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1.
JACC Basic Transl Sci ; 4(1): 83-94, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30847422

RESUMO

Myocardial infarction (MI)-induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia-reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

2.
Science ; 348(6238): 1036-9, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25977370

RESUMO

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.


Assuntos
Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Telomerase/genética , Alelos , Linhagem Celular Tumoral , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica
3.
Acta Neuropathol ; 129(4): 597-607, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25724300

RESUMO

Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/complicações , Distúrbios no Reparo do DNA/tratamento farmacológico , Dacarbazina/análogos & derivados , Glioma/complicações , Neoplasias Encefálicas/tratamento farmacológico , Estudos de Coortes , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Distúrbios no Reparo do DNA/etiologia , Dacarbazina/uso terapêutico , Progressão da Doença , Feminino , Glioma/tratamento farmacológico , Humanos , Masculino , Mutação/genética , Receptores Imunológicos/genética , Estatísticas não Paramétricas , Temozolomida , Proteínas Supressoras de Tumor/genética
4.
Genome Res ; 24(5): 761-74, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24709822

RESUMO

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.


Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Mutação , Regiões Promotoras Genéticas , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Telomerase/genética , Telomerase/metabolismo , Ativação Transcricional , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Gli3 com Dedos de Zinco
5.
Science ; 343(6167): 189-193, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24336570

RESUMO

Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct from those in the initial tumor. To explore this hypothesis, we sequenced the exomes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF; this suggests that recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt-mTOR (mammalian target of rapamycin) pathways that bore the signature of TMZ-induced mutagenesis.


Assuntos
Antineoplásicos Alquilantes/efeitos adversos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Dacarbazina/análogos & derivados , Glioma/tratamento farmacológico , Glioma/patologia , Recidiva Local de Neoplasia/induzido quimicamente , Recidiva Local de Neoplasia/genética , Antineoplásicos Alquilantes/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/genética , DNA Helicases/genética , Análise Mutacional de DNA , Dacarbazina/efeitos adversos , Dacarbazina/uso terapêutico , Glioma/genética , Humanos , Mutagênese/efeitos dos fármacos , Gradação de Tumores , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genética , Temozolomida , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Nuclear Ligada ao X
6.
Neuro Oncol ; 16(3): 361-71, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24311636

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) contains a population of cells that exhibit stem cell phenotypes. These cancer stem cells (CSCs) may be a source of therapeutic resistance, although support for this important concept is limited. METHODS: We determined whether early-passage GBM CSCs respond differently than patient-matched, genotypically similar non-CSCs to clinically relevant single or serial doses of temozolomide (TMZ), radiation therapy (XRT), or alternating TMZ treatment and XRT, which is the standard of care for GBM patients. RESULTS: Despite the phenotypic differences, including the presence of stem cell markers and formation of intracranial tumors, the CSCs and matched non-CSCs were equally resistant to TMZ in a majority of patients, using 2 independent assays. TMZ response was consistent with methylated O(6)-DNA methylguanine-methyltransferase (MGMT) and MGMT protein levels in both culture types. In contrast, CSCs were unexpectedly more responsive to XRT compared with matched non-CSCs from 2 patients despite having relatively equal resistance to TMZ. However, for the majority of culture pairs from individual patients, responses in CSCs were indistinguishable from non-CSC cultures. CONCLUSIONS: In our patient-matched primary cultures, response to TMZ was tightly linked to the individual tumor's MGMT status and independent of their phenotypic differences. TMZ and XRT together revealed no additive benefit compared with monotherapy for either culture type, in contrast to the notion that the CSC population is more resistant to XRT. If the tumor cell response in vitro mirrors therapeutic response in larger patient cohorts, these rapid assays in primary cultures could allow -empirical selection of efficacious therapeutic agents on a patient-specific basis.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/terapia , Dacarbazina/análogos & derivados , Glioblastoma/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Antígeno AC133 , Antígenos CD/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia , Dacarbazina/uso terapêutico , Glicoproteínas/metabolismo , Humanos , Peptídeos/metabolismo , Fenótipo , Temozolomida , Células Tumorais Cultivadas
7.
Neuro Oncol ; 15(11): 1518-31, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23887941

RESUMO

BACKGROUND: Mechanisms of glioma invasion remain to be fully elucidated. Glioma cells within glioblastoma multiforme (GBM) range from well-differentiated tumor cells to less-differentiated brain tumor-initiating cells (BTICs). The ß2-subunit of Na(+)/K(+)-ATPase, called the adhesion molecule on glia (AMOG), is highly expressed in normal glia but is thought to be universally downregulated in GBM. To test our hypothesis that expression of AMOG is heterogeneous in GBM and confers a less invasive phenotype, we compared it between BTICs and differentiated cells from patient-matched GBM and then tested GBM invasion in vitro after AMOG overexpression. METHODS: Immunohistochemistry, immunoblotting, and real-time PCR were used to characterize AMOG protein and mRNA expression in tumor samples, BTICs, and differentiated cells. Matrigel invasion assay, scratch assay, and direct cell counting were used for testing in vitro invasion, migration, and proliferation, respectively. RESULTS: While AMOG expression is heterogeneous in astrocytomas of grades II-IV, it is lost in most GBM. BTICs express higher levels of AMOG mRNA and protein compared with patient-matched differentiated tumor cells. Overexpression of AMOG decreased GBM cell and BTIC invasion without affecting migration or proliferation. Knockdown of AMOG expression in normal human astrocytes increased invasion. CONCLUSIONS: AMOG expression inhibits GBM invasion. Its downregulation increases invasion in glial cells and may also represent an important step in BTIC differentiation. These data provide compelling evidence implicating the role of AMOG in glioma invasion and provide impetus for further investigation.


Assuntos
Adenosina Trifosfatases/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Neoplasias Encefálicas/mortalidade , Regulação para Baixo , Glioblastoma/mortalidade , Humanos , Invasividade Neoplásica , Taxa de Sobrevida , Células Tumorais Cultivadas
8.
Cancer Cell ; 23(6): 711-3, 2013 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-23763996

RESUMO

Activated STAT3 and increased expression of the histone methyltransferase EZH2 are independently associated with the most malignant subset of gliomas. In this issue of Cancer Cell, Kim and colleagues discover that EZH2 enhances STAT3 activation by trimethylating lysine180 in STAT3 and does so preferentially in glioma stem-like cells.


Assuntos
Glioblastoma/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos
9.
Adv Exp Med Biol ; 754: 313-38, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22956508

RESUMO

Accurate detection of epimutations in tumor cells is crucial for -understanding the molecular pathogenesis of cancer. Alterations in DNA methylation in cancer are functionally important and clinically relevant, but even this well-studied area is continually re-evaluated in light of unanticipated results, such as the strong association between aberrant DNA methylation in adult tumors and polycomb group profiles in embryonic stem cells, cancer-associated genetic mutations in epigenetic regulators such as DNMT3A and TET family genes, and the discovery of altered 5-hydroxymethylcytosine, a product of TET proteins acting on 5-methylcytosine, in human tumors with TET mutations. The abundance and distribution of covalent histone modifications in primary cancer tissues relative to normal cells is an important but largely uncharted area, although there is good evidence for a mechanistic role of cancer-specific alterations in histone modifications in tumor etiology, drug response, and tumor progression. Meanwhile, the discovery of new epigenetic marks continues, and there are many useful methods for epigenome analysis applicable to primary tumor samples, in addition to cancer cell lines. For DNA methylation and hydroxymethylation, next-generation sequencing allows increasingly inexpensive and quantitative whole-genome profiling. Similarly, the refinement and maturation of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) has made possible genome-wide mapping of histone modifications, open chromatin, and transcription factor binding sites. Computational tools have been developed apace with these epigenome methods to better enable accurate interpretation of the profiling data.


Assuntos
Biomarcadores Tumorais/genética , Epigênese Genética/genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Animais , Humanos
10.
Cancer Cell ; 22(1): 21-35, 2012 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-22789536

RESUMO

Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor cell invasion through enhanced recruitment of the protein tyrosine phosphatase 1B (PTP1B) to a MET/VEGFR2 heterocomplex, thereby suppressing HGF-dependent MET phosphorylation and tumor cell migration. Consequently, VEGF blockade restores and increases MET activity in GBM cells in a hypoxia-independent manner, while inducing a program reminiscent of epithelial-to-mesenchymal transition highlighted by a T-cadherin to N-cadherin switch and enhanced mesenchymal features. Inhibition of MET in GBM mouse models blocks mesenchymal transition and invasion provoked by VEGF ablation, resulting in substantial survival benefit.


Assuntos
Neoplasias Encefálicas/patologia , Transição Epitelial-Mesenquimal/fisiologia , Glioblastoma/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Bevacizumab , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
PLoS One ; 7(6): e38881, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22719973

RESUMO

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.


Assuntos
Hibridização Genômica Comparativa , Fragmentação do DNA , Humanos
12.
Nat Biotechnol ; 28(10): 1097-105, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20852635

RESUMO

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.


Assuntos
Alelos , Metilação de DNA/genética , Epigênese Genética , Análise de Sequência de DNA/métodos , Linhagem Celular , Ilhas de CpG/genética , Citosina/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Sulfitos/metabolismo
13.
Epigenomics ; 2(1): 105-17, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20657796

RESUMO

The haploid human genome contains approximately 29 million CpGs that exist in a methylated, hydroxymethylated or unmethylated state, collectively referred to as the DNA methylome. The methylation status of cytosines in CpGs and occasionally in non-CpG cytosines influences protein­DNA interactions, gene expression, and chromatin structure and stability. The degree of DNA methylation at particular loci may be heritable transgenerationally and may be altered by environmental exposures and diet, potentially contributing to the development of human diseases. For the vast majority of normal and disease methylomes however, less than 1% of the CpGs have been assessed, revealing the formative stage of methylation mapping techniques. Thus, there is significant discovery potential in new genome-scale platforms applied to methylome mapping, particularly oligonucleotide arrays and the transformative technology of next-generation sequencing. Here, we outline the currently used methylation detection reagents and their application to microarray and sequencing platforms. A comparison of the emerging methods is presented, highlighting their degrees of technical complexity, methylome coverage and precision in resolving methylation. Because there are hundreds of unique methylomes to map within one individual and interindividual variation is likely to be significant, international coordination is essential to standardize methylome platforms and to create a full repository of methylome maps from tissues and unique cell types.


Assuntos
Ilhas de CpG/genética , Metilação de DNA/fisiologia , Genoma Humano/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Teorema de Bayes , Metilação de DNA/genética , Enzimas de Restrição do DNA , Humanos , Imunoprecipitação/métodos , Polimorfismo de Nucleotídeo Único/genética
14.
Nature ; 466(7303): 253-7, 2010 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-20613842

RESUMO

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.


Assuntos
Encéfalo/metabolismo , Sequência Conservada/genética , Metilação de DNA , Regiões Promotoras Genéticas/genética , Animais , Encéfalo/anatomia & histologia , Encéfalo/citologia , Proteínas de Transporte/genética , Linhagem Celular , Ilhas de CpG/genética , DNA Intergênico/genética , DNA Intergênico/metabolismo , Lobo Frontal/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso , Especificidade de Órgãos , Transcrição Gênica/genética
15.
Future Oncol ; 5(10): 1615-29, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20001799

RESUMO

Epigenetic mechanisms involving DNA methylation, histone modifications and noncoding RNAs regulate and maintain gene-expression states. Similar to genetic mutations, alterations in epigenetic regulation can lead to uncontrolled cell division, tumor initiation and growth, invasiveness and metastasis. Research in brain cancer, particularly gliomas, has uncovered global and gene-specific DNA hypomethylation, local DNA hypermethylation of gene promoters and the de-regulation of microRNA expression. Understanding epigenetic dysregulation in brain cancers has provided new tools for prognostication, as well as suggesting new approaches to therapy. There is significant interest in new sequencing-based technologies that map genetic and epigenetic alterations comprehensively and at high resolution. These methods are being applied to brain tumors, and will better define the contribution of epigenetic defects to tumorigenesis.


Assuntos
Neoplasias Encefálicas/genética , Epigênese Genética , Animais , Humanos
16.
Methods Mol Biol ; 568: 203-16, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19582429

RESUMO

Aberrant DNA methylation is one of the major characteristics of tumor cells in addition to genetic and other epigenetic alterations. Evidence shows that both regional hypermethylation and global hypomethylation can occur in cancer cells. Increased DNA methylation can be found at select tumor-suppressor gene promoters, causing the silencing of these genes in tumorigenic cells. At the same time, a global decrease in DNA methylation is frequently observed in cancer cells, which may contribute to genome instability. Unlike genetic mutations, hypermethylation at tumor-suppressor gene promoters can be reversed with epigenetic therapy by using DNA demethylating agents.To better understand the mechanisms of cancer initiation and progression, and to better assess the effects of epigenetic therapy, a reliable high-throughput method for genome-wide DNA methylation analysis is needed. Recently, the process of coupling methylated DNA immunoprecipitation (mDIP) with microarray hybridization has been proven to be a successful strategy to map genome-wide DNA methylation patterns in different cell types.


Assuntos
Metilação de DNA/genética , Genoma Humano/genética , Imunoprecipitação/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Humanos , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e Rotulagem
17.
Cell Stem Cell ; 2(2): 160-9, 2008 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-18371437

RESUMO

We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on approximately 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression.


Assuntos
Antígenos de Diferenciação/metabolismo , Ilhas de CpG , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Animais , Antígenos de Diferenciação/genética , Proliferação de Células , Ilhas de CpG/genética , Células-Tronco Embrionárias/citologia , Epigênese Genética , Perfilação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Metilação , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas do Grupo Polycomb , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
18.
Proc Natl Acad Sci U S A ; 105(12): 4709-14, 2008 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-18339804

RESUMO

X chromosome inactivation (XCI) is an essential mechanism for dosage compensation of X-linked genes in female cells. We report that subcultures from lines of female human embryonic stem cells (hESCs) exhibit variation (0-100%) for XCI markers, including XIST RNA expression and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on the inactive X chromosome (Xi). Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a monoallelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have already completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already not random. Moreover, XIST gene expression in subsets of cultured female hESCs is unstable and subject to stable epigenetic silencing by DNA methylation. In the absence of XIST expression, approximately 12% of X-linked promoter CpG islands become hypomethylated and a portion of X-linked alleles on the Xi are reactivated. Because alterations in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine.


Assuntos
Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Inativação do Cromossomo X/genética , Células Cultivadas , Ilhas de CpG/genética , Metilação de DNA , DNA Complementar/genética , Mecanismo Genético de Compensação de Dose , Células-Tronco Embrionárias/citologia , Feminino , Genes Ligados ao Cromossomo X , Marcadores Genéticos , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Development ; 132(15): 3345-56, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16014513

RESUMO

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.


Assuntos
Astrócitos/citologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Transdução de Sinais , Animais , Sequência de Bases , Encéfalo/embriologia , Diferenciação Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/deficiência , DNA (Citosina-5-)-Metiltransferases/genética , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ratos , Células-Tronco/citologia , Transativadores/metabolismo , Transfecção
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