RESUMO
A majority of breast cancers are driven by estrogen via estrogen receptor-α (ERα). Our previous studies indicate that hypoxia-inducible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. However, whether ERα is implicated in the direct regulation of HIF-1α and the role of HIF-1α in endocrine therapy response are unknown. In this study we found that a subpopulation of HIF-1α targets, many of them bearing both hypoxia response elements and estrogen response elements, are regulated by ERα in normoxia and hypoxia. Interestingly, the HIF-1α gene itself also bears an estrogen response element, and its expression is directly regulated by ERα. Clinical data revealed that expression of the HIF-1α gene or a hypoxia metagene signature is associated with a poor outcome to endocrine treatment in ERα(+) breast cancer. HIF-1α was able to confer endocrine therapy resistance to ERα(+) breast cancer cells. Our findings define, for the first time to our knowledge, a direct regulatory pathway between ERα and HIF-1α, which might modulate hormone response in treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Transdução de Sinais , Tamoxifeno/uso terapêutico , Transcrição Gênica/fisiologiaRESUMO
MicroRNAs are important posttranscriptional regulators of gene expression, which have been shown to fine-tune innate immune responses downstream of pattern recognition receptor (PRR) signaling. This study identifies miR-650 as a novel PRR-responsive microRNA that is downregulated upon stimulation of primary human monocyte-derived dendritic cells (MDDCs) with a variety of different microbe-associated molecular patterns. A comprehensive target search combining in silico analysis, transcriptional profiling, and reporter assays reveals that miR-650 regulates several well-known interferon-stimulated genes, including IFIT2 and MXA. In particular, downregulation of miR-650 in influenza A infected MDDCs enhances the expression of MxA and may therefore contribute to the establishment of an antiviral state. Together these findings reveal a novel link between miR-650 and the innate immune response in human MDDCs.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Vírus da Influenza A/imunologia , MicroRNAs/imunologia , Proteínas de Resistência a Myxovirus/biossíntese , Células Cultivadas , Células Dendríticas/metabolismo , Regulação para Baixo , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/imunologia , Immunoblotting , MicroRNAs/genética , Microscopia Confocal , Proteínas de Resistência a Myxovirus/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , TransfecçãoRESUMO
RATIONALE: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited. OBJECTIVE: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state. METHODS AND RESULTS: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft. CONCLUSIONS: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.
Assuntos
Prótese Vascular , Fibroblastos/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/citologia , Miócitos de Músculo Liso/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Separação Celular/métodos , Quimiocinas , Feto/citologia , Fibroblastos/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos de Músculo Liso/metabolismo , Ativação Transcricional/fisiologia , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismoRESUMO
The generation of induced pluripotent stem (iPS) cells is an important tool for regenerative medicine. However, the main restriction is the risk of tumor development. In this study we found that during the early stages of somatic cell reprogramming toward a pluripotent state, specific gene expression patterns are altered. Therefore, we developed a method to generate partial-iPS (PiPS) cells by transferring four reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) to human fibroblasts for 4 d. PiPS cells did not form tumors in vivo and clearly displayed the potential to differentiate into endothelial cells (ECs) in response to defined media and culture conditions. To clarify the mechanism of PiPS cell differentiation into ECs, SET translocation (myeloid leukemia-associated) (SET) similar protein (SETSIP) was indentified to be induced during somatic cell reprogramming. Importantly, when PiPS cells were treated with VEGF, SETSIP was translocated to the cell nucleus, directly bound to the VE-cadherin promoter, increasing vascular endothelial-cadherin (VE-cadherin) expression levels and EC differentiation. Functionally, PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. Furthermore, PiPS-ECs displayed good attachment, stabilization, patency, and typical vascular structure when seeded on decellularized vessel scaffolds. These findings indicate that reprogramming of fibroblasts into ECs via SETSIP and VEGF has a potential clinical application.
Assuntos
Reprogramação Celular , Células Endoteliais/citologia , Fibroblastos/metabolismo , Neovascularização Patológica , Engenharia Tecidual/métodos , Animais , Antígenos CD/genética , Aorta/patologia , Caderinas/genética , Diferenciação Celular , Células Cultivadas , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos SCID , Modelos Genéticos , Regiões Promotoras Genéticas , Células-Tronco/citologia , Estresse MecânicoRESUMO
BACKGROUND: Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. RESULTS: Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. CONCLUSIONS: Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.
Assuntos
Hibridização Genômica Comparativa , Deleção de Sequência/genética , Arabidopsis/genética , DNA de Plantas/análise , DNA de Plantas/metabolismo , Genoma de Planta , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.
Assuntos
Genoma , Proteína HMGB1/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica , Animais , DNA/genética , DNA/metabolismo , Dano ao DNA , Epigênese Genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteína HMGB1/genética , Células HeLa , Histonas/genética , Humanos , Camundongos , Modelos Teóricos , RNA/genética , RNA/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP-chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections.
Assuntos
Encéfalo/embriologia , Fatores de Transcrição Forkhead/genética , Redes Reguladoras de Genes , Neuritos/metabolismo , Proteínas Repressoras/genética , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Corpo Estriado/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The exit of antigen-presenting cells and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such an exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels are unknown. We show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LECs), leading to expression of the key leukocyte adhesion receptors intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of dendritic cells (DCs) via afferent lymphatics. Lastly, we show that tumor necrosis factor alpha stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking monoclonal antibodies. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for antiinflammatory therapy.
Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Leucócitos/imunologia , Leucócitos/patologia , Vasos Linfáticos/imunologia , Adulto , Animais , Células Cultivadas , Dermatite de Contato/metabolismo , Endotélio Linfático/imunologia , Endotélio Linfático/metabolismo , Endotélio Linfático/patologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/fisiologia , Leucócitos/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
There is much evidence that T cells may be activated via mechanisms that act independently of direct TCR ligation. Despite this, the question of whether such forms of bystander T cell activation occur during immune responses is hotly debated. To address some outstanding questions, we set up an in vitro system within which to analyze bystander T cell activation in human T cells, in the absence of the possibility for TCR cross-reactivity. In addition, we have investigated the genetic, phenotypic, and functional characteristics of bystander-activated T cells. In this study, we show that bystander T cell activation is, indeed, observed during a specific immune response, and that it occurs preferentially among CD4(+) memory T cells. Furthermore, bystander-activated T cells display a distinct gene expression profile. The mechanism for bystander T cell activation involves soluble factors, and the outcome is an elevated level of apoptosis. This may provide an explanation for the attrition of T cell memory pools of heterologous specificity during immune responses to pathogens such as viruses.
Assuntos
Apoptose/imunologia , Efeito Espectador/imunologia , Linfócitos T CD4-Positivos/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RATIONALE: Approximately 60 to 70% of patients with pulmonary sarcoidosis have disease that resolves spontaneously; the rest follow a chronic course with varying levels of fibrosis. It is unclear why some patients progress and if treatment affects outcome. OBJECTIVES: To determine differential gene expression profile in lungs of patients with self-limiting sarcoidosis compared to those with progressive-fibrotic disease, and to analyze the biological relevance of these differentially expressed genes. METHODS: We examined microarray expression of 26,626 genes in transbronchial biopsies of granulomatous areas in lungs of patients with active but self-limiting (n = 8) versus those with active, progressive (+/- fibrotic) pulmonary disease (n = 7). MEASUREMENTS AND MAIN RESULTS: Three hundred thirty-four genes were differentially expressed between the two groups (P < 0.01, Bayesian moderated t test). Gene Set Enrichment Analysis showed over-representation of gene-sets (defined by Gene Ontology) related to host immune activation, proliferation, and defense, among genes up-regulated in the progressive-fibrotic group (FDR q < 0.0001 for the top 43 gene sets), and a marked enrichment of, and similarity in gene expression profiles between, progressive-fibrotic sarcoidosis and hypersensitivity pneumonitis (HP), (q < 0.001), but not idiopathic pulmonary fibrosis (IPF). CONCLUSIONS: The findings suggest that patients with progressive/fibrotic pulmonary sarcoidosis have intense immune activity related to host defense in their lungs, with processes more similar to HP than IPF. The study also demonstrates that transbronchial lung biopsy samples can provide good-quality RNA for gene expression profiling, supporting its potential use as a prognostic classifier for pulmonary sarcoidosis.
Assuntos
Expressão Gênica/genética , Pulmão/patologia , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/patologia , Adulto , Idoso , Biópsia , Brônquios/patologia , Progressão da Doença , Feminino , Fibroblastos , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regulação para Cima/genéticaRESUMO
BACKGROUND: Renal cell carcinoma (RCC) is histopathologically heterogeneous with clear cell and papillary the most common subtypes. The most frequent molecular abnormality in clear cell RCC is VHL inactivation but promoter methylation of tumour suppressor genes is common in both subtypes of RCC. To investigate whether RCC CpG methylation status was influenced by histopathology and VHL status we performed high-throughput epigenetic profiling using the Illumina Goldengate Methylation Array in 62 RCC (29 RCC from von Hippel-Lindau (VHL) disease patients, 20 sporadic clear cell RCC with wild type VHL and 13 sporadic papillary RCC). RESULTS: 43 genes were methylated in >20% of primary RCC (range 20-45%) and most (37/43) of these had not been reported previously to be methylated in RCC. The distribution of the number of methylated CpGs in individual tumours differed from the expected Poisson distribution (p < 0.00001; log-likelihood G test) suggesting that a subset of RCC displayed a CpG Island Methylator Phenotype. Comparison of RCC subtypes revealed that, on average, tumour specific CpG methylation was most prevalent in papillary RCC and least in VHL RCC. Many of the genes preferentially methylated in pRCC were linked to TGFbeta or ERK/Akt signalling. CONCLUSION: These findings demonstrate differing patterns of tumour-specific CpG methylation in VHL and non VHL clear cell RCC and papillary RCC, and identify multiple novel potential CpG methylation biomarkers for RCC.
Assuntos
Carcinoma de Células Renais/genética , Ilhas de CpG/genética , Metilação de DNA , Neoplasias Renais/genética , Doença de von Hippel-Lindau/genética , Adulto , Idoso , Análise por Conglomerados , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Neoplasias Renais/complicações , Pessoa de Meia-Idade , Distribuição de Poisson , Reprodutibilidade dos Testes , Estatísticas não ParamétricasRESUMO
Hypoxia-inducible factor 1α is a key regulator of the hypoxia response in normal and cancer tissues. It is well recognized to regulate glycolysis and is a target for therapy. However, how tumor cells adapt to grow in the absence of HIF1α is poorly understood and an important concept to understand for developing targeted therapies is the flexibility of the metabolic response to hypoxia via alternative pathways. We analyzed pathways that allow cells to survive hypoxic stress in the absence of HIF1α, using the HCT116 colon cancer cell line with deleted HIF1α versus control. Spheroids were used to provide a 3D model of metabolic gradients. We conducted a metabolomic, transcriptomic, and proteomic analysis and integrated the results. These showed surprisingly that in three-dimensional growth, a key regulatory step of glycolysis is Aldolase A rather than phosphofructokinase. Furthermore, glucose uptake could be maintained in hypoxia through upregulation of GLUT14, not previously recognized in this role. Finally, there was a marked adaptation and change of phosphocreatine energy pathways, which made the cells susceptible to inhibition of creatine metabolism in hypoxic conditions. Overall, our studies show a complex adaptation to hypoxia that can bypass HIF1α, but it is targetable and it provides new insight into the key metabolic pathways involved in cancer growth. IMPLICATIONS: Under hypoxia and HIF1 blockade, cancer cells adapt their energy metabolism via upregulation of the GLUT14 glucose transporter and creatine metabolism providing new avenues for drug targeting.
Assuntos
Neoplasias do Colo/genética , Metabolismo Energético/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias do Colo/patologia , Creatina/genética , Creatina/metabolismo , Frutose-Bifosfato Aldolase/genética , Glucose/metabolismo , Glicólise/genética , Células HCT116 , Humanos , Esferoides Celulares/metabolismo , Hipóxia Tumoral/genéticaRESUMO
Regulatory T (T(R)) cells are integral to the maintenance of intestinal homeostasis, where an intricate balance between tolerance and immunity must be maintained. Recently, studies have focused on the identification of molecules involved in the function and/or development of T(R) cells. One such molecule, the G-protein coupled receptor Gpr83, has been identified through gene expression analysis as being overexpressed within thymic and peripheral naturally arising regulatory T (nT(R)) cell populations. The aim of this study was to further define the characteristics of Gpr83 expression and to investigate the role of Gpr83 in T(R)-cell development and function through the generation and analysis of Gpr83-deficient mice. Following activation, naïve CD4(+) T cells induce Gpr83 expression in a transforming growth factor (TGF)-beta dependent manner. Rather than being a general marker of activation, Gpr83 expression could only be detected in cells also expressing forkhead/winged helix transcription factor (Foxp3), further supporting the association of Gpr83 with the regulatory cell phenotype. Mice deficient in Gpr83 expression developed normally and did not display signs of inflammatory disease. Thymic nT(R)-cell development was unaffected by a lack of Gpr83 expression and peripheral nT(R)-cell homeostasis was normal when compared with that of wild-type mice. Gpr83 expression was dispensable for the regulatory activity of nT(R) cells as Gpr83-deficient nT(R) cells could suppress the development of disease in a T-cell transfer model of colitis. These results suggest a redundant role for Gpr83 in the function of T(R) cells in this model of disease. Further studies are required to determine the role of Gpr83 in T(R)-cell biology.
Assuntos
Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Receptores Acoplados a Proteínas G/imunologia , Linfócitos T Reguladores/imunologia , Animais , Células da Medula Óssea/imunologia , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Homeostase/imunologia , Imunidade nas Mucosas , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Receptores Acoplados a Proteínas G/deficiência , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disorder caused by dystrophin deficiency. Previous work suggested that increased expression of the dystrophin-related protein utrophin in the mdx mouse can reduce the dystrophic pathophysiology. Physiological tests showed that the transgenic mouse muscle functioned in a way similar to normal muscle. More recently, it has become possible to analyse disease pathways using microarrays, a sensitive method to evaluate the efficacy of a therapeutic approach. We thus examined the gene expression profile of mdx mouse muscle compared to wild-type mouse muscle and compared the data with that obtained from the transgenic line overexpressing utrophin. The data confirm that the expression of utrophin in the mdx mouse muscle results in a global gene expression profile more similar to that seen for the wild-type mouse. This study confirms that a strategy to up-regulate utrophin is likely to be beneficial in dystrophin deficiency.
Assuntos
Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Análise de Sequência com Séries de Oligonucleotídeos , Utrofina/genética , Análise de Variância , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapiaRESUMO
The prostanoid biosynthetic enzyme cyclooxygenase-2 (Cox-2) is up-regulated in several neuroendocrine tumors. The aim of the current study was to employ a neuroendocrine cell (PC12) model of Cox-2 overexpression to identify gene products that might be implicated in the oncogenic and/or inflammatory actions of this enzyme in the setting of neuroendocrine neoplasia. Expression array and real-time PCR analysis demonstrated that levels of the neuroendocrine marker chromogranin A (CGA) were 2- and 3.2-fold higher, respectively, in Cox-2 overexpressing cells (PCXII) vs. their control (PCMT) counterparts. Immunocytochemical and immunoblotting analyses confirmed that both intracellular and secreted levels of CGA were elevated in response to Cox-2 induction. Moreover, exogenous addition of prostaglandin E(2) (1 microm) mimicked this effect in PCMT cells, whereas treatment of PCXII cells with the Cox-2 selective inhibitor NS-398 (100 nm) reduced CGA expression levels, thereby confirming the biospecificity of this finding. Levels of neuron-specific enolase were similar in the two cell lines, suggesting that the effect of Cox-2 on CGA expression was specific and not due to a global enhancement of neuroendocrine marker expression/differentiation. Cox-2-dependent CGA up-regulation was associated with significantly increased chromaffin granule number and intracellular and secreted levels of dopamine. CGA promoter-driven reporter gene expression studies provided evidence that prostaglandin E(2)-dependent up-regulation required a proximal cAMP-responsive element (-71 to -64 bp). This study is the first to demonstrate that Cox-2 up-regulates both CGA expression and bioactivity in a neuroendocrine cell line and has major implications for the role of this polypeptide in the pathogenesis of neuroendocrine cancers in which Cox-2 is up-regulated.
Assuntos
Cromogranina A/genética , Cromogranina A/metabolismo , Ciclo-Oxigenase 2/genética , Dinoprostona/farmacologia , Dinoprostona/fisiologia , Animais , AMP Cíclico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Células PC12 , Reação em Cadeia da Polimerase , RNA Neoplásico/genética , Ratos , TransfecçãoRESUMO
Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 (PEBP1)/Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low PEBP1 expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of PEBP1 after, but not prior to, DC vaccination. Moreover, the change in PEBP1 expression upon vaccination correlated well with survival. Further analyses revealed that PEBP1 expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including STAT3, NOTCH1, and MAPK1. Concordantly, PEBP1 inversely correlated with the myeloid/lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease.
RESUMO
AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Bradicardia/genética , Cálcio/metabolismo , Frequência Cardíaca/genética , Sarcolema/metabolismo , Nó Sinoatrial/metabolismo , Adulto , Animais , Bradicardia/metabolismo , Eletrocardiografia Ambulatorial , Exercício Físico , Coração/diagnóstico por imagem , Humanos , Imagem Cinética por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Condicionamento Físico Animal , Resistência Física , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Nó Sinoatrial/patologiaRESUMO
Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles. In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.
RESUMO
Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. Here, we report that hypoxia promotes tumour heterogeneity through the epigenetic regulation of CAIX. Based on hypoxic CAIX expression we identify and characterize two distinct populations of tumour cells, one that has inducible expression of CAIX and one that does not. The CAIX+ve population is enriched with cells expressing cancer stem cell markers and which have high self-renewal capacity. We show that differential CAIX expression is due to differences in chromatin structure. To further investigate the relationship between chromatin organization and hypoxic induction of CAIX expression we investigated the effect of JQ1 an inhibitor of BET bromodomain proteins and A366 a selective inhibitor of the H3K9 methyltransferase G9a/GLP. We identified that these drugs were able to modulate hypoxic CAIX expression induction. This further highlights the role of epigenetic modification in adaption to hypoxia and also in regulation of heterogeneity of cells within tumours. Interestingly, we identified that the two subpopulations show a differential sensitivity to HDAC inhibitors, NaBu or SAHA, with the CAIX positive showing greater sensitivity to treatment. We propose that drugs modulating chromatin regulation of expression may be used to reduce heterogeneity induced by hypoxia and could in combination have significant clinical consequences.
Assuntos
Antígenos de Neoplasias/biossíntese , Anidrases Carbônicas/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Animais , Antígenos de Neoplasias/genética , Anidrase Carbônica IX , Anidrases Carbônicas/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Indução Enzimática , Feminino , Células HCT116 , Xenoenxertos , Humanos , Isoenzimas/biossíntese , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologiaRESUMO
Following exposure to vaccines, antigen-specific CD8(+) T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8(+) T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8(+) T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8(+) T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.