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1.
J Immunol ; 193(1): 111-9, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24879793

RESUMO

IL-13 can bind to two distinct receptors: a heterodimer of IL-13Rα1/IL-4Rα and IL-13Rα2. Whereas IL-13Rα1/IL-4Rα engagement by IL-13 leads to the activation of STAT6, the molecular events triggered by IL-13 binding to IL-13Rα2 remain incompletely understood. IL-4 can bind to and signal through the IL-13Rα1/IL-4Rα complex but does not interact with IL-13Rα2. Idiopathic pulmonary fibrosis is a progressive and generally fatal parenchymal lung disease of unknown etiology with no current pharmacologic treatment options that substantially prolong survival. Preclinical models of fibrotic diseases have implicated IL-13 activity on multiple cell types, including macrophages and fibroblasts, in initiating and perpetuating pathological fibrosis. In this study, we show that IL-13, IL-4, IL-13Rα2, and IL-13-inducible target genes are expressed at significantly elevated levels in lung tissue from patients with idiopathic pulmonary fibrosis compared with control lung tissue. IL-4 and IL-13 induce virtually identical transcriptional responses in human monocytes, macrophages, and lung fibroblasts. IL-13Rα2 expression can be induced in lung fibroblasts by IL-4 or IL-13 via a STAT6-dependent mechanism, or by TNF-α via a STAT6-independent mechanism. Endogenously expressed IL-13Rα2 decreases, but does not abolish, sensitivity of lung fibroblasts to IL-13 and does not affect sensitivity to IL-4. Genome-wide transcriptional analyses of lung fibroblasts stimulated with IL-13 in the presence of Abs that selectively block interactions of IL-13 with IL-13Rα1/IL-4Rα or IL-13Rα2 show that endogenously expressed IL-13Rα2 does not activate any unique IL-13-mediated gene expression patterns, confirming its role as a decoy receptor for IL-13 signaling.


Assuntos
Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Fibrose Pulmonar Idiopática/imunologia , Subunidade alfa2 de Receptor de Interleucina-13/imunologia , Interleucina-13/imunologia , Pulmão/imunologia , Transdução de Sinais/imunologia , Feminino , Fibroblastos/patologia , Estudo de Associação Genômica Ampla , Humanos , Fibrose Pulmonar Idiopática/patologia , Subunidade alfa1 de Receptor de Interleucina-13/imunologia , Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/imunologia , Pulmão/patologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Monócitos/imunologia , Monócitos/patologia , Fator de Transcrição STAT6/imunologia , Fator de Necrose Tumoral alfa/imunologia
2.
J Infect Dis ; 209(10): 1542-50, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24286981

RESUMO

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Transcriptoma , Abscesso/microbiologia , Animais , Humanos , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Análise Serial de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Dermatopatias Bacterianas/microbiologia , Virulência
3.
Nat Genet ; 48(4): 407-16, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26928227

RESUMO

We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.


Assuntos
Neoplasias Pulmonares/genética , Mesotelioma/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias Pleurais/genética , Processamento Alternativo , Linhagem Celular Tumoral , Análise Mutacional de DNA , Exoma , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Mesotelioma/metabolismo , Mesotelioma/mortalidade , Mesotelioma Maligno , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/mortalidade , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 22(11): 1797-803, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12426207

RESUMO

OBJECTIVE: This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. METHODS AND RESULTS: We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. CONCLUSIONS: The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.


Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Linfocinas/fisiologia , Veias Umbilicais/química , Veias Umbilicais/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Linfocinas/genética , Linfocinas/farmacologia , Mutação/genética , Mutação/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Receptores CXCR4/biossíntese , Fatores de Tempo , Veias Umbilicais/citologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular
5.
Nat Genet ; 47(1): 13-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25401301

RESUMO

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.


Assuntos
Carcinoma de Células Renais/classificação , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Mutação , Adenoma Oxífilo/classificação , Adenoma Oxífilo/genética , Adenoma Oxífilo/patologia , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , DNA de Neoplasias , Dosagem de Genes , Instabilidade Genômica , Humanos , Neoplasias Renais/classificação , Neoplasias Renais/patologia , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/fisiologia , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/genética , Translocação Genética
6.
Physiol Genomics ; 11(3): 245-51, 2002 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-12388793

RESUMO

Mechanical forces modulate endothelial cell functions through several mechanisms including regulation of gene transcription. In the present study, gene transcription by human umbilical vein endothelial cells (HUVEC) either maintained under control pressure (that is, standard cell culture conditions equivalent to 0.15 mmHg sustained hydrostatic pressure) or exposed to 60/20 mmHg sinusoidal pressures at 1 Hz were compared using Affymetrix GeneChip microarrays to identify cellular/molecular mechanisms associated with endothelial cell responses to cyclic pressure. Cyclic pressure selectively affected transcription of 14 genes that included a set of mechanosensitive proteins involved in hemostasis (tissue plasminogen activator), cell adhesion (integrin-alpha2), and cell signaling (Rho B, cytosolic phospholipase A2), as well as a unique subset of cyclic pressure-sensitive genes such as vascular endothelial growth factor (VEGF)-C and transforming growth factor (TGF)-beta2. The present study also provided first evidence that VEGF-C, the most highly induced gene under 60/20 mmHg, mediated HUVEC proliferation in response to this cyclic pressure. Cyclic pressure is, therefore, a mechanical force that modulates endothelial cell functions (such as proliferation) by activating a specific transcriptional program.


Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/metabolismo , Transcrição Gênica , Divisão Celular , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Periodicidade , Pressão , RNA Mensageiro/biossíntese , Estresse Mecânico , Fator C de Crescimento do Endotélio Vascular
7.
Physiol Genomics ; 10(1): 13-20, 2002 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-12118101

RESUMO

The objective of this study was to use gene expression data from well-defined cell culture models, in combination with expression data from diagnostic samples of human diseased tissues, to identify potential therapeutic targets and markers of disease. Using Affymetrix oligonucleotide array technology, we identified a common profile of genes upregulated during endothelial morphogenesis into tubelike structures in three in vitro models of angiogenesis. Rigorous data selection criteria were used to identify a list of over 1,000 genes whose expression was increased more than twofold over baseline at either 4, 8, 24, 40 or 50 h. To further refine and prioritize this list, we used standard bioinformatic algorithms to identify potential transmembrane and secreted proteins. We then overlapped this gene set with genes upregulated in colon tumors vs. normal colon, resulting in a subset of 128 genes in common with our endothelial list. We removed from this list those genes expressed in 6 different colon tumor lines, resulting in a list of 24 putative, vascular-specific angiogenesis-associated genes. Three genes, gp34, stanniocalcin-1 (STC-1), and GA733-1, were expressed at levels 10-fold or more in colon tumors compared with normal mucosa. We validated the vascular-specific expression of one of these genes, STC-1, by in situ hybridization. The ability to combine in vitro and in vivo data sets should permit one to identify putative angiogenesis target genes in various tumors, chronic inflammation, and other disorders where therapeutic manipulation of angiogenesis is a desirable treatment modality.


Assuntos
Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Linhagem Celular , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Marcação de Genes/métodos , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Veias Umbilicais
8.
Physiol Genomics ; 11(3): 263-72, 2002 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-12399448

RESUMO

DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes. Muscle function was depressed for 3 days postligation, but returned to normal by day 7. Decreased muscle function was accompanied by reduced expression of genes involved in mitochondrial energy production, muscle contraction, and calcium handling. The induction of MyoD on day 1 denoted the beginning of muscle regeneration and was followed by the reemergence of the embryonic forms of muscle contractile proteins, which peaked at day 7. Transcriptional analysis indicated that the ischemic skeletal muscle may transition through a functional adaptation stage with recovery of contractile force prior to full regeneration. Several members of the insulin-like growth factor axis were coordinately induced in a time frame consistent with their playing a role in the regenerative process.


Assuntos
Isquemia/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Regeneração , Doença Aguda , Animais , Citocinas/biossíntese , Citocinas/genética , Artéria Femoral/cirurgia , Perfilação da Expressão Gênica , Isquemia/metabolismo , Isquemia/patologia , Cinética , Ligadura , Extremidade Inferior , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Esquelético/patologia , Miosinas/biossíntese , Miosinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Receptores de Citocinas/biossíntese , Receptores de Citocinas/genética , Somatomedinas/biossíntese , Somatomedinas/genética , Transcrição Gênica
9.
Sci Signal ; 6(271): ra25, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23592840

RESUMO

The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and also represents a major therapeutic target for tumors and intraocular neovascular disorders. The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define the VEGF-regulated phosphoproteome and its temporal dynamics in human umbilical vein endothelial cells and then used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses. The PI3K (phosphatidylinositol 3-kinase)-mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central in activating VEGF-regulated phosphorylation and increasing endothelial cell viability by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1), an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases. This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival but also rendered cells more susceptible to PI3K inhibition. Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and reprogramming of receptor tyrosine kinases in human endothelial cells.


Assuntos
Ativação Enzimática/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Complexos Multiproteicos/metabolismo , Fosfopeptídeos/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose/fisiologia , Proteína Forkhead Box O1 , Células Endoteliais da Veia Umbilical Humana , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosforilação , Proteômica
10.
Mol Cancer Ther ; 11(5): 1143-54, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22402123

RESUMO

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genes ras , Humanos , Concentração Inibidora 50 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos
11.
Inflamm Bowel Dis ; 16(10): 1717-28, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20848455

RESUMO

BACKGROUND: Genome-wide microarray expression analysis creates a comprehensive picture of gene expression at the cellular level. The aim of this study was to investigate differential intestinal gene expression in patients with Crohn's disease (CD) and controls with subanalysis of confirmed CD susceptibility genes, associated pathways, and cell lineage. METHODS: In all, 172 biopsies from 53 CD and 31 control subjects were studied. Paired endoscopic biopsies were taken at ileocolonoscopy from five specific anatomical locations including the terminal ileum (TI) for RNA extraction and histology. The 41,058 expression sequence tags were analyzed using the Agilent platform. RESULTS: Analysis of all CD biopsies versus controls showed 259 sequences were upregulated and 87 sequences were downregulated. Upregulated genes in CD included SAA1 (fold change [FC] +7.5, P = 1.47 × 10(-41)) and REGL (FC +7.3, P = 2.3 × 10(-16)), whereas cellular detoxification genes including-SLC14A2 (FC-2.49, P = 0.00002) were downregulated. In the CD TI biopsies diubiquitin (FC+11.3, P < 1 × 10(-45)), MMP3 (FC+7.4, P = 1.3 × 10(-11)), and IRTA1 (FC-11.4, P = 4.7 × 10(-12)) were differentially expressed compared to controls. In the colon SAA1 (FC+6.3, P = 5.3 × 10(-8)) was upregulated and thymic stromal lymphopoietin (TSLP) (FC-2.3, P = 2.7 × 10(-6)) was downregulated comparing noninflamed CD and control biopsies, and the colonic inflammatory CD signature was characterized by downregulation of the organic solute carriers-SLC38A4, SLC26A2, and OST alpha. Of CD susceptibility genes identified by genome-wide association scan IL-23A, JAK2, and STAT3 were upregulated in the CD group, confirming the dysregulation of Th17 signaling. CONCLUSIONS: These data characterize the dysregulation of a series of specific inflammatory pathways highlighting potential pathogenic mechanisms as well as areas for translation to therapeutic targets.


Assuntos
Biomarcadores/metabolismo , Doença de Crohn/genética , Perfilação da Expressão Gênica , Mucosa Intestinal/metabolismo , Adulto , Estudos de Casos e Controles , Colo/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Feminino , Seguimentos , Estudo de Associação Genômica Ampla , Humanos , Íleo/metabolismo , Íleo/patologia , Intestinos/patologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Cancer Res ; 68(13): 5380-9, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-18593940

RESUMO

Breast cancer is a heterogeneous disease with distinct molecular subtypes characterized by differential response to targeted and chemotherapeutic agents. Enhanced understanding of the genetic alterations characteristic of different subtypes is needed to pave the way for more personalized administration of therapeutic agents. We have taken a functional genomics approach using a well-characterized panel of breast cancer cell lines to identify putative biomarkers of resistance to antimitotic agents such as paclitaxel and monomethyl-auristatin-E (MMAE). In vitro studies revealed a striking difference in sensitivity to these agents between cell lines from different subtypes, with basal-like cell lines being significantly more sensitive to both agents than luminal or HER2-amplified cell lines. Genome-wide association studies using copy number data from Affymetrix single nucleotide polymorphism arrays identified amplification of the chromosome 17q21 region as being highly associated with resistance to both paclitaxel and MMAE. An unbiased approach consisting of RNA interference and high content analysis was used to show that amplification and concomitant overexpression of the gene encoding the ABCC3 drug transporter is responsible for conferring in vitro resistance to paclitaxel and MMAE. We also show that amplification of ABCC3 is present in primary breast tumors and that it occurs predominantly in HER2-amplified and luminal tumors, and we report on development of a specific fluorescence in situ hybridization assay that may have utility as a predictive biomarker of taxane resistance in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Carcinoma Ductal de Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Genes erbB-2 , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Taxoides/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos/análise , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico/métodos , Análise por Conglomerados , Amplificação de Genes/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Análise em Microsséries , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Paclitaxel/uso terapêutico , Interferência de RNA
13.
Genomics ; 83(3): 508-17, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14962677

RESUMO

Various approaches have been developed for the preparation of samples for gene expression monitoring. For Affymetrix chips, a standard protocol is widely used; however, this is inefficient for small samples such as laser capture microdissections. Several amplification procedures for such samples already exist, and our goal was to test two of them: the first is based on random PCR amplification, and the second, linear amplification, involves performing the standard protocol twice. We analyzed a dilution of a commercially available mouse brain total RNA preparation and microdissections from mouse hippocampus and striatum. We evaluated the quality of microarray data by analyzing several chip parameters and performing multiple comparisons. At the biological level, brain microdissections prepared with either method gave similar expression results. At the technical level, analysis of the commercial sample showed that random PCR amplification is more reproducible, requires smaller RNA input, and generates cRNA of higher quality than linear amplification.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Animais , Encéfalo/metabolismo , Estudos de Avaliação como Assunto , Feminino , Perfilação da Expressão Gênica , Camundongos , Microdissecção/métodos , Modelos Biológicos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Complementar/metabolismo , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade
14.
Microcirculation ; 10(1): 63-81, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12610664

RESUMO

The process of endothelial differentiation into a network of tube-like structures with patent lumens requires an integrated program of gene expression. To identify genes upregulated in endothelial cells during the process of tube formation, RNA was prepared from several different time points (0, 4, 8, 24, 40, and 48 hours) and from three different experimental models of human endothelial tube formation: in collagen gels and fibrin gels driven by the combination of PMA (80), bFGF (40 ng/ml) and bFGF (40 ng/ml) or in collagen gels driven by the combination of HGF (40 ng/ml) and VEGF (40 ng/ml). Gene expression was evaluated using Affymetrix Gene Chip oligonucleotide arrays. Over 1000 common genes were upregulated greater than twofold over baseline at one or more time points in the three different models. In the present study, we discuss the identified genes that could be assigned to major functional classes: apoptosis, cytoskeleton, proteases, matrix, and matrix turnover, pumps and transporters, membrane lipid turnover, and junctional molecules or adhesion proteins.


Assuntos
Endotélio Vascular/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica/fisiologia , Animais , Apoptose/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Diferenciação Celular/genética , Células Cultivadas/metabolismo , Colágeno , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Fibrina , Géis , Perfilação da Expressão Gênica , Substâncias de Crescimento/farmacologia , Humanos , Lipídeos de Membrana/biossíntese , Neovascularização Fisiológica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Acetato de Tetradecanoilforbol/farmacologia
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