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1.
J Biol Chem ; 291(41): 21829-21835, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27563064

RESUMO

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function.


Assuntos
DNA/química , Sequências de Repetição em Tandem , Telômero/química , Proteína 1 de Ligação a Repetições Teloméricas/química , Animais , DNA/metabolismo , Camundongos , Domínios Proteicos , Células Sf9 , Spodoptera , Tanquirases/química , Tanquirases/genética , Tanquirases/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/química , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
2.
BMC Genomics ; 16: 403, 2015 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-25997541

RESUMO

BACKGROUND: Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression. RESULTS: Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events. CONCLUSIONS: Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.


Assuntos
Bases de Dados Genéticas , Genoma Humano , Neoplasias da Bexiga Urinária/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Cromossomos Humanos X , Classe I de Fosfatidilinositol 3-Quinases , Análise por Conglomerados , Variações do Número de Cópias de DNA , Feminino , Instabilidade Genômica , Humanos , Masculino , Fosfatidilinositol 3-Quinases/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/patologia , Proteínas ras/genética
3.
Proc Natl Acad Sci U S A ; 107(48): 20780-5, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21078999

RESUMO

Malignant tumors result from the accumulation of genetic alterations in oncogenes and tumor suppressor genes. Much less is known about the genetic changes in benign tumors. Seborrheic keratoses (SK) are very frequent benign human epidermal tumors without malignant potential. We performed a comprehensive mutational screen of genes in the FGFR3-RAS-MAPK and phosphoinositide 3-kinase (PI3K)-AKT pathways from 175 SK, including multiple lesions from each patient. SK commonly harbored multiple bona fide oncogenic mutations in FGFR3, PIK3CA, KRAS, HRAS, EGFR, and AKT1 oncogenes but not in tumor suppressor genes TSC1 and PTEN. Despite the occurrence of oncogenic mutations and the evidence for downstream ERK/MAPK and PI3K pathway signaling, we did not find induction of senescence or a DNA damage response. Array comparative genomic hybridization (aCGH) analysis revealed that SK are genetically stable. The pattern of oncogenic mutations and X chromosome inactivation departs significantly from randomness and indicates that spatially independent lesions from a given patient share a clonal relationship. Our findings show that multiple oncogenic mutations in the major signaling pathways involved in cancer are not sufficient to drive malignant tumor progression. Furthermore, our data provide clues on the origin and spread of oncogenic mutations in tissues, suggesting that apparently independent (multicentric) adult benign tumors may have a clonal origin.


Assuntos
Ceratose Seborreica/genética , Ceratose Seborreica/patologia , Mutação/genética , Oncogenes/genética , Apoptose/genética , Biomarcadores Tumorais , Proliferação de Células , Senescência Celular , Células Clonais , Análise Mutacional de DNA , Testes Genéticos , Genoma Humano/genética , Genótipo , Humanos , Ceratose Seborreica/enzimologia , Proteínas Quinases Ativadas por Mitógeno/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/genética
6.
Chem Biol Drug Des ; 95(1): 66-78, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31469231

RESUMO

High-throughput assays are a common strategy for the identification of compounds able to modulate a certain cellular activity. Here, we show an automatized analysis platform for the quantification of nuclear foci as inhibitory effect of compounds on a target protein labeled by fluorescent antibodies. Our experience led us to a fast analysis platform that combines cell-based assays, high-content screening, and confocal microscopy, with an automatic and user-friendly statistical analysis of plate-based assays including positive and negative controls, able to identify inhibitory effect of compounds tested together with the Z-prime and Window of individual plate-based assays to assess the reliability of the results. The platform integrates a web-based tool implemented in Pipeline Pilot and R, and allows computing the inhibition values of different parameters obtained from the high-content screening and confocal microscopy analysis. This facilitates the exploration of the results using the different parameters, providing information at different levels as the number of foci observed, the sum of intensity of foci, area of foci, etc, the detection and filtering of outliers over the assay plate, and finally providing a set of statistics of the parameters studied together with a set of plots that we believe significantly helps to the interpretation of the assay results.


Assuntos
Imunofluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Anticorpos/imunologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/química , Humanos , Microscopia Confocal , Imagem Óptica , Reprodutibilidade dos Testes , Telômero/metabolismo , Telômero/ultraestrutura
7.
Nat Commun ; 8(1): 1278, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097657

RESUMO

Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.


Assuntos
Envelhecimento/genética , Neoplasias da Mama/genética , Dano ao DNA/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Classe I de Fosfatidilinositol 3-Quinases , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Transplante de Neoplasias , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Estabilidade Proteica , Telômero/efeitos dos fármacos , Proteína 1 de Ligação a Repetições Teloméricas/efeitos dos fármacos , Tiazóis/farmacologia
8.
EMBO Mol Med ; 7(7): 930-49, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25971796

RESUMO

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.


Assuntos
Dano ao DNA , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Mutantes/genética , Proteína Oncogênica p21(ras)/genética , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/antagonistas & inibidores , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Deleção de Genes , Neoplasias Pulmonares/patologia , Camundongos , Mutação de Sentido Incorreto , Análise de Sobrevida , Proteína 1 de Ligação a Repetições Teloméricas/genética , Resultado do Tratamento
9.
J Clin Invest ; 125(2): 824-30, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25574842

RESUMO

NOTCH signaling suppresses tumor growth and proliferation in several types of stratified epithelia. Here, we show that missense mutations in NOTCH1 and NOTCH2 found in human bladder cancers result in loss of function. In murine models, genetic ablation of the NOTCH pathway accelerated bladder tumorigenesis and promoted the formation of squamous cell carcinomas, with areas of mesenchymal features. Using bladder cancer cells, we determined that the NOTCH pathway stabilizes the epithelial phenotype through its effector HES1 and, consequently, loss of NOTCH activity favors the process of epithelial-mesenchymal transition. Evaluation of human bladder cancer samples revealed that tumors with low levels of HES1 present mesenchymal features and are more aggressive. Together, our results indicate that NOTCH serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal and invasive features.


Assuntos
Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Receptor Notch1/genética , Receptor Notch2/genética , Fatores de Transcrição HES-1 , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
10.
J Natl Cancer Inst ; 104(24): 1897-904, 2012 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-23108201

RESUMO

BACKGROUND: Previous evidence suggests that 25-hydroxyvitamin D(3) [25(OH)D(3)] protects against several cancers. However, little is known regarding urothelial bladder cancer (UBC). We analyzed the association between plasma 25(OH)D(3) and overall risk of UBC, as well as according to stage and FGFR3 molecular subphenotypes. METHODS: Plasma concentrations of 25(OH)D(3) in 1125 cases with UBC and 1028 control subjects were determined by a chemiluminescence immunoassay. FGFR3 mutational status and expression in tumor tissue were assessed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression adjusting for potential confounders. Analyses were further stratified by tumor invasiveness and grade, FGFR3 expression, and smoking status. Cell proliferation was measured in human UBC cell lines cultured with 1α,25-dihydroxyvitamin D(3). RESULTS: A statistically significantly increased risk of UBC was observed among subjects presenting the lowest concentrations of 25(OH)D(3) (OR(adj) = 1.83; 95% CI = 1.19 to 2.82; P = .006), showing a dose-response effect (P (trend) = .004). The association was stronger for patients with muscle-invasive tumors, especially among low-FGFR3 expressers (OR(adj) = 5.94; 95% CI = 1.72 to 20.45; P = .005). The biological plausibility of these associations is supported by the fact that, in vitro, 1α,25-dihydroxyvitamin D(3) upregulates FGFR3 expression in UBC cell lines with low levels of wild-type FGFR3. CONCLUSION: These findings support a role of vitamin D in the pathogenesis of UBC and show that 25(OH)D(3) levels are associated with FGFR3 expression in the tumor. Because FGFR3 mutation and overexpression are markers of better outcome, our findings suggest that individuals with low levels of plasma 25(OH)D(3) may be at high risk of more aggressive forms of UBC.


Assuntos
Anticarcinógenos/sangue , Biomarcadores Tumorais/metabolismo , Calcifediol/sangue , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Neoplasias da Bexiga Urinária/epidemiologia , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Western Blotting , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Europa (Continente)/epidemiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoensaio/métodos , Modelos Logísticos , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Razão de Chances , RNA Mensageiro/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Medição de Risco , Fatores de Risco , Espanha/epidemiologia , Regulação para Cima , Neoplasias da Bexiga Urinária/sangue , Neoplasias da Bexiga Urinária/química , Neoplasias da Bexiga Urinária/prevenção & controle
11.
Endocrinology ; 150(11): 5143-52, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19819978

RESUMO

The thyroid hormone receptor (TR)-alpha is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRalpha is posttranslationally modified by acetylation in response to its own ligand (T(3)). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRalpha decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRalpha mutant lost the ability to transactivate even at high T(3) concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRalpha interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRalpha suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRalpha.


Assuntos
Receptores alfa dos Hormônios Tireóideos/química , Receptores alfa dos Hormônios Tireóideos/metabolismo , Acetilação , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Transformação Celular Neoplásica , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Receptores alfa dos Hormônios Tireóideos/genética , Ativação Transcricional
12.
Regen Med ; 1(4): 575-87, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17465851

RESUMO

The differentiation of a stem cell is dependent on the environmental cues that it receives and can be modulated by the expression of different master regulators or by secreted factors or inducers. The use of genetically modified stem cells to express the required factors can direct differentiation along the requisite pathway. This approach to the engineering of stem cells is important, as control of the pluripotentiality of stem cells is necessary in order to avoid unwanted growth, migration or differentiation to nontarget tissues. The authors provide an overview of the stem cell engineering field, highlighting challenges and solutions, and focusing on recent developments in therapeutic applications in areas such as autoimmunity, CNS lesions, bone and joint diseases, cancer and myocardial infarction.


Assuntos
Células-Tronco Pluripotentes/citologia , Engenharia Tecidual , Animais , Diferenciação Celular , Transplante de Células , Humanos
13.
EMBO J ; 22(12): 3102-12, 2003 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-12805224

RESUMO

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein-protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein-protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Transcrição Gênica , Animais , Proteína de Ligação a CREB , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Genes Reporter , Humanos , Ácidos Hidroxâmicos/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Hipófise/citologia , Hipófise/metabolismo , Regiões Promotoras Genéticas , Inibidores da Síntese de Proteínas/metabolismo , Receptores dos Hormônios Tireóideos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Tri-Iodotironina/metabolismo
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