumors, we stained tissue sections of human TGCTs (including seminomas, embryonal carcinomas, and mixed germ cell tumors) with an anti-NS antibody. NS expression was detected in 18/28 (64%) embryonal carcinomas and in 52/86 (60%) seminomas (Figure 1, Supplemental Figure S1, and Supplemental Table S1). NS protein was localized mainly in nuclei, and was not present in the cytoplasm. In most NS+ cells, NS protein was detected in both the nucleoplasm and nucleoli (Figure 1 and Supplemental Figure S1). There were no remarkable differences in expression pattern or abundance of NS between embryonal carcinomas and seminomas. NS expression was detected in undifferentiated components, including the embryonal carcinoma portion of all mixed germ cell tumors, and most tumor cells in the embryonal carcinoma component highly expressed NS (Figure 2, A and B). NS was not expressed in most interstitial cells, but was expressed in a few undifferentiated mesenchymal cells within the embryonal carcinoma (Figure 2, A and B). NS expression was not detected in the well-differentiated intestine-like component of a mature teratoma (Figure 2C), but weak nucleolar expression was seen in epidermal squamous cells (Figure 2D). Thus, NS was highly expressed in the embryonal carcinoma component, but not in well-differentiated mature teratoma tissues. Because it has been reported that OCT3/4, which is a critical regulator of the undifferentiated status of ES cells, controls the malignant fate of germ cell tumors,27Gidekel S. Pizov G. Bergman Y. Pikarsky E. Oct-3/4 is a dose-dependent oncogenic fate determinant.Cancer Cell. 2003; 4: 361-370Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar we next compared NS expression with OCT3/4 expression. OCT3/4 protein was detected in all mixed germ cell tumors. It was highly expressed in the embryonal carcinoma component (Figure 2E), but not in the well-differentiated mature teratoma components, including the intestine-like cells and epidermal cells (Supplemental Figure S2). Double staining with anti-OCT3/4 and anti-NS antibodies clearly showed that both proteins were expressed in the same cells (Figure 2F). Thus, coexpression of NS and OCT3/4 was observed mainly in undifferentiated germ cell tumors (embryonal carcinomas).Figure 2Abundant expression of NS in undifferentiated cells in human mixed-type germ cell tumors. Expression of NS and OCT3/4 in human germ cell tumors of mixed type (both embryonal carcinoma and mature teratoma cells). Sections were stained with an anti-NS or anti-OCT3/4 antibody, followed by a DAB peroxidase reaction; sections were counterstained with hematoxylin. A: Immunohistochemistry for NS in the embryonal carcinoma component. Most tumor cells of the embryonal carcinoma are positive for NS (asterisk), as are a few undifferentiated mesenchymal cells (double asterisk). B: Higher magnification of a portion of A. NS is expressed in the nucleoli of embryonal carcinoma cells (asterisk) and undifferentiated mesenchymal cells (double asterisk). C and D: Immunohistochemistry for NS in the intestine-like component (C) and in squamous cells of the epidermis (D). No positive cells were found in the intestine-like component. Weak nucleolar expression was found in the epidermal squamous cells. The insets show digitally enlarged images of the boxed regions. E: Immunohistochemistry for OCT3/4. Many OCT3/4+ cells were seen in the embryonal carcinoma (asterisk) but none in the undifferentiated mesenchymal components (double asterisk). F: Double immunohistochemical staining for NS (brown) and OCT3/4 (green). Most embryonal carcinoma cells express both NS and OCT3/4. Original magnification: ×200 (A and C); ×400 (B and D–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because TGCTs possibly originate from PGCs, we evaluated NS expression in developing gonadal tissues in mice. Previously, we had generated transgenic reporter mice expressing GFP under the control of the NS promoter (NS-GFP Tg mice) and reported that the GFP successfully reflects NS expression in the developing testes.23Ohmura M. Naka K. Hoshii T. Muraguchi T. Shugo H. Tamase A. Uema N. Ooshio T. Arai F. Takubo K. Nagamatsu G. H