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1.
Commun Biol ; 6(1): 640, 2023 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-37316562

RESUMO

Human genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB)1. To identify factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)2 on sorted erythroid lineage cells derived from bone marrow (BM) or cord blood (CB), representing adult and fetal states, respectively. BM to CB cell ATAC-seq profile comparisons revealed genome-wide enrichment of NFI DNA binding motifs and increased NFIX promoter chromatin accessibility, suggesting that NFIX may repress HBG. NFIX knockdown in BM cells increased HBG mRNA and fetal hemoglobin (HbF) protein levels, coincident with increased chromatin accessibility and decreased DNA methylation at the HBG promoter. Conversely, overexpression of NFIX in CB cells reduced HbF levels. Identification and validation of NFIX as a new target for HbF activation has implications in the development of therapeutics for hemoglobinopathies.


Assuntos
Cromatina , Hemoglobina Fetal , Adulto , Humanos , Cromatina/genética , Hemoglobina Fetal/genética , Linhagem da Célula/genética , Bioensaio , Células da Medula Óssea , Fatores de Transcrição NFI/genética
2.
PLoS One ; 12(1): e0169128, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28060870

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and inhibitors of DNA methyltransferase or histone deacetylase (HDAC) enzymes have exhibited activity in preclinical AML models. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have potential beneficial clinical activity in AML, however the toxicity profiles of non-selective HDAC inhibitors in the combination setting limit their clinical utility. In this work, we describe the preclinical development of selective inhibitors of HDAC1 and HDAC2, which are hypothesized to have improved safety profiles, for combination therapy in AML. We demonstrate that selective inhibition of HDAC1 and HDAC2 is sufficient to achieve efficacy both as a single agent and in combination with azacitidine in preclinical models of AML, including established AML cell lines, primary leukemia cells from AML patient bone marrow samples and in vivo xenograft models of human AML. Gene expression profiling of AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the combination of both have identified a list of genes involved in transcription and cell cycle regulation as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients.


Assuntos
Antineoplásicos/farmacologia , Azacitidina/farmacologia , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/metabolismo , Animais , Biomarcadores , Células da Medula Óssea , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Terapia de Alvo Molecular , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS One ; 11(4): e0153767, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27073918

RESUMO

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and ß-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult ß-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.


Assuntos
Anemia Falciforme/metabolismo , Células Eritroides/efeitos dos fármacos , Hemoglobina Fetal/metabolismo , Fator de Transcrição GATA2/metabolismo , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Anemia Falciforme/genética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Eritroides/metabolismo , Fator de Transcrição GATA2/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Globinas beta/genética , Globinas beta/metabolismo
4.
Oncotarget ; 6(7): 4863-87, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25605023

RESUMO

Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor - B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells.


Assuntos
Reparo do DNA , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Complexo Repressor Polycomb 2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste , Células HeLa , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Complexo Repressor Polycomb 2/genética , Transfecção , Ubiquitina-Proteína Ligases/genética
5.
Biol Open ; 1(7): 658-66, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23213459

RESUMO

The integrin α6ß4 is defined as an adhesion receptor for laminins. Referred to as 'ß4', this integrin plays a key role in the progression of various carcinomas through its ability to orchestrate key signal transduction events and promote cell motility. To identify novel downstream effectors of ß4 function in breast cancer, microRNAs (miRNAs) were examined because of their extensive links to tumorigenesis and their ability to regulate gene expression globally. Two breast carcinoma cell lines and a collection of invasive breast carcinomas with varying ß4 expression were used to assess the effect of this integrin on miRNA expression. A novel miRNA microarray analysis termed quantitative Nuclease Protection Assay (qNPA) revealed that ß4 expression can significantly alter miRNA expression and identified two miRNA families, miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, that are consistently downregulated by expression of this integrin. Analysis of published Affymetrix GeneChip data identified 54 common targets of miR-92ab and miR-99ab/100 within the subset of ß4-regulated mRNAs, revealing several genes known to be key components of ß4-regulated signaling cascades and effectors of cell motility. Gene ontology classification identified an enrichment in genes associated with cell migration within this population. Finally, gene set enrichment analysis of all ß4-regulated mRNAs revealed an enrichment in targets belonging to distinct miRNA families, including miR-92ab and others identified by our initial array analyses. The results obtained in this study provide the first example of an integrin globally impacting miRNA expression and provide evidence that select miRNA families collectively target genes important in executing ß4-mediated cell motility.

6.
J Biol Chem ; 287(13): 9835-9844, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22308039

RESUMO

The α6ß4 integrin (referred to as "ß4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in ß4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of ß4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates ß4-mediated invasion. Expression of ß4 in ß4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous ß4, miR-29a expression is low and ß4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that ß4 can regulate SPARC expression and that SPARC is an effector of ß4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.


Assuntos
Regulação Neoplásica da Expressão Gênica , Integrina beta4/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Osteonectina/biossíntese , Biossíntese de Proteínas , Linhagem Celular Tumoral , Humanos , Integrina beta4/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Osteonectina/genética
7.
Science ; 334(6057): 799-802, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22076376

RESUMO

In the mammalian genome, 5'-CpG-3' dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific ß-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.


Assuntos
Metilação de DNA , Eritroblastos/metabolismo , Eritropoese , Animais , Ilhas de CpG , Replicação do DNA , Fosfatos de Dinucleosídeos/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Fígado/embriologia , Região de Controle de Locus Gênico , Elementos Nucleotídeos Longos e Dispersos , Camundongos , Fase S , Análise de Sequência de DNA , Transcrição Gênica , Globinas beta/genética
8.
J Vis Exp ; (54)2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21847081

RESUMO

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.


Assuntos
Antígenos CD/análise , Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Citometria de Fluxo/métodos , Receptores da Transferrina/análise , Animais , Células Precursoras Eritroides/química , Feminino , Masculino , Camundongos , Gravidez
9.
PLoS Biol ; 8(9)2010 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-20877475

RESUMO

Hematopoietic progenitors undergo differentiation while navigating several cell division cycles, but it is unknown whether these two processes are coupled. We addressed this question by studying erythropoiesis in mouse fetal liver in vivo. We found that the initial upregulation of cell surface CD71 identifies developmentally matched erythroblasts that are tightly synchronized in S-phase. We show that DNA replication within this but not subsequent cycles is required for a differentiation switch comprising rapid and simultaneous committal transitions whose precise timing was previously unknown. These include the onset of erythropoietin dependence, activation of the erythroid master transcriptional regulator GATA-1, and a switch to an active chromatin conformation at the ß-globin locus. Specifically, S-phase progression is required for the formation of DNase I hypersensitive sites and for DNA demethylation at this locus. Mechanistically, we show that S-phase progression during this key committal step is dependent on downregulation of the cyclin-dependent kinase p57(KIP2) and in turn causes the downregulation of PU.1, an antagonist of GATA-1 function. These findings therefore highlight a novel role for a cyclin-dependent kinase inhibitor in differentiation, distinct to their known function in cell cycle exit. Furthermore, we show that a novel, mutual inhibition between PU.1 expression and S-phase progression provides a "synchromesh" mechanism that "locks" the erythroid differentiation program to the cell cycle clock, ensuring precise coordination of critical differentiation events.


Assuntos
Ciclo Celular , Eritropoese , Proteínas Proto-Oncogênicas/metabolismo , Fase S , Transativadores/metabolismo , Animais , Antígenos CD/metabolismo , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Metilação de DNA , Replicação do DNA , Regulação para Baixo , Camundongos , Receptores da Transferrina/metabolismo
10.
Gastroenterology ; 136(3): 912-23, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19109961

RESUMO

BACKGROUND AND AIMS: Tumor necrosis factor (TNF) superfamily members have attracted attention as new therapeutic targets for treating inflammatory disease. TNF-like weak inducer of apoptosis (TWEAK) is a unique, multifunctional TNF family cytokine that signals through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). The role of this pathway in the intestine has not been previously reported. METHODS: The 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model was conducted in TWEAK- or Fn14-deficient mice or in normal mice treated with a TWEAK-blocking monoclonal antibody, and clinical severity, histopathology, immunohistochemistry for cell infiltrates, TWEAK and Fn14, gene expression profiling in the colon, and systemic adaptive immunity were assessed. The effect of TWEAK on colon epithelial cell production of inflammatory mediators was analyzed in vitro. The gamma-irradiation injury model was conducted in TWEAK- or Fn14-deficient mice, and crypt epithelial death was assessed. RESULTS: Colitis severity and histologic scores were significantly reduced by TWEAK pathway deficiency or TWEAK-blocking monoclonal antibody. Neutrophil and macrophage infiltrates, chemokines, cytokines, and matrix metalloproteinase expression were reduced in the TWEAK-deficient colon after TNBS administration; however, systemic adaptive immune responses to trinitrophenyl were not altered. Fn14 is expressed on colon epithelial cells in TNBS colitis, and TWEAK induces epithelial production of pathogenic mediators. TWEAK also regulates intestinal epithelial turnover, as evidenced by reduced epithelial cell death after gamma-irradiation injury in TWEAK and Fn14 knockout mice. CONCLUSIONS: Our studies elucidate a nonredundant TWEAK-intestinal epithelial cell axis and suggest that blocking TWEAK may dampen chronic intestinal inflammation and allow normal epithelial repair.


Assuntos
Colite/metabolismo , Colite/patologia , Mucosa Intestinal/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Colite/imunologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Citocina TWEAK , Modelos Animais de Doenças , Raios gama , Sistema Imunitário/fisiologia , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Neutrófilos/patologia , RNA Mensageiro/metabolismo , Receptor de TWEAK , Fatores de Necrose Tumoral/genética , Úlcera/imunologia , Úlcera/metabolismo , Úlcera/patologia
11.
Arthritis Rheum ; 54(9): 3011-21, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16947635

RESUMO

OBJECTIVE: Previously published studies have demonstrated that a majority of systemic sclerosis (SSc) fibroblasts exhibit elevated levels of transforming growth factor beta type I receptor (TGFbetaRI). An experimental model that recapitulates this condition was established in control dermal fibroblasts by titrating the dose of adenovirus vector expressing TGFbetaRI (AdTGFbetaRI). The present study was undertaken to determine the functional consequences of increased levels of TGFbetaRI in SSc. METHODS: Gene array analysis of control dermal fibroblasts transduced with AdTGFbetaRI was performed using GeneChip expression arrays. Gene validation was done by Northern blot, quantitative reverse transcriptase-polymerase chain reaction, and Western blot techniques. TGFbeta blockade was performed using soluble TGFbeta receptor. TGFbetaRI kinase/activin receptor-like kinase 5 was inhibited with pharmacologic inhibitors. TGFbetaRI and TGFbetaRII protein levels and collagen production were examined by Western blotting in primary dermal fibroblasts from 9 SSc patients and 9 healthy adults. Endogenous TGFbetaRI levels were suppressed in control and SSc fibroblasts using specific small interfering RNA (siRNA). RESULTS: Global gene analysis indicated that a 2-fold increase in TGFbetaRI levels in control fibroblasts resulted in profibrotic changes that closely resembled the phenotype of SSc fibroblasts. A total of 125 genes were up-regulated, including COL1A1, COL1A2, and connective tissue growth factor, and 206 genes were down-regulated. Elevated production of collagen in cells transduced with AdTGFbetaRI was dependent on the autocrine TGFbeta, but not TGFbetaRI kinase activity. Eight of the 9 SSc strains exhibited increased levels of TGFbetaRI protein, which correlated with increased collagen synthesis. Treatment of SSc and matched control fibroblasts with siRNA that normalizes TGFbetaRI levels reverted collagen protein production in SSc fibroblasts to the levels observed in control fibroblasts. CONCLUSION: Our findings demonstrate that aberrantly expressed TGFbetaRI may drive an autocrine loop involved in the up-regulation of collagen and other matrix-related genes in SSc fibroblasts.


Assuntos
Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Escleroderma Sistêmico/genética , Pele/patologia , Northern Blotting , Colágeno/genética , Primers do DNA , Fibroblastos/patologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escleroderma Sistêmico/patologia
12.
Nat Biotechnol ; 24(7): 832-40, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16823376

RESUMO

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and 'in-house' platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by quantitative real-time (QRT)-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent preprocessing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms.


Assuntos
Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sondas de DNA/química , Sondas de DNA/classificação , Análise em Microsséries/classificação , Reprodutibilidade dos Testes
13.
Arthritis Rheum ; 54(6): 1961-73, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16736506

RESUMO

OBJECTIVE: To determine whether biopsy specimens obtained from systemic sclerosis (SSc) lesions show a distinctive gene profile, whether that gene profile is maintained in fibroblasts cultured from SSc skin biopsy specimens, and whether results from tissue obtained from multiple clinical centers can be combined to yield useful observations in this rare disease. METHODS: Biopsy samples and passaged fibroblasts were stored in RNAlater solution prior to processing for RNA. RNA from SSc and control skin biopsy specimens, as well as SSc and control explanted passage 4 fibroblasts, from 9 patients and 9 controls was hybridized to Affymetrix HG-U133A arrays. Data were analyzed using the BRB ArrayTools system. When appropriate, findings were followed up with immunohistochemical analysis or TaqMan studies. RESULTS: Biopsy samples obtained from patients with SSc had a robust and distinctive gene profile, with approximately 1,800 qualifiers distinguishing normal skin from SSc skin at a significant level. The SSc phenotype was the major driver of sample clusters, independent of origin. Alterations in transforming growth factor beta and Wnt pathways, extracellular matrix proteins, and the CCN family were prominent. Explanted fibroblasts from SSc biopsy samples showed a far smaller subset of changes that were relatively variable between samples, suggesting that either nonfibroblast cell types or other aspects of the dermal milieu are required for full expression of the SSc phenotype. CONCLUSION: SSc has a distinct gene profile that is not confounded by geographic location, indicating that extended multicenter studies may be worthwhile to identify distinct subsets of disease by transcript profiling. Explanted SSc fibroblasts show an incomplete reflection of the SSc phenotype.


Assuntos
Fibroblastos/química , Perfilação da Expressão Gênica , Escleroderma Sistêmico/genética , Pele/citologia , Adulto , Biópsia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo
14.
Genomics ; 88(1): 111-21, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16624518

RESUMO

We have developed a total RNA amplification and labeling strategy for use with Affymetrix GeneChips. Our protocol, which we denote BIIB, employs two rounds of linear T7 amplification followed by Klenow labeling to generate a biotinylated cDNA. In benchmarking studies using a titration of mouse universal total RNA, BIIB outperformed commercially available kits in terms of sensitivity, accuracy, and amplified target length, while providing equivalent results for technical reproducibility. BIIB maintained 50 and 44% present calls from 100 and 50 pg of total RNA, respectively. Inter- and intrasample precision studies indicated that BIIB produces an unbiased and complete expression profile within a range of 5 ng to 50 pg of starting total RNA. From a panel of spiked exogenous transcripts, we established the BIIB linear detection limit to be 20 absolute copies. Additionally, we demonstrate that BIIB is sensitive enough to detect the stochastic events inherent in a highly diluted sample. Using RNA isolated from whole tissues, we further validated BIIB accuracy and precision by comparison of 224 expression ratios generated by quantitative real-time PCR. The utility of our method is ultimately illustrated by the detection of biologically expected trends in a T cell/B cell titration of 100 primary cells flow sorted from a healthy mouse spleen.


Assuntos
Linfócitos B/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/genética , Linfócitos T/metabolismo , Transcrição Gênica , Animais , Biotinilação , Separação Celular , Citometria de Fluxo , Camundongos , Técnicas de Amplificação de Ácido Nucleico/normas , Análise de Sequência com Séries de Oligonucleotídeos , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Baço/citologia
15.
Genomics ; 85(3): 309-21, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15718098

RESUMO

Generation of tissue-specific, normalized and subtracted cDNA libraries has the potential to characterize the expression of rare transcriptional units not represented on Affymetrix GeneChips. Initial sequence analysis of our murine cDNA clone collections showed that as much as 86, 45, and 30% of clones are not represented on the Affymetrix Mu11k, MG-U74, and MG-430 chip sets, respectively. A detailed study that compared EST sequences of a subtracted library generated from mouse retina to those of MG-430 consensus sequences was undertaken, using UniGene build 124 as the common reference. A set of 1111 nonredundant transcript regions, not represented on the commercial array, was identified. These clusters were used as the primary filter for analyzing a data set produced by assaying samples from the Pde6b(rd1) mouse model of retinal degeneration on a 12,325-feature retinal cDNA microarray. QRT-PCR validated eight unique transcripts identified by microarray. Seven of the transcripts showed retina-specific expression. Full-length cloning strategies were applied to two of the ESTs. The genes discovered by this approach are the full-length mouse homologue of guanylate cyclase 2F (GUCY2F) and a carboxy-truncated splice variant of retinal S-antigen (SAG), known as regulators of the visual phototransduction G-protein-coupled receptor-mediated signaling pathway. These sequences have been assigned GenBank Accession Nos. and , respectively.


Assuntos
Genômica , Degeneração Retiniana/genética , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Modelos Animais de Doenças , Guanilato Ciclase/genética , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
16.
Biotechniques ; 32(5): 1051-2, 1054, 1056-7, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12019778

RESUMO

The use of microarrays to monitor gene expression has become a standard research tool at both academic and industrial research institutions. Quality control of common printing defects during DNA deposition onto glass substrates is critical to maintaining data integrity and preventing the needless consumption of precious RNA, labeling reagents, and time. Here we demonstrate a nondestructive method for monitoring the quality of every spot on every chip of a microarray production run. We have identified many common manufacturing defects, while not perturbing the attachment of our oligonucleotide target to the substrate or altering further hybridization. This protocol is simple, fast, and inexpensive.


Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/normas , Controle de Qualidade , Carbocianinas , Corantes Fluorescentes , Hibridomas , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linfócitos T
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