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1.
Oncol Rep ; 34(5): 2251-8, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26352760

RESUMO

The model animal of renal cell carcinoma (RCC), the Eker rat, has a germline mutation in the tuberous sclerosis 2 (Tsc2) gene. Heterozygous mutants develop RCCs by second hit in the wild-type Tsc2 allele, whereas homozygous mutants are embryonic lethal. In the present study, a new cell differentiation model was developed to study the mechanism of Tsc2 mutation-associated pathogenesis by generating Tsc2-deficient embryonic stem cells (ESCs) from Eker rats. Tsc2+/+, Tsc2+/- and Tsc2-/- ESCs were all capable of generating three germ layers: mesoderm, ectoderm, and endoderm. Interestingly, epithelial tumor-like abnormal ductal structures were reproducibly observed in Tsc2-/- teratomas from different ESC lines. Immunohistochemical analysis revealed that mammalian target of rapamycin complex 1 (mTORC1) signaling was activated in abnormal ducts of Tsc2-/- teratomas, on the basis of positive staining for p-S6 and p-4EBP1. In these abnormal ducts, expression levels of epithelial markers (i.e., megalin and cubilin) and the cytoplasmic localization of E-cadherin and ß-catenin were similar to those in Eker rat RCCs. Moreover, a transcription factor regulated by mTORC1, named TFE3, was located in the nuclei of abnormal ducts and Eker rat RCCs. As a negative regulator of ESC differentiation, TFE3 may result in tissue-specific differentiation defects related to tumorigenesis in Eker rats and Tsc2-/- teratomas. The present study suggests that ESCs derived from Eker rats constitute a novel experimental tool with which to analyze differentiation defects and cell-type specific pathogenesis associated with Tsc2 deficiency.


Assuntos
Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Teratoma/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Caderinas/metabolismo , Carcinogênese/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Núcleo Celular/metabolismo , Células-Tronco Embrionárias , Técnicas de Inativação de Genes , Neoplasias Renais/genética , Neoplasias Renais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Transporte Proteico , Ratos , Teratoma/metabolismo , Teratoma/patologia , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismo
2.
Int J Oncol ; 46(5): 1944-52, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25738543

RESUMO

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by TSC1 or TSC2 mutations. TSC causes the development of tumors in various organs such as the brain, skin, kidney, lung, and heart. The protein complex TSC1/2 has been reported to have an inhibitory function on mammalian target of rapamycin complex 1 (mTORC1). Treatment with mammalian target of rapamycin (mTOR) inhibitors has demonstrated tumor­reducing effects in patients with TSC but is also associated with various adverse effects. In recent years, experiments involving in vivo differentiation of pluripotent stem cells have been reported as useful in elucidating mechanisms of pathogenesis and discovering new therapeutic targets for several diseases. To reveal the molecular basis of the pathogenesis caused by the Tsc2 mutation, we derived embryonic stem cells (ESCs) from Eker rats, which have the Tsc2 mutation and develop brain lesions and renal tumors. Although several studies have reported the necessity of Tsc1 and Tsc2 regulation to maintain ESCs and hematopoietic stem cells, we successfully established not only Tsc2+/+ and Tsc2+/- ESCs but also Tsc2-/- ESCs. We confirmed that these cells express pluripotency markers and retain the ability to differentiate into all three germ layers. Comprehensive gene expression analysis of Tsc2+/+ and Tsc2+/- ESCs revealed similar profiles, whereas the profile of Tsc2-/- ESCs was distinct from these two. In vitro differentiation experiments using these ESCs combined with in vivo experiments may reveal the mechanism of the tissue­specific pathogenesis caused by the Tsc2 mutation and identify specific new therapeutic targets.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Supressoras de Tumor/deficiência , Animais , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Técnicas de Genotipagem , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Complexos Multiproteicos/metabolismo , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos BN , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
3.
Hum Genet ; 110(2): 157-65, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11935322

RESUMO

Determination of left-right axis is a precocious embryonic event, and all phenotypic anomalies resulting from disruption of the normal lateralization process are collectively referred to as the lateralization defect. A transgenic mouse with lateralization defect and hepatic, kidney, and pancreatic anomalies has resulted from disruption of the inv gene by insertion of a transgene. The human ortholog is thus a good candidate for lateralization defect in humans, in particular in cases with associated hepatic anomalies. Here, we have identified, mapped, and characterized the INV human gene and screened a series of heterotaxic patients (with or without biliary anomalies) for mutation in this gene. In a German family of Turkish origin, we have found that all available affected and unaffected individuals are heterozygous for a mutation in the splicing donor site of intron 12 in the INV gene resulting in two different aberrant splicing isoforms. This can be explained either by a randomization of lateralization defects or, as suggested earlier, di- or trigenic inheritance, although we have been unable to detect, in this family, a mutation in genes known to be involved in the human lateralization defect ( LEFTY1, LEFTY2, ACVR2B, NODAL, ZIC3, and CFC1). In contrast to the mouse, the affected individuals have no biliary anomalies, and the absence of mutation in a series of seven cases with lateralization defect and biliary anomalies demonstrates that INV is not frequently involved in such a phenotype in humans.


Assuntos
Atresia Biliar/genética , Mapeamento Cromossômico , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/embriologia , Química Encefálica , Análise Mutacional de DNA , Primers do DNA , Desenvolvimento Embrionário e Fetal , Feminino , Feto , Lateralidade Funcional/genética , Biblioteca Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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