RESUMEN
Bromodomain-containing proteins are vital for controlling the expression of many pro-inflammatory genes. Consequently, compounds capable of inhibiting specific bromodomain-facilitated protein-protein interactions would be predicted to alleviate inflammation, making them valuable agents in the treatment of diseases caused by dysregulated inflammation, such as age-related macular degeneration. Here, we assessed the ability of known inhibitors JQ-1, PFI-1, and IBET-151 to protect from the inflammation and cell death caused by etoposide exposure in the human retinal pigment epithelial cell line, ARPE-19. The potential anti-inflammatory effects of the bromodomain inhibitors were assessed by ELISA (enzyme-linked immunosorbent assay) profiling. The involvement of NF-κB and SIRT1 in inflammatory signaling was monitored by ELISA and western blotting. Furthermore, SIRT1 was knocked down using a specific siRNA or inhibited by EX-527 to elucidate its role in the inflammatory reaction. The bromodomain inhibitors effectively decreased etoposide-induced release of IL-6 and IL-8. This anti-inflammatory effect was not related to SIRT1 activity, although all bromodomain inhibitors decreased the extent of acetylation of p53 at the SIRT1 deacetylation site. Overall, since bromodomain inhibitors display anti-inflammatory properties in human retinal pigment epithelial cells, these compounds may represent a new way of alleviating the inflammation underlying the onset of age-related macular degeneration.
Asunto(s)
Antiinflamatorios/farmacología , Compuestos de Azabiciclo/farmacología , Azepinas/farmacología , Benzodiazepinas/farmacología , Células Epiteliales/metabolismo , Etopósido/antagonistas & inhibidores , Piridinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Carbazoles/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Etopósido/farmacología , Regulación de la Expresión Génica , Humanos , Inflamación , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Interleucina-8/metabolismo , Modelos Biológicos , FN-kappa B/genética , FN-kappa B/metabolismo , Dominios Proteicos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , Sirtuina 1/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The effects of maternal cigarette smoking on the transcriptome of human full-term placentas were investigated by a microarray analysis. QPCR was performed for a selected set of metabolizing genes. Differentially expressed genes were selected by fold change (+/-1.5-fold) and analysis of variance (P<0.05) between the control and smoker groups. The expression of 174 probe sets was affected significantly. Chronic cigarette smoking induced the expression of CYP1A1. A trend toward a decrease in the expression of several steroid hormone-metabolizing enzymes, including CYP19A1, was detected. The expression of phase II enzymes was not altered, and no enriched categories were observed among the regulated genes, except for aryl hydrocarbon receptor (AhR)-CYP1A1. The unaltered expression of phase II enzymes may result in an increase in the levels of active metabolites and elevated oxidative chemical stress in the placenta and the fetus. On the basis of our results, it seems that cigarette smoke acts as a hormone disrupter in the placenta.
Asunto(s)
Aromatasa/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Análisis por Micromatrices , Placenta/metabolismo , Complicaciones del Embarazo/metabolismo , Fumar/metabolismo , Adolescente , Adulto , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1 , Regulación hacia Abajo , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Embarazo , Complicaciones del Embarazo/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Artemisinin (a sesquiterpene lactone endoperoxide) has become important in multi-drug treatment of malaria. There is evidence that artemisinin induces drug metabolism which could result in drug-drug interactions. The objective of this study was to characterize the inductive properties of artemisinin on drug-metabolizing cytochrome P450 (CYP450) enzymes. The possibility of artemisinin to induce CYP450 was studied in artemisinin-treated (orally for four days) and vehicle-treated rats using reverse transcriptase polymerase chain reaction (RT-PCR). The effect on enzymatic activities in mouse microsomes from multiple artemisinin administration (intraperitonally) to mice were also studied as well as the effect on the expression in mouse primary hepatocytes and HEK293 cells. Increased CYP2B1 mRNA levels in rats could be seen after artemisinin treatment as well as a weak but reproducible increase in the intensity of CYP1A2. Administration of artemisinin to mice up-regulated hepatic CYP2B10-dependent, and to a lesser extent, CYP2A5-dependent enzyme activities. In primary hepatocyte culture, artemisinin significantly increased the CYP2B10 mRNA levels whereas the CYP2A5 mRNA levels were increased to a lesser extent. No significant changes were seen in the levels of other CYP enzymes. Artemisinin was an activator of constitutive androstane receptor (CAR) but not pregnane X receptor (PXR) in HEK293 cells. The results demonstrate that the drug exerts its effects on drug metabolism via the CAR receptor that results in up-regulation of genes such as the Cyp2b. The weaker up-regulation of CYP2A5 might also be CAR-dependent or alternatively, a consequence of artemisinin toxicity. The results of this study are of importance when predicting potential drug-drug interactions in multi-drug therapies with artemisinin.
Asunto(s)
Antiinfecciosos/administración & dosificación , Artemisininas/administración & dosificación , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Sesquiterpenos/administración & dosificación , Factores de Transcripción/efectos de los fármacos , Administración Oral , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Células Cultivadas , Receptor de Androstano Constitutivo , Citocromo P-450 CYP1A2/efectos de los fármacos , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP2B1/efectos de los fármacos , Familia 2 del Citocromo P450 , Interacciones Farmacológicas , Inducción Enzimática/efectos de los fármacos , Hepatocitos , Masculino , Ratones , Oxigenasas de Función Mixta/metabolismo , Receptor X de Pregnano , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de Esteroides/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
PURPOSE: To develop and characterize a new drug-regulated gene expression system based on the nuclear receptor constitutive androstane receptor (CAR). METHODS: Both transient and stable transfection into HEK293 cells of luciferase plasmids under the control of either drug- and steroid-responsive nuclear receptor CAR or the tetracycline-sensitive transactivator tTA were used in development of stable cell lines. RESULTS: A stable first-generation cell line that expresses luciferase gene under the control of nuclear receptor CAR was developed. The luciferase expression in CAR-producing cells could be suppressed by androstanes and reactivated by structurally unrelated drugs chlorpromazine, metyrapone, phenobarbital, and clotrimazole. The kinetics of luciferase expression in CAR-producing cells and the tTA system were comparable. The overall regulation of CAR system was improved by modifications to the DNA binding domain and site. CONCLUSIONS: Because of its wide ligand selectivity and transferable ligand binding domain, CAR expands the repertoire of regulated gene expression systems.
Asunto(s)
Biotecnología/métodos , Regulación de la Expresión Génica , Genes Reporteros , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Androstenoles/farmacología , Sitios de Unión , Células Cultivadas , Receptor de Androstano Constitutivo , ADN/efectos de los fármacos , ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Humanos , Ligandos , Luciferasas/genética , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.
Asunto(s)
Técnicas de Transferencia de Gen , Glicosaminoglicanos/fisiología , Liposomas/química , Adyuvantes Inmunológicos/farmacología , Medios de Contraste/farmacología , ADN/metabolismo , ADN/farmacocinética , Citometría de Flujo , Fluoresceína/farmacología , Genes Reporteros , Terapia Genética/métodos , Proteínas Fluorescentes Verdes , Heparitina Sulfato/farmacología , Ácido Hialurónico/farmacología , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/farmacocinética , Microscopía Confocal , Plásmidos/metabolismo , Unión Proteica , TransfecciónRESUMEN
Recent findings indicate that induction of drug metabolism is regulated by activation of specific members of the nuclear receptor gene family. This minireview deals with the mechanisms by which phenobarbital and phenobarbital-type chemicals induce cytochrome P450 and other genes, and summarises the knowledge on the role of the constitutively active receptor CAR in the induction process. The potential implications of CAR-mediated induction for drug research and possible uses of CAR are also discussed.
Asunto(s)
Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Fenobarbital/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos de Respuesta/fisiología , Transactivadores/metabolismo , Factores de Transcripción , Animales , Anticonvulsivantes/farmacología , Biofarmacia , Química Farmacéutica , Receptor de Androstano Constitutivo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Humanos , Oxidorreductasas N-Desmetilantes/efectos de los fármacos , Oxidorreductasas N-Desmetilantes/metabolismo , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacosRESUMEN
Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA. To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared. The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer. Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene. Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method. Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol). Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL. In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1). The polymeric systems were ineffective. The effect of the complexation medium was further studied on CV-1 cells. Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM). Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size. Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes. In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides.
Asunto(s)
Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Compuestos Organotiofosforados/administración & dosificación , Línea Celular , Portadores de Fármacos , Humanos , Lípidos/química , Liposomas , Luciferasas/genética , Luciferasas/metabolismo , Oligonucleótidos Antisentido/síntesis química , Compuestos Organotiofosforados/química , Compuestos Organotiofosforados/farmacocinética , Tamaño de la Partícula , Transfección/genética , Transfección/métodosRESUMEN
Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Dimerización , Retroalimentación , Humanos , Isoenzimas/genética , Ligandos , Receptor Cross-Talk , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Elementos de Respuesta/genética , Procesos de Determinación del SexoRESUMEN
Eighteen peptide-oligonucleotide phosphorothioate conjugates were prepared in good yield and thoroughly characterized with electrospray ionization mass spectra. When applied to the living cells, conjugates exhibiting membrane translocation and nuclear localization properties displayed efficient intracellular penetration but failed to show any serious antisense effect. Studies on the intracellular distribution of the fluorescein-labeled conjugates revealed their trapping in endosomes.
Asunto(s)
Núcleo Celular/química , Oligonucleótidos/química , Oligopéptidos/química , Compuestos Organotiofosforados/química , Membrana Celular/química , Reactivos de Enlaces Cruzados , Disulfuros/química , Electroforesis en Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato , Expresión Génica/genética , Hidrólisis , Luciferasas/biosíntesis , Luciferasas/genética , Espectrometría de Masas , Microscopía Confocal , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/química , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Compuestos Organotiofosforados/síntesis química , Compuestos Organotiofosforados/metabolismo , Ribonucleasa HRESUMEN
The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between -1733 and -1683 bp in the gene's 5'-flanking region. An in vitro translated CAR acting as a retinoid X receptor alpha heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the human CYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Oxidorreductasas N-Desmetilantes/genética , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Transactivadores/genética , Factores de Transcripción , Secuencia de Bases , Receptor de Androstano Constitutivo , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Esteroide Hidroxilasas , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Receptor de Androstano Constitutivo , Familia 2 del Citocromo P450 , Dimerización , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fenobarbital/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Receptores X Retinoide , Factores de Tiempo , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
By extending previous studies of the phenobarbital (PB)-responsive 132-base pair (bp) enhancer sequence in the CYP2B10 gene, we have delimited a 51-bp enhancer element that is fully inducible by PB in mouse primary hepatocytes. Sixteen structurally unrelated phenobarbital-type inducers activated the 51-bp enhancer element in transient transfection assays. The results thus indicate that most PB-type inducers, if not all inducers, increase the transcription of the CYP2B10 gene by activating this 51-bp element, now designated PB-responsive enhancer module or PBREM.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Elementos de Facilitación Genéticos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Fenobarbital/farmacología , Esteroide Hidroxilasas , Xenobióticos/farmacología , Animales , Composición de Base , Alcanfor/farmacología , Familia 2 del Citocromo P450 , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Plaguicidas/farmacología , Bifenilos Policlorados/farmacología , Piridinas/farmacología , Solventes/farmacologíaRESUMEN
We cloned cDNAs which encode a mouse liver nuclear protein with an apparent molecular mass of 51 kDa, using sequences derived from a purified protein as the basis for designing specific primers. The deduced amino acid sequences revealed that the 51-kDa protein contains characteristic subdomain structures of a protein kinase. The bacterially expressed recombinant 51-kDa protein catalyzed phosphorylation of general substrates such as casein and was autophosphorylated at serine residue(s). This 51-kDa protein kinase, designated 51PK, is 40% identical to the 34-kDa protein kinase encoded by the vaccinia virus B1 gene and 25% identical to the casein kinase I isoforms, including yeast HRR25. The 51PK mRNA was expressed as two splice variants and the 51PK protein was exclusively localized in nuclei. Northern hybridization showed that 51PK mRNA was expressed in various tissues, with highest levels in testis, spleen, lung, and liver. These results, therefore, indicate that 51PK is a nuclear serine/threonine kinase and a novel distinct member of the protein kinase superfamily.
Asunto(s)
Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caseína Quinasas , Núcleo Celular/enzimología , Clonación Molecular , ADN Complementario/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Virus Vaccinia/enzimología , Virus Vaccinia/genética , Proteínas Virales/genéticaRESUMEN
Using a primary hepatocyte culture in which the mouse Cyp2b10 gene transcription is activated by phenobarbital (PB)-type inducers, we examined the cellular signalling mechanisms associated with PB induction. Low nanomolar concentrations of protein serine/threonine phosphatase inhibitors okadaic acid (OA) and calyculin A blocked the induction of CYP2B10 mRNA. Nuclear run-on assays indicated that OA suppressed Cyp2b10 gene transcription. Pretreatment of the cells with an inhibitor of Ca2+/calmodulin-dependent protein kinases ¿1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62)¿ or with a flavonoid, naringin, were completely or partly protective respectively against the OA-mediated suppression of CYP2B10 mRNA. Several other established modulators of protein kinase activities did not greatly affect the induction of CYP2B10 mRNA, nor could they prevent the suppressive effect of OA. Our results indicate that specific protein phosphorylation-dephosphorylation is required for the induction of Cyp2b10 gene expression, which is modulated through multiple endogenous and exogenous signals.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , Fenobarbital/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Esteroide Hidroxilasas , Transcripción Genética/efectos de los fármacos , Animales , Células Cultivadas , Familia 2 del Citocromo P450 , Ratones , Ratones Endogámicos C57BL , Ácido Ocadaico/farmacología , ARN Mensajero/metabolismoRESUMEN
This article reviews recent progress in characterizing cis-acting DNA elements of the phenobarbital-inducible CYP2B genes. Whereas proximal DNA elements such as the C/EBP binding site regulate basal transcription activity, phenobarbital-responsive enhancer activity is governed by the distal element (designated phenobarbital-responsive enhancer module, PBREM) residing about -2.3 kbp upstream from the transcription start site. Proximal elements are not required to enhance the phenobarbital-inducible transcription, since the PBREM can confer the inducibility to several heterologous promoters. Repression of the basal transcription by a negative element upstream of the -0.8 kbp region, however, may be necessary for the proper regulation of the CYP2B genes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Fenobarbital/farmacología , Secuencias Reguladoras de Ácidos Nucleicos , Bacillus megaterium/genética , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción EnzimáticaRESUMEN
Induction of drug- and carcinogen-metabolizing cytochrome P450s by xenobiotic chemicals is a common cellular defense mechanism, usually leading to increased detoxification of xenobiotics but sometimes, paradoxically, to formation of more toxic and carcinogenic metabolites. Phenobarbital (PB) is an archetypal representative for chemicals including industrial solvents, pesticides, plant products, and clinically used drugs that induce several genes within CYP subfamilies 2B, 2A, 2C, and 3A in rodents and humans. Although the transcription of these CYP genes is activated by PB, the associated molecular mechanisms have not yet been elucidated. Here we have analyzed, in detail, enhancer activity of a far upstream region of mouse Cyp2b10 gene and report a 132-base pair PB-responsive enhancer module (PBREM) with a 33-base pair core element containing binding sites for nuclear factor I- and nuclear receptor-like factors. Mutations of these binding sites abolish the ability of PBREM to respond to inducers in mouse primary hepatocytes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Proteínas Potenciadoras de Unión a CCAAT , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Elementos de Facilitación Genéticos/efectos de los fármacos , Hígado/enzimología , Fenobarbital/farmacología , Esteroide Hidroxilasas , Animales , Composición de Base , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Familia 2 del Citocromo P450 , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Inducción Enzimática , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFI , Proteínas Nucleares , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Recombinantes/biosíntesis , Factores de Transcripción/metabolismo , Transfección , Proteína 1 de Unión a la Caja YAsunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450 , Hígado/enzimología , Preparaciones Farmacéuticas/metabolismo , Esteroide Hidroxilasas , Animales , Citocromo P-450 CYP2A6 , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/fisiología , Inducción Enzimática , Epitelio/enzimología , Femenino , Predicción , Regulación de la Expresión Génica , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/metabolismo , Humanos , Pulmón/enzimología , Masculino , Oxigenasas de Función Mixta/metabolismo , Mutagénesis , Especificidad de Órganos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/fisiología , Especificidad por SustratoRESUMEN
The mouse phenobarbital (PB)-inducible Cyp2b10 gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405-base pair (bp) Cyp2bl0 promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (-123/-82 bp) into the middle of a consensus sequence to the so-called "Barbie box." In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10 promoter identified a basal transcription element at -64/-34 bp and a negative element at -971/-775 bp. Sequences contained within the -1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the -1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene 158, 263-268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10 gene are located in the distal promoter.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Fenobarbital/farmacología , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Secuencia de Consenso , Citocromo P-450 CYP2B1 , Cartilla de ADN , Inducción Enzimática , Intrones , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Piridinas/farmacología , Ratas , Proteínas Recombinantes/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , TATA Box , TransfecciónRESUMEN
The effects of daily cocaine administration for up to 14 days were studied in terms of hepatic morphology and the expression of cytochrome P450 (CYP) enzymes in the mouse liver. Daily intraperitoneal doses of 60 mg/kg of cocaine for 3 days induced severe hepatocellular necrosis in the pericentral zone and decreased activities of CYP1A2, CYP2A4/5, and CYP2Cx. The microsomal CYP2B10 protein content was increased by about 2.5-fold, but 2B10-dependent 7-pentoxyresorufin O-dealkylase (PROD) activity was barely detectable. Five or seven daily cocaine doses caused prominent pericentral inflammation and a significant (up to 14-fold) increase in the microsomal protein content and PROD activity. An increase in microsomal CYP3A was also evident, but CYP2A5 and CYP1A2 still remained at a low level. Immunohistochemical examination showed that the relative induction of CYP2B10 and CYP3A after treatment with cocaine was strongest in perivenous hepatocytes. Immunoinhibition experiments showed that CYP2B10 accounted for catalysis of only 15% to 20% of the enhanced microsomal cocaine N-demethylase (CNDM) activity, which correlated well with immunoreactive 3A protein, and could be blocked 70% to 90% by triacetyloleandomycin. After 10 or 14 daily doses of cocaine, regenerative changes with hepatocyte ballooning were observed, coinciding with increases in CYP1A2, CYP2A4/5, and CYP3A. These results suggest the following: (1) cocaine enhances its own cytochrome P450-dependent metabolism; (2) increased production of norcocaine in microsomes is catalyzed mainly by CYP3A enzyme(s), whereas 2B10, although markedly increased by cocaine treatment, has only a minor role in cocaine hepatotoxicity; and (3) despite increased microsomal CNDM activity, cocaine-induced liver injury is reversible in mice.