RESUMEN
[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (Cmax of 7.1 µM), JNJ-2482272 had plasma concentrations beneath the lower limit of quantification (3 ng/ml) after 6 consecutive days of dosing. To determine if JNJ-2482272 is an autoinducer in rats, plated rat hepatocytes were treated with JNJ-2482272 for 2 days. The major hydroxylated metabolites of JNJ-2482272 were isolated and characterized by mass spectrometry and NMR analyses. Compared with the vehicle-treated cells, a concentration-dependent increase was observed in the formation of phase I- and II-mediated metabolites coinciding with greater expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in rat hepatocytes. CYP1A1, CYP1A2, CYP1B1, and UGT1A6 transcripts were predominantly induced, suggesting that JNJ-2482272 is an activator of the aryl hydrocarbon receptor (AhR). In a human AhR reporter assay, JNJ-2482272 demonstrated potent AhR activation with an EC50 value of 0.768 nM, a potency more comparable to the strong AhR activator and toxin 2,3,7,8-tetrachloro-dibenzodioxin than to weaker AhR activators 3-methylcholanthrene, ß-naphthoflavone, and omeprazole. In plated human hepatocytes, JNJ-2482272 induced CYP1A1 gene expression with an EC50 of 20.4 nM and increased CYP1A activity >50-fold from basal levels. In human recombinant P450s, JNJ-2482272 was exclusively metabolized by the CYP1 family of enzymes and most rapidly by CYP1A1. The summation of these in vitro findings bridges the in vivo conclusion that JNJ-2482272 is a strong autoinducer in rats and potentially in humans through potent AhR activation. SIGNIFICANCE STATEMENT: Drugs that induce their own metabolism (autoinducers) can lack sustained exposures for pharmacology and safety assessment hindering their development. JNJ-2482272 is demonstrated herein as a strong aryl hydrocarbon receptor (AhR) activator and CYP1A autoinducer, explaining its near complete loss of exposure after repeat administration in rat, which is likely translatable to human (if progressed further) considering its nanomolar potency comparable to "classical" AhR ligands like 2,3,7,8-tetrachloro-dibenzo-dioxin despite bearing a "nonclassical" drug structure.
Asunto(s)
Citocromo P-450 CYP1A1 , Receptores de Hidrocarburo de Aril , Aminas , Animales , Citocromo P-450 CYP1A1/metabolismo , Humanos , Pirimidinas/farmacología , Ratas , Receptores de Hidrocarburo de Aril/metabolismo , Tiazoles/farmacologíaRESUMEN
BACKGROUND: Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress-induced pathology. OBJECTIVE: We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. METHODS: Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. RESULTS: Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-ß-induced ASM cell proliferation and CXCL8 release. CONCLUSIONS: Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.
Asunto(s)
Antioxidantes/farmacología , Mitocondrias/metabolismo , Músculo Liso/metabolismo , Compuestos Organofosforados/farmacología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sistema Respiratorio/metabolismo , Ubiquinona/análogos & derivados , Adulto , Anciano , Remodelación de las Vías Aéreas (Respiratorias)/genética , Animales , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/tratamiento farmacológico , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/patología , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Estrés Oxidativo/efectos de los fármacos , Ozono , Neumonía/inducido químicamente , Neumonía/tratamiento farmacológico , Neumonía/genética , Neumonía/patología , Enfermedad Pulmonar Obstructiva Crónica/inducido químicamente , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/patología , Transducción de Señal , Fumar/metabolismo , Fumar/fisiopatología , Ubiquinona/farmacologíaRESUMEN
The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.
Asunto(s)
Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Esteroles/farmacología , Células Th17/citología , Animales , Diferenciación Celular , Colestanotriol 26-Monooxigenasa/metabolismo , Interleucina-17/biosíntesis , Ligandos , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Esteroles/metabolismoRESUMEN
To demonstrate the benefits of RNA-Seq over microarray in transcriptome profiling, both RNA-Seq and microarray analyses were performed on RNA samples from a human T cell activation experiment. In contrast to other reports, our analyses focused on the difference, rather than similarity, between RNA-Seq and microarray technologies in transcriptome profiling. A comparison of data sets derived from RNA-Seq and Affymetrix platforms using the same set of samples showed a high correlation between gene expression profiles generated by the two platforms. However, it also demonstrated that RNA-Seq was superior in detecting low abundance transcripts, differentiating biologically critical isoforms, and allowing the identification of genetic variants. RNA-Seq also demonstrated a broader dynamic range than microarray, which allowed for the detection of more differentially expressed genes with higher fold-change. Analysis of the two datasets also showed the benefit derived from avoidance of technical issues inherent to microarray probe performance such as cross-hybridization, non-specific hybridization and limited detection range of individual probes. Because RNA-Seq does not rely on a pre-designed complement sequence detection probe, it is devoid of issues associated with probe redundancy and annotation, which simplified interpretation of the data. Despite the superior benefits of RNA-Seq, microarrays are still the more common choice of researchers when conducting transcriptional profiling experiments. This is likely because RNA-Seq sequencing technology is new to most researchers, more expensive than microarray, data storage is more challenging and analysis is more complex. We expect that once these barriers are overcome, the RNA-Seq platform will become the predominant tool for transcriptome analysis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Perfilación de la Expresión Génica , Transcriptoma , Análisis de Varianza , Secuencia de Bases , Células Cultivadas , Humanos , Activación de Linfocitos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: MicroRNAs (MiRNAs) are short non-coding RNAs that control protein expression through various mechanisms. Their altered expression has been shown to be associated with various cancers. The aim of this study was to profile miRNA expression in colorectal cancer (CRC) and to analyze the function of specific miRNAs in CRC cells. MirVana miRNA Bioarrays were used to determine the miRNA expression profile in eight CRC cell line models, 45 human CRC samples of different stages, and four matched normal colon tissue samples. SW620 CRC cells were stably transduced with miR-143 or miR-145 expression vectors and analyzed in vitro for cell proliferation, cell differentiation and anchorage-independent growth. Signalling pathways associated with differentially expressed miRNAs were identified using a gene set enrichment analysis. RESULTS: The expression analysis of clinical CRC samples identified 37 miRNAs that were differentially expressed between CRC and normal tissue. Furthermore, several of these miRNAs were associated with CRC tumor progression including loss of miR-133a and gain of miR-224. We identified 11 common miRNAs that were differentially expressed between normal colon and CRC in both the cell line models and clinical samples. In vitro functional studies indicated that miR-143 and miR-145 appear to function in opposing manners to either inhibit or augment cell proliferation in a metastatic CRC model. The pathways targeted by miR-143 and miR-145 showed no significant overlap. Furthermore, gene expression analysis of metastatic versus non-metastatic isogenic cell lines indicated that miR-145 targets involved in cell cycle and neuregulin pathways were significantly down-regulated in the metastatic context. CONCLUSION: MiRNAs showing altered expression at different stages of CRC could be targets for CRC therapies and be further developed as potential diagnostic and prognostic analytes. The identified biological processes and signalling pathways collectively targeted by co-expressed miRNAs in CRC provide a basis for understanding the functional role of miRNAs in cancer.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/fisiopatología , Perfilación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Metástasis de la NeoplasiaRESUMEN
Functional alterations in striatal projection neurons play a critical role in the development of motor symptoms in Parkinson's disease (PD), but their molecular adaptation to dopamine depletion remains poorly understood. In particular, type and extent of regulation in postsynaptic signal transduction pathways that determine the responsiveness of striatal projection neurons to incoming stimuli, are currently unknown. Using cell-type-specific transcriptome analyses in a rodent model of chronic dopamine depletion, we identified large-scale gene expression changes, including neurotransmitter receptors, signal transduction cascades, and target proteins of dopamine signaling in striatonigral and striatopallidal neurons. Within the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) cascade of enzymes that plays a central role in signal integration of dopaminoceptive neurons multiple catalytic and regulatory subunits change their mRNA expression levels. In addition to the number of genes the fact that the alterations occur at multiple levels stresses the biological relevance of transcriptional regulation for adaptations of postsynaptic signaling pathways. The overall pattern of changes in both striatonigral and striatopallidal neurons is compatible with homeostatic mechanisms. In accordance with the distinct biological effects of dopamine D(1) and D(2) receptor stimulation, the alterations of the transcriptional profiles most likely result in prodopaminergic phosphorylation patterns. Our data provide insight into the disease-related plasticity of functional genomic networks in vivo that might contribute to the protracted preclinical phase of PD. In addition, the data have potential implications for the symptomatic treatment of the disease.
Asunto(s)
Cuerpo Estriado/citología , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Dopamina/deficiencia , Regulación de la Expresión Génica/fisiología , Neuronas/fisiología , Transducción de Señal/fisiología , Sustancia Negra/citología , Adrenérgicos/toxicidad , Análisis de Varianza , Animales , Cuerpo Estriado/efectos de los fármacos , Dopaminérgicos/farmacología , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Levodopa/farmacología , Masculino , Análisis por Micromatrices , Microdisección/métodos , Vías Nerviosas/lesiones , Neuronas/efectos de los fármacos , Oxidopamina/toxicidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada/metabolismoRESUMEN
Motor symptoms in Parkinson's disease (PD) are associated with complex changes of firing properties in basal ganglia output neurons (BGON). The abnormalities are generally attributed to altered synaptic input and potential post-synaptic mechanisms are currently unknown. Our cell-type selective transcriptome analyses of BGON in the rat 6-hydroxydopamine (6-OHDA) model of PD identified the ion channel HCN3 as a likely contributor to altered neuronal excitability. Quantitative PCR experiments confirmed the HCN3 upregulation in the rat and mouse 6-OHDA models and also demonstrated selectivity of the effect for HCN3. In accordance with the mRNA expression data, in vitro whole cell patch-clamp recordings in BGON showed increased HCN3 current amplitudes and increased rebound excitability in BGON of 6-OHDA treated rats. These data establish HCN3 up-regulation as a novel candidate mechanism that might contribute to the in vivo changes of electrical activity in basal ganglia output neurons of the parkinsonian brain.
Asunto(s)
Ganglios Basales/fisiopatología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Dopamina/deficiencia , Neuronas/fisiología , Trastornos Parkinsonianos/fisiopatología , Canales de Potasio/metabolismo , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/antagonistas & inhibidores , Modelos Animales de Enfermedad , Expresión Génica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Oxidopamina , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
Manufacturing cell line development involves transfection of therapeutic antibody genes into host cell lines and isolation of primary transfectomas that upon subcloning yield high expressing cell lines secreting the desired antibody. In an attempt to increase productivity of these cell lines, we set out to identify cellular genes whose expression level may affect antibody productivity. For this purpose, three different sets of mouse myeloma production cell lines expressing variable levels of three different therapeutic antibodies were subjected to microarray analysis using Murine GeneChip MG_U74Av2 arrays. A total of 456 genes were identified showing significant differential expression between at least one high expresser versus the control or its corresponding low expresser. Among these, 161 genes were common among at least one set of cell lines, and 26 genes were common among two or more sets of cell lines. Functional classification revealed that a majority of these genes have biological process function related to cell metabolism and cell growth. A subset of the 26 genes that were identified as commonly regulated among any two or all three sets of cell lines were selected (by several criteria) for quantitative PCR confirmation of the microarray methodology. The expression level of two genes, Secretory Leukocyte Protease Inhibitor (SLPI) and Cell Division Cycle-6 (Cdc6), correlated with antibody productivity in at least two sets of cell lines, suggesting that they can potentially be utilized as targets for engineering a superior transfection host cell line. Additionally, these genes may be used for screening murine myeloma production cell lines for superior productivity.
Asunto(s)
Anticuerpos/química , Biotecnología/métodos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma , Mieloma Múltiple/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Ratones , Mieloma Múltiple/terapia , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción GenéticaRESUMEN
Toxicogenomics technology defines toxicity gene expression signatures for early predictions and hypotheses generation for mechanistic studies, which are important approaches for evaluating toxicity of drug candidate compounds. A large gene expression database built using cDNA microarrays and liver samples treated with over one hundred paradigm compounds was mined to determine gene expression signatures for nongenotoxic carcinogens (NGTCs). Data were obtained from male rats treated for 24 h. Training/testing sets of 24 NGTCs and 28 noncarcinogens were used to select genes. A semiexhaustive, nonredundant gene selection algorithm yielded six genes (nuclear transport factor 2, NUTF2; progesterone receptor membrane component 1, Pgrmc1; liver uridine diphosphate glucuronyltransferase, phenobarbital-inducible form, UDPGTr2; metallothionein 1A, MT1A; suppressor of lin-12 homolog, Sel1h; and methionine adenosyltransferase 1, alpha, Mat1a), which identified NGTCs with 88.5% prediction accuracy estimated by cross-validation. This six genes signature set also predicted NGTCs with 84% accuracy when samples were hybridized to commercially available CodeLink oligo-based microarrays. To unveil molecular mechanisms of nongenotoxic carcinogenesis, 125 differentially expressed genes (P<0.01) were selected by Student's t-test. These genes appear biologically relevant, of 71 well-annotated genes from these 125 genes, 62 were overrepresented in five biochemical pathway networks (most linked to cancer), and all of these networks were linked by one gene, c-myc. Gene expression profiling at early time points accurately predicts NGTC potential of compounds, and the same data can be mined effectively for other toxicity signatures. Predictive genes confirm prior work and suggest pathways critical for early stages of carcinogenesis.
Asunto(s)
Carcinógenos/toxicidad , Transformación Celular Neoplásica/inducido químicamente , Perfilación de la Expresión Génica , Genes Relacionados con las Neoplasias/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Animales , Transformación Celular Neoplásica/genética , Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Neoplasias Hepáticas Experimentales/genética , Masculino , Pruebas de Mutagenicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , ToxicogenéticaRESUMEN
Macrophage activators (MA), peroxisome proliferators (PP), and oxidative stressors/reactive metabolites (OS/RM) all produce oxidative stress and hepatotoxicity in rats. However, these three classes of hepatotoxicants give three distinct gene transcriptional profiles on cDNA microarrays, an indication that rat hepatocytes respond/adapt quite differently to these three classes of oxidative stressors. The differential gene responses largely reflect differential activation of transcription factors: MA activate Stat-3 and NFkB, PP activate PPARa, and OS/RM activate Nrf2. We have used gene signature profiles for each of these three classes of hepatotoxicants to categorize over 100 paradigm (and 50+ in-house proprietary) compounds as to their oxidative stress potential in rat liver. In addition to a role for microarrays in predictive toxicology, analyses of small subsets of these signature profiles, genes within a specific pathway, or even single genes often provide important insights into possible mechanisms involved in the toxicities of these compounds.
Asunto(s)
Genómica , Hígado/efectos de los fármacos , Estrés Oxidativo , Toxicología , Animales , Perfilación de la Expresión Génica , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
Formation of free radicals and other reactive molecules is responsible for the adverse effects produced by a number of hepatotoxic compounds. cDNA microarray technology was used to compare transcriptional profiles elicited by training and testing sets of 15 oxidant stressors/reactive metabolite treatments to those produced by approximately 85 other paradigm compounds (mostly hepatotoxicants) to determine a shared signature profile for oxidant stress-associated hepatotoxicity. Initially, 100 genes were chosen that responded significantly different to oxidant stressors/reactive metabolites (OS/RM) compared to other samples in the database, then a 25-gene subset was selected by multivariate analysis. Many of the selected genes (e.g., aflatoxin aldehyde reductase, diaphorase, epoxide hydrolase, heme oxgenase and several glutathione transferases) are well-characterized oxidant stress/Nrf-2-responsive genes. Less than 10 other compounds co-cluster with our training and testing set compounds and these are known to generate OS/RMs as part of their mechanisms of toxicity. Using OS/RM signature gene sets, compounds previously associated with macrophage activation formed a distinct cluster separate from OS/RM and other compounds. A 69-gene set was chosen to maximally separate compounds in control, macrophage activator, peroxisome proliferator and OS/RM classes. The ease with which these 'oxidative stressor' classes can be separated indicates a role for microarray technology in early prediction and classification of hepatotoxicants. The ability to rapidly screen the oxidant stress potential of compounds may aid in avoidance of some idiosyncratic drug reactions as well as overtly toxic compounds.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Perfilación de la Expresión Génica , Hígado/fisiología , Factores Activadores de Macrófagos/metabolismo , Estrés Oxidativo/genética , Proliferadores de Peroxisomas/metabolismo , Transactivadores/biosíntesis , Animales , Proteínas de Unión al ADN/genética , Factores Activadores de Macrófagos/genética , Factor 2 Relacionado con NF-E2 , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-Dawley , Transactivadores/genéticaRESUMEN
Macrophage activation contributes to adverse effects produced by a number of hepatotoxic compounds. Transcriptional profiles elicited by two macrophage activators, LPS and zymosan A, were compared to those produced by 100 paradigm compounds (mostly hepatotoxicants) using cDNA microarrays. Several hepatotoxicants previously reported to activate liver macrophages produced transcriptional responses similar to LPS and zymosan, and these were used to construct a gene signature profile for macrophage activators in the liver. Measurement of cytokine mRNAs in the same liver samples by RT-PCR independently confirmed that these compounds are associated with macrophage activation. In addition to expected effects on acute phase proteins and metabolic pathways that are regulated by LPS and inflammation, a strong induction was observed for many endoplasmic reticulum-associated stress/chaperone proteins. Additionally, many genes in our macrophage activator signature profile were well-characterized PPARalpha-induced genes which were repressed by macrophage activators. A shared gene signature profile for peroxisome proliferators was determined using a training set of clofibrate, WY 14643, diethylhexylphthalate, diisononylphthalate, perfluorodecanoic acid, perfluoroheptanoic acid, and perfluorooctanoic acid. The signature profile included macrophage activator-induced genes that were repressed by peroxisome proliferators. NSAIDs comprised an interesting pharmacological class in that some compounds, notably diflunisal, co-clustered with peroxisome proliferators whereas several others co-clustered with macrophage activators, possibly due to endotoxin exposure secondary to their adverse effects on the gastrointestinal system. While much of these data confirmed findings from the literature, the transcriptional patterns detected using this toxicogenomics approach showed relationships between genes and biological pathways requiring complex analysis to be discerned.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Proliferadores de Peroxisomas/farmacología , Animales , Citocinas/genética , Expresión Génica , Perfilación de la Expresión Génica , Hígado/citología , Hígado/efectos de los fármacos , Activación de Macrófagos , Macrófagos/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Generating gene-expression profiles from laser-captured cells requires the successful combination of laser-capture microdissection, RNA extraction, RNA amplification, and microarray analysis. To permit single-cell gene-expression profiling, the RNA amplification method has to be sufficiently powerful to bridge the gap between the amount of RNA available from a single cell to what is required by the microarray, a gap that spans 5 to 6 orders of magnitude. This chapter focuses on the amplification of RNA using a two-round T7 RNA amplification method. The protocols described are adapted for laser-captured material and have been used to generate gene expression profiles from single laser-captured cells.
Asunto(s)
Rayos Láser , Microdisección/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/genética , Escherichia coli/genética , Perfilación de la Expresión Génica/métodos , Microdisección/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Laser capture microdissection in combination with microarrays allows for the expression analysis of thousands of genes in selected cells. Here we describe single-cell gene expression profiling of CA1 neurons in the rat hippocampus using a combination of laser capture, T7 RNA amplification, and cDNA microarray analysis. Subsequent cluster analysis of the microarray data identified two different cell types: pyramidal neurons and an interneuron. Cluster analysis also revealed differences among the pyramidal neurons, indicating that even a single cell type in vivo is not a homogeneous population of cells at the gene expression level. Microarray data were confirmed by quantitative RT-PCR and in situ hybridization. We also report on the reproducibility and sensitivity of this combination of methods. Single-cell gene expression profiling offers a powerful tool to tackle the complexity of the mammalian brain.
Asunto(s)
Hipocampo/metabolismo , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Bacteriófago T7/genética , Recuento de Células , Análisis por Conglomerados , Femenino , Hipocampo/citología , Hibridación in Situ , Interneuronas/química , Interneuronas/metabolismo , Rayos Láser , Neuronas/química , Células Piramidales/química , Células Piramidales/metabolismo , ARN sin Sentido/análisis , ARN sin Sentido/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
DNA microarrays provide a global view of the physiological state of the cell by parallel analysis of the expression levels of all the genes in an organism. The effects of four bactericidal agents on the expression pattern of Escherichia coli MG1655 were assessed. Compounds were chosen on the basis of their different mechanisms of action and included inhibitors of DNA replication and recombination, translation, transcription and cell wall biosynthesis. The addition of rifampin resulted in increased expression of the target, rpoB, as well as several genes involved in nucleotide salvage and purine biosynthesis. The addition of ampicillin resulted in overall changes in gene expression that showed some similarity to changes induced by rifampin. The addition of the antibiotics kanamycin or norfloxacin resulted in the induction of unique gene expression signatures: a heat shock response to kanamycin and an SOS response to norfloxacin. Several genes of unknown function showed expression profiles similar to the genes associated with the SOS or the heat shock response. Thus, these profiles define families of genes with similar expression phenotypes that can be tested for related function.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Expresión Génica/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ampicilina/farmacología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana , Norfloxacino/farmacología , Rifampin/farmacología , Transcripción GenéticaRESUMEN
Erythropoietin (EPO) is the primary modulator of red blood cell production. Recently EPO has received considerable attention for its functions outside of hematopoiesis, including its effects in the nervous system where it has been shown to act as a neuroprotectant. To understand the function of EPO in the nervous system and to determine if EPO functions through the same signaling pathways identified in hematopoietic cells, we used cDNA array hybridization and RT-PCR to investigate the changes in gene expression induced by EPO in the neuronal-like PC-12 cell line. PC-12 cells cultured in the presence of EPO (10 U/ml) showed significant changes in gene expression by 3 h with a return to basal expression levels for the vast majority of genes by 24 h. The genes influenced by EPO included genes with known functions in cell proliferation, differentiation and apoptosis. Semi-quantitative RT-PCR confirmed that 24 h pre-treatment with EPO (10 pM) resulted in a 2.5-fold increase in the expression of the anti-apoptotic gene bcl(XL) and a 4-fold decrease in the expression of the pro-apoptotic gene bak. In addition to supporting the current models of EPO function these results suggest previously unidentified mechanisms by which EPO may function in neurons.
Asunto(s)
Diferenciación Celular/genética , División Celular/genética , Supervivencia Celular/genética , Eritropoyetina/metabolismo , Regulación de la Expresión Génica/fisiología , Neuronas/metabolismo , Transducción de Señal/genética , Animales , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eritropoyetina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/genética , Neuronas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína bcl-XRESUMEN
Information on the neuroanatomical expression of a given gene is critical to understanding its function in the central nervous system. The integration of laser capture microdissection (LCM), T7-based RNA amplification and cDNA microarrays allows for this information to be simultaneously generated for thousands of genes. To validate this integrative approach, we catalogued the gene expression profiles of seven rat brain nuclei or subnuclei. A hundred cells from the following seven brain nuclei were analyzed: locus coeruleus (LC), dorsal raphe nucleus (DR), parvocellular division (PA) and magnocellular division (MG) of the hypothalamic paraventricular nucleus (PVN) and CA1, CA3 and dentate gyrus (DG) divisions of the hippocampal formation. Of the 2145 genes investigated, 1402 genes (65%) gave a hybridization signal statistically different from the background level that was defined by non-specific hybridizations to 15 different plant genes. Validation of our microarray data on four arbitrarily selected genes was confirmed by Real-Time PCR. Previous research showing expression patterns of 'signature' genes (n=17) for specific brain nuclei are consistent with our findings. For example, as previously shown, enriched mRNA expression encoding the serotonin transporter or tyrosine hydroxylase was found in DR and LC cells, respectively. Interestingly, expression of the serotonin 5-HT(2B) receptor mRNA was also found in DR cells. We confirmed this new finding by in-situ hybridization. The hierarchical clustering analysis of gene expression shows that the two divisions of the PVN (PA and MG) are closely related to each other, as well as the three regions of the hippocampal formation (CA1, CA3 and DG), which also showed similar gene expression profiles. This study demonstrates the importance, feasibility and utility of cellular brain nuclei profiling.
Asunto(s)
Química Encefálica/genética , Hipocampo/metabolismo , Mesencéfalo/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Animales , Análisis por Conglomerados , Expresión Génica/fisiología , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Marcadores Genéticos , Hipocampo/química , Hibridación in Situ , Masculino , Mesencéfalo/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Núcleo Hipotalámico Paraventricular/química , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT2B , Receptores de Serotonina/biosíntesis , Receptores de Serotonina/genéticaRESUMEN
Many aspects of physiology and behavior are temporally organized into daily 24 hr rhythms, driven by an endogenous circadian clock. Studies in eukaryotes have identified a network of interacting genes forming interlocked autoregulatory feedback loops which underlie overt circadian organization in single cells. While in mammals the master oscillator resides in the suprachiasmatic nuclei of the hypothalamus, semiautonomous circadian oscillators also exist in peripheral tissues and in immortalized fibroblasts, where rhythmicity is induced following a serum shock. We used this model system in combination with high-density cDNA microarrays to examine the magnitude and quality of clock control of gene expression in mammalian cells. Supported by application of novel bioinformatics tools, we find approximately 2% of genes, including expected canonical clock genes, to show consistent rhythmic circadian expression across five independent experiments. Rhythmicity in most of these genes is novel, and they fall into diverse functional groups, highlighted by a predominance of transcription factors, ubiquitin-associated factors, proteasome components, and Ras/MAPK signaling pathway components. When grouped according to phase, 68% of the genes were found to peak during estimated subjective day, 32% during estimated subjective night, with a tendency to peak at a phase corresponding to anticipation of dawn or dusk.