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1.
Methods Mol Biol ; 2311: 25-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033075

RESUMO

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Neuroblastoma/metabolismo , Neurogênese , Neurônios/patologia , Feocromocitoma/patologia , Teratocarcinoma/patologia , ADP Ribose Transferases/farmacologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Técnicas de Cultura de Células , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Neurogênese/efeitos dos fármacos , Crescimento Neuronal , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Fenótipo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Ratos , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Transfecção , Tretinoína/farmacologia
2.
Mol Biol Rep ; 48(2): 1985-1994, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33619662

RESUMO

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.


Assuntos
Proteínas Ligadas por GPI/metabolismo , Genes Supressores de Tumor , Neurogênese/genética , Teratocarcinoma/metabolismo , Animais , Sítios de Ligação , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica/genética , Camundongos , Células PC12 , Regiões Promotoras Genéticas , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Teratocarcinoma/genética , Tubulina (Proteína)/metabolismo , Regulação para Cima
3.
Int J Mol Med ; 47(1): 397-407, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416104

RESUMO

Immature ovarian teratocarcinoma (IOT) is a rare and malignant type of ovarian teratoma, and the molecular mechanisms underlying the pathogenesis and malignant phenotype of IOT remain uncharacterized. The present study examined a long non­coding RNA (lncRNA), long­chain intergenic non­coding RNA324 (LINC00324), which may serve a crucial role in pathogenesis of IOT. According to the results, LINC00324 was upregulated in IOT tissues and cells, as determined by reverse transcription­quantitative PCR, and its depletion impaired cell proliferation ability and improved cell apoptosis ability in IOT. Furthermore, LINC00324 acted as a miR­214­5p sponge to derepress cyclin dependent kinase 6 (CDK6), cyclin D1 (CCND1), murine double minute homolog 2 (MDM2), and mouse double minute 4 (MDM4) expression, thus increasing IOT cell proliferation and repressing apoptosis. Taken together, these results demonstrated that LINC00324 could serve as a competing endogenous RNA to facilitate IOT cell proliferation by regulation of miR­214­5p­CDK6/CCND1/MDM2/MDM4 network, which possibly provide a novel therapeutic target for IOT.


Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Neoplasias Ovarianas/metabolismo , RNA Longo não Codificante/metabolismo , Teratocarcinoma/metabolismo , Adolescente , Adulto , Linhagem Celular Tumoral , Criança , Feminino , Humanos , MicroRNAs/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , Teratocarcinoma/genética , Teratocarcinoma/patologia
4.
Cell Reprogram ; 21(4): 171-180, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31298562

RESUMO

Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in both human embryonic stem cells (hESCs) and cancer stem cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency. This article evaluates the expression of variants of the main stem cell-specific transcription factors NANOG and OCT4 critically and accurately with specific primers designed for identifying the most important variants that maintain stemness. We have examined four variants of NANOG along with a processed pseudogene and seven variants of OCT4 in human teratocarcinoma cell lines (NTERA2D1, SuSa, GCT-27, and 833KE), hESCs, and ovarian cancer cells by reverse transcriptase-polymerase chain reaction. In addition, we have examined their expression in NTERA2D1 cells on differentiation with all-trans-retinoic-acid. We show that NANOG1 is expressed in all teratocarcinoma cells and can be distinguished from NANOGP8, which is an expressed pseudogene. NANOG2 was not expressed in any of the cell lines, including ESCs. OCT4A was expressed in all cells, whereas the variant OCT4B-variant 3 was expressed only in NTERA2D1 cells. On differentiation of NTERA2D1 with retinoic acid, only NANOGP8 and OCT4A were expressed. In ovarian cancer cells, only 3/6 expressed NANOG1 and OCT4A. All malignant cells from patients with ovarian cancer (N = 6) expressed NANOG1 and OCT4A. These results demonstrate the necessity to precisely evaluate the expression of stem cell transcription factors when defining stemness.


Assuntos
Processamento Alternativo , Células-Tronco Embrionárias Humanas/metabolismo , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Teratocarcinoma/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Isoformas de Proteínas , Fatores de Transcrição SOXB1/genética , Teratocarcinoma/genética , Teratocarcinoma/patologia
5.
Oncol Rep ; 39(2): 851-859, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29207191

RESUMO

The pituitary sex hormones (SexHs): follicle­stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) regulate several functions crucial for reproduction, including oogenesis, spermatogenesis, and lactation. An important source of prolactin-like hormones, known as lactogens, is the placenta, and lactogens bind to the PRL receptor (PRLR) with high affinity and thereby mimic the actions of PRL. Recently, it has been demonstrated that pituitary SexHs were involved in metastatic lung cancer, certain sarcomas, and leukemia. In the present study we aimed to investigate whether FSH, LH, and PRL were able to stimulate stem cells involved in early development. To address this issue we employed a murine embryonic stem cell line (ES-D3) as well as two teratocarcinoma cell lines, P19 (murine) and NTera2 (human). We determined that all these cells expressed SexH receptors at the mRNA and protein levels and that stimulation of these receptors induced phosphorylation of p42/44 MAPK, p38 MAPK, and AKT. Moreover, ES-D3, P19, and NTera2 cells responded with increased migration and adhesion to physiological concentrations of pituitary SexHs. In view of these findings we proposed that maternal-derived pituitary SexHs regulate the biology of stem cells involved in early development.


Assuntos
Células-Tronco Embrionárias/citologia , Gonadotropinas Hipofisárias/farmacologia , Receptores da Gonadotropina/metabolismo , Teratocarcinoma/metabolismo , Neoplasias Testiculares/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Hormônio Foliculoestimulante/farmacologia , Humanos , Hormônio Luteinizante/farmacologia , Masculino , Camundongos , Prolactina/farmacologia , Receptores da Gonadotropina/genética , Transdução de Sinais/efeitos dos fármacos , Teratocarcinoma/genética , Neoplasias Testiculares/genética
6.
Genes Dev ; 31(12): 1195-1201, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28765161

RESUMO

Epigenetic programs regulate the development and maintenance of organisms over a lifetime. These programs are carried out through chemical modifications of DNA and proteins such as histones and transcription factors. These epigenetic modifications are less stable than genetic alterations and even reversible under a variety of circumstances, such as developmental changes, regeneration of tissues, cell divisions, aging, and pathological conditions observed in many cancers. The p53 protein not only enforces the stability of the genome by the prevention of genetic alterations in cells but also plays a role in regulating the epigenetic changes that can occur in cells. The full-length p53 protein is largely inactive in stem cells but, when activated, helps to commit these cells to developmental lineages through a series of epigenetic changes. Just as p53 impacts epigenetic change, the enzyme activities that carry out epigenetic protein modifications act on the p53 protein and its splice variants in stem and progenitor cells to silence or activate its transcriptional activities. Thus, there is a great deal of cross-talk between the p53 protein and epigenetic programs. This review collects the diverse experimental evidence that leads to these conclusions. This in turn permits new ideas and directions for the treatment of cancers, reactivating developmental pathways for tissue regeneration and responses to the impact of aging.


Assuntos
Epigênese Genética , Células-Tronco/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Reprogramação Celular/genética , Humanos , Neoplasias/genética , Neoplasias/fisiopatologia , Neoplasias/terapia , Regeneração/genética , Células-Tronco/patologia , Teratocarcinoma/genética , Teratocarcinoma/fisiopatologia , Proteína Supressora de Tumor p53/metabolismo
7.
Biochem Cell Biol ; 95(2): 251-262, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28177772

RESUMO

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-ß-catenin pathway. The upregulation of Wnt6 and activation of ß-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-ß-catenin signaling, thereby allowing PE cells to differentiate.


Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Teratocarcinoma/genética , Proteínas Wnt/genética , beta Catenina/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Endoderma/metabolismo , Endoderma/patologia , Retroalimentação Fisiológica , Receptores Frizzled , Genes Reporter , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Tretinoína/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
8.
Proc Natl Acad Sci U S A ; 113(35): 9822-7, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27535933

RESUMO

TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53's transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Metilação , Domínios Proteicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Proteína Supressora de Tumor p53/metabolismo
9.
Stem Cells Transl Med ; 5(5): 694-702, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26987352

RESUMO

UNLABELLED: Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. SIGNIFICANCE: Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature ß cell phenotype would lead to safe islet replacement therapy for diabetes.


Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Diabetes Mellitus Tipo 2/cirurgia , Células-Tronco Pluripotentes Induzidas/transplante , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/cirurgia , Queratinócitos/transplante , Regeneração , Teratocarcinoma/prevenção & controle , Adulto , Idoso , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Xenoenxertos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/efeitos adversos , Queratinócitos/metabolismo , Queratinócitos/patologia , Lentivirus/genética , Masculino , Camundongos SCID , Fenótipo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Transfecção
10.
Nucleic Acids Res ; 44(10): 4665-83, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26883630

RESUMO

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.


Assuntos
Elementos Alu , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Diferenciação Celular/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Hidrocarboneto Arílico/fisiologia , Teratocarcinoma/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Camundongos , MicroRNAs/metabolismo , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Teratocarcinoma/enzimologia , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Teratoma/genética , Teratoma/metabolismo , Transcrição Gênica , Tretinoína/farmacologia
11.
Anticancer Res ; 34(11): 6443-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25368244

RESUMO

BACKGROUND/AIM: Defensins are basic peptides involved in non-immune bio-defense mechanisms in a normal epithelium. Human oral squamous cell carcinoma cells (OSCC) also produce human beta-defensins (HBDs), although their exact function is not clear. This study aimed to analyze the variation in gene expression levels of hBDs in co-cultures of OSCC with murine cells. MATERIALS AND METHODS: Two OSCC cell lines (HSC-3, HSC-4) were co-cultured with mouse embryonic fibroblasts, NIH/3T3 or a mouse chondrogenic cell line derived from teratocarcinoma, ATDC5, for 1.5 days. Expression patterns of the hBD genes were investigated by real-time polymerase chain reaction (RT-PCR). RESULTS: hBD1 expression increased when co-cultured with NIH/3T3 but decreased when co-cultured with ATDC5. Expression of hBD2 and hBD4 tended to decrease. OSCC cells formed colonies when co-cultured with NIH/3T3 but were scattered when co-cultured with ATDC5. CONCLUSION: hBDs expression in OSCC is dependent on the type of co-cultured cells and differences in gene expression may be responsible for the morphological differences observed. OSCC may produce HBDs for purposes other than bio-defense by surrounding cells.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Microambiente Tumoral , beta-Defensinas/metabolismo , Animais , Apoptose , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Camundongos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Células NIH 3T3 , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , beta-Defensinas/genética
12.
Nat Commun ; 5: 4825, 2014 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-25198699

RESUMO

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs, inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation, differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs, mainly as a consequence of increased replication. Furthermore, trisomy 12 increases the tumorigenicity of hPSCs in vivo, inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last, a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together, these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications.


Assuntos
Carcinogênese/genética , Proliferação de Células/genética , Cromossomos Humanos Par 12/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/metabolismo , Trissomia/genética , Aneuploidia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Células-Tronco de Carcinoma Embrionário/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Neoplasias Embrionárias de Células Germinativas/genética , Teratocarcinoma/genética , Teratoma/genética
13.
PLoS One ; 8(12): e80555, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24324608

RESUMO

OBJECT: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo. STUDY METHOD: Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model. RESULTS: Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3. SIGNIFICANCE: α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.


Assuntos
Inibidores da Angiogênese/farmacologia , Colágeno Tipo IV/farmacologia , Proteína Ligante Fas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Cutâneas/irrigação sanguínea , Teratocarcinoma/irrigação sanguínea , Animais , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Proteína Ligante Fas/agonistas , Proteína Ligante Fas/metabolismo , Humanos , Camundongos , Neovascularização Patológica , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Isoformas de Proteínas/farmacologia , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Teratocarcinoma/tratamento farmacológico , Teratocarcinoma/genética , Teratocarcinoma/patologia , Receptor fas/genética , Receptor fas/metabolismo
14.
Pathol Int ; 63(8): 408-14, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23957916

RESUMO

Embryonic stem (ES) cells deficient in poly(ADP-ribose) polymerase-1 (Parp-1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp-1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild-type (Parp-1(+/+) ) and Parp-1(-/-) ES cells. The weights of the tumors derived from Parp-1(-/-) ES cells were lower than those of the tumors derived from Parp-1(+/+) ES cells (P < 0.05). The Parp-1(-/-) tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp-1(-/-) tumors, but not for the Parp-1(+/+) tumors (P < 0.05). Invasions were more frequently observed with the Parp-1(-/-) tumors compared with the Parp-1(+/+) tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp-1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.


Assuntos
Células-Tronco Embrionárias/patologia , Poli(ADP-Ribose) Polimerases/genética , Teratocarcinoma/patologia , Animais , Transformação Celular Neoplásica , Feminino , Genótipo , Células Gigantes/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Poli(ADP-Ribose) Polimerase-1 , Deleção de Sequência , Teratocarcinoma/genética , Trofoblastos/patologia , Útero/patologia
15.
Sci Rep ; 2: 743, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077727

RESUMO

Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.


Assuntos
Fibrossarcoma/genética , Células Germinativas/fisiologia , Células-Tronco/fisiologia , Teratocarcinoma/genética , Animais , Linhagem Celular Tumoral , Fibrossarcoma/patologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Camundongos , Camundongos Endogâmicos C3H , Células-Tronco/citologia
16.
Anat Rec (Hoboken) ; 295(12): 2104-13, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22976998

RESUMO

Aberrant activation of the Wnt/ß-catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/ß-catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3ß specific inhibitor (SB216763) was used to activate Wnt/ß-catenin signaling. All trans-retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real-time PCR, and Western-blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c-myc expression. The results showed that SB216763 induced the nuclear translocation of ß-catenin, upregulated the expression of c-myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans-RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c-myc overexpression. C-myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/ß-catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/ß-catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c-myc expression.


Assuntos
Proliferação de Células , Células-Tronco de Carcinoma Embrionário/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Teratocarcinoma/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/patologia , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Teratocarcinoma/genética , Teratocarcinoma/patologia , Fatores de Tempo , Transfecção , Tretinoína/farmacologia , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos
17.
Int J Dev Biol ; 56(4): 197-206, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22562197

RESUMO

The pluripotent potential of embryonic stem cells has often seen them touted as the future of regenerative medicine. The road to any therapeutic success however, must stretch back to teratocarcinoma, the tumour from which pluripotent stem cells (embryonal carcinoma cells) were first derived. This 2011 meeting in Cardiff acted as a historical perspective from which the impact of embryonal carcinoma cell research on the present pluripotent stem cell landscape could be observed, with many of the early luminaries in this field still very active. The meeting addressed the genetic and epigenetic make-up of pluripotent stem cells, the mechanisms which control their fate, and their relationship to the early embryo proper. With each speaker tasked with revisiting previous questions, this meeting demonstrated how far has been travelled, yet how far is left to go.


Assuntos
Células-Tronco Embrionárias/patologia , Células-Tronco Pluripotentes/patologia , Teratocarcinoma/patologia , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco de Carcinoma Embrionário/patologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Proteínas de Homeodomínio/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Teratocarcinoma/genética
18.
Cell Prolif ; 45(2): 140-7, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22288737

RESUMO

OBJECTIVES: Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)-cell system was prepared. MATERIALS AND METHODS: Diploid, tetraploid, pentaploid, hexaploid, octaploid and decaploid H1 (ES) cells (2H1, 4H1, 5H1, 6H1, 8H1 and 10H1 cells, respectively) were cultured for about 460 days in L15F10 medium without leukaemia inhibitory factor (LIF). The cells cultured under LIF-free conditions were denoted as 2H1(-), 4H1(-), 5H1(-), 6H1(-), 8H1(-) and 10H1(-) cells, respectively. Pluripotency and gene expression were examined. RESULTS: Ploidy alteration of H1(-) cells was similar to that of H1 cells. The polyploid H1(-) cells showed positive activity of alkaline phosphatase, suggesting that they maintained pluripotency in vitro without LIF. The polyploid H1(-) cells formed teratocarcinomas in mouse abdomen, suggesting they could differentiate in mouse abdomen in vivo. 2H1, 4H1 and polyploid H1(-) cells expressed nanog, oct3/4 and sox2 genes, suggesting that they fulfilled the criteria of ES cells. Nanog gene was significantly over-expressed in 4H1 and polyploid H1(-) cells, suggesting that overexpression of nanog gene was a characteristic of polyploid H1 cells. CONCLUSION: Polyploid H1 (ES) cells retained pluripotency in vitro, without LIF with nanog over-expression.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Poliploidia , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Diploide , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Expressão Gênica , Proteínas de Homeodomínio/genética , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , RNA/genética , RNA/metabolismo , Teratocarcinoma/etiologia , Teratocarcinoma/genética , Teratocarcinoma/patologia
19.
BMC Cancer ; 12: 35, 2012 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-22273460

RESUMO

BACKGROUND: Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-ß signaling and activation of the ß-catenin pathway can induce EMT in intratumoral cancer cells. METHODS: We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC). RESULTS: Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating ß-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-ß was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-ß levels and tumor grade. CONCLUSIONS: Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.


Assuntos
Transição Epitelial-Mesenquimal , Macrófagos/patologia , Teratocarcinoma/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/fisiologia , Camundongos , Invasividade Neoplásica/patologia , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia
20.
J Neurosci Res ; 89(10): 1685-97, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21674570

RESUMO

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.


Assuntos
Astrócitos/patologia , Química Encefálica/genética , Moduladores de Receptores de Canabinoides/genética , Moduladores de Receptores de Canabinoides/metabolismo , Neurônios/patologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fenótipo , Teratocarcinoma/química , Teratocarcinoma/genética , Teratocarcinoma/patologia
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