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1.
Mol Cancer Ther ; 18(12): 2207-2219, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31530649

RESUMEN

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.


Asunto(s)
Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Antineoplásicos/farmacología , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Femenino , Células HeLa , Humanos , Masculino
2.
Cancer Discov ; 9(2): 248-263, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30373917

RESUMEN

Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.


Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas de Unión a Retinoblastoma/genética , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Hepatology ; 46(5): 1597-610, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17668877

RESUMEN

UNLABELLED: Multidrug resistance-associated proteins (Mrps) are adenosine triphosphate-dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin-glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor-E2-related factor-2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte-specific glutamate-cysteine ligase catalytic subunit-null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild-type and Nrf2-null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2-dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert-butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [-185 base pairs (bp)], Mrp3 (-9919 bp), and Mrp4 (-3767 bp). CONCLUSION: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps.


Asunto(s)
Glutatión/metabolismo , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/fisiología , Región de Flanqueo 5' , Animales , Antioxidantes/farmacología , Hidroxianisol Butilado/farmacología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Hepatocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Factor 2 Relacionado con NF-E2/genética , Regiones Promotoras Genéticas , Pirazinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Tionas , Tiofenos
4.
Cell Stress Chaperones ; 11(4): 356-63, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17278884

RESUMEN

Bile duct ligation (BDL) causes hepatocellular oxidative stress and injury. The transcription factor nuclear factor-E2-related factor (Nrf2) induces expression of numerous genes including NAD(P)H:quinone oxidoreductase 1 (Nqo1) during periods of oxidative stress. Therefore, we hypothesized that BDL increases liver expression of mouse antioxidant genes in an Nrf2-dependent manner. BDL or sham surgeries were performed on male C57BL/6, Nrf2-null, and wild-type mice. Livers were collected at 1, 3, and 7 days after surgery for analysis of messenger ribonucleic acid (mRNA) levels of Nrf2-responsive genes as well as Nqo1 protein and activity. BDL increased mRNA expression of multiple Nrf2 genes in mouse liver, compared to sham-operated controls. Follow-up studies investigating protein expression, enzyme activity, and Nrf2 dependency were limited to Nqo1. Nqo1 protein expression and activity in mouse livers was increased 2- to 3-, and 4- to 5-fold at 3 and 7 days after BDL, respectively. Studies also showed that BDL increases Nqol mRNA, protein expression, and enzyme activity in livers from wild-type mice, but not in Nrf2-null mice. In conclusion, expression of Nrf2-dependent genes is increased during cholestasis. These studies also demonstrate that Nqo1 expression and activity in mouse liver are induced via an Nrf2-dependent mechanism.


Asunto(s)
Colestasis/enzimología , Hígado/metabolismo , NADPH Deshidrogenasa/metabolismo , NADP/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Alanina Transaminasa/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/cirugía , Colestasis/metabolismo , Glutatión Transferasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , NAD(P)H Deshidrogenasa (Quinona) , Factor 2 Relacionado con NF-E2/genética , ARN Mensajero/metabolismo
5.
Free Radic Biol Med ; 35(4): 351-67, 2003 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12899938

RESUMEN

Whereas ch/ch wild-type mice and ch/14CoS heterozygotes are viable, 14CoS/14CoS mice homozygous for a 3800 kb deletion on chromosome 7 die during the first day postpartum. Death is caused by disruption of the fumarylacetoacetate hydrolase (Fah) gene; absence of FAH, final enzyme in the tyrosine catabolism pathway, leads to accumulation of reactive electrophilic intermediates. In this study, we kept 14CoS/14CoS mice alive for 60 d with oral 2-(2-nitro-4-trifluoromethyl-benzyol)-1,3-cyclohexanedione (NTBC), an inhibitor of p-hydroxyphenylpyruvate dioxygenase, second enzyme in the tyrosine catabolic pathway. The 70% of NTBC-treated 14CoS/14CoS mice that survived 60 d showed poor growth and developed corneal opacities, compared with ch/14CoS littermates; NTBC-rescued Fah(-/-) knockout mice did not show growth retardation or ocular toxicity. NTBC-rescued 14CoS/14CoS mice also exhibited a striking oxidative stress response in liver and kidney, as measured by lower GSH levels and mRNA induction of four genes: glutamate cysteine ligase catalytic (Gclc) and modifier (Gclm) subunits, NAD(P)H:quinone oxidoreductase (Nqo1), and heme oxygenase-1 (Hmox1). Withdrawal of NTBC for 24-48 h from rescued adult 14CoS/14CoS mice resulted in severe apoptosis of the liver, detected histologically and by cytochrome c release from the mitochondria, increased caspase 3-like activity, and further decreases in GSH content. In kidney, proximal tubular epithelial cells were abnormal. Human hereditary tyrosinemia type I (HT1), caused by mutations in the FAH gene, is an autosomal recessive disorder in which the patient usually dies of liver fibrosis and cirrhosis during early childhood; NTBC treatment is known to prolong HT1 children's lives-although liver fibrosis, cirrhosis, hepatocarcinoma, and corneal opacities sometimes occur. The mouse data in the present study are consistent with the possibility that endogenous oxidative stress-induced apoptosis may be the underlying cause of liver pathology seen in NTBC-treated HT1 patients.


Asunto(s)
Apoptosis , Hepatocitos/metabolismo , Hepatocitos/patología , Estrés Oxidativo , Animales , Caspasa 3 , Caspasas/metabolismo , Ciclohexanonas/farmacología , Radicales Libres , Glutamato-Cisteína Ligasa/química , Glutatión/metabolismo , Heterocigoto , Humanos , Hidrolasas/genética , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica , Modelos Químicos , Nitrobenzoatos/farmacología , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Tiempo , Tirosina/metabolismo , Tirosinemias/metabolismo
6.
Biochem Pharmacol ; 63(9): 1739-54, 2002 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-12007577

RESUMEN

Glutamate-cysteine ligase is a heterodimer comprising a modifier (GCLM) and a catalytic (GCLC) subunit. In mouse Hepa-1c1c7 hepatoma cell cultures, we found that tert-butylhydroquinone (tBHQ; 50 microM) induces the GCLM and GCLC mRNAs approximately 10- and approximately 2-fold, respectively, and that these increases primarily reflect de novo transcription. We determined that the mouse Gclm gene has seven exons, spanning 22.3 kb; all exons, intron-exon junctions, and 4.7 kb of 5'-flanking region were sequenced. By RNase protection analysis, we identified two major and several minor transcription start-site clusters over a 300-bp region. The Gclm 5'-flanking region is GC-rich and lacks a canonical TATA box. Transient and stable transfection studies, using luciferase reporter constructs containing incremental Gclm 5'-flanking deletions (4.7-0.5 kb), showed high basal activity but only modest ( approximately 2-fold) inducibility by tBHQ. The only candidate motif for oxidative stress regulation (in the 4.7-kb region we sequenced) is a putative inverted electrophile response element (EPRE) 9 bp upstream from the 5'-most transcription start-site. Site-directed mutagenesis of this -9 EPRE demonstrated minimal (30-40%) decreases in tBHQ induction and no effect on basal activity-suggesting that this EPRE might be necessary but not sufficient. The nuclear erythroid factor-2 (NEF2)-related factor-2 (NRF2) is known to transactivate via EPRE motifs. In the presence of co-transfected NRF cDNA expression vector, however, no increase in Gclm promoter activity was observed. Thus, the endogenous Gclm gene shows robust transcriptional activation by tBHQ in the intact Hepa-1 cell, but reporter constructs containing up to 4.7 kb of promoter (having only the one EPRE at -9) demonstrate a disappointing response, indicating that the major tBHQ-responsive regulatory element of the mouse Gclm gene must exist either further 5'- or 3'-ward of the 4.7-kb region studied.


Asunto(s)
Antioxidantes/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , Hidroquinonas/farmacología , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Cadmio/farmacología , ADN/análisis , Genoma , Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Datos de Secuencia Molecular , Estrés Oxidativo , Regiones Promotoras Genéticas/efectos de los fármacos , Subunidades de Proteína , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , TATA Box , Sitio de Iniciación de la Transcripción , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Vitamina K 3/farmacología
7.
Toxicology ; 187(2-3): 217-28, 2003 May 03.
Artículo en Inglés | MEDLINE | ID: mdl-12699910

RESUMEN

Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression.


Asunto(s)
Adenocarcinoma/patología , Modelos Animales de Enfermedad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas/inmunología , Proteínas/metabolismo , Uteroglobina , Animales , Antígenos Transformadores de Poliomavirus/fisiología , Bronquios/inmunología , Bronquios/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Transformación Celular Viral/genética , Expresión Génica , Genotipo , Heterocigoto , Hiperplasia/patología , Inmunohistoquímica , Neoplasias Pulmonares/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Factores de Tiempo
8.
Toxicol Sci ; 108(2): 482-91, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19211616

RESUMEN

The hepatobiliary disposition of thyroxine (T4) was evaluated in Groningen Yellow transport deficient (TR(-)) rats lacking functional multidrug resistance-associated protein 2 (Mrp2; Abcc2). Male Wistar and TR(-) rats were dosed orally (4 days) with phenobarbital (PB; 100 mg/kg) or DMP 904 (200 mg/kg), after which T4 homeostasis and hepatic cytochromes P450, UDP-glucuronosyltransferase, xenobiotic transporters, and T4 glucuronidation were determined. Serum concentrations of T4 were approximately 50% higher in control TR(-) rats than Wistars. PB and DMP 904 increased hepatic levels of P450s and T4-glucuronidation (T4-G), and these changes were associated with decreased serum T4 levels in both strains. In Wistar but not TR(-) rats, DMP 904 increased thyroid stimulating hormone levels twofold. Hepatobiliary clearance of T4 was determined after intravenous infusion of [(125)I]T4 to rats dosed with PB and DMP 904 (4 days). PB and DMP 904 increased plasma clearance and hepatic uptake of [(125)I]T4 equivalents in Wistar but not TR(-) rats. Total biliary clearance (Cl(bile)) was approximately 0.85 and 0.2 ml/h in Wistar and TR(-) rats, respectively, with virtually no T4-G excreted in bile in TR(-) rats. Biliary clearance of unconjugated T4 was also lower in control TR(-) rats than in Wistars, although DMP 904 increased its biliary clearance in both strains. These results suggest that Mrp2 is likely to be responsible for biliary excretion of T4-G and contributes in part to excretion of T4. Decreased biliary clearance of T4 and metabolites in TR(-) rats mitigated but did not prevent drug-induced changes in serum T4, suggesting that other factors contribute to changes in T4 homeostasis in these rats.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Bilis/metabolismo , Sistema Biliar/metabolismo , Hipotiroidismo/inducido químicamente , Hipotiroidismo/metabolismo , Hígado/metabolismo , Receptores de Hormona Tiroidea/genética , Hormonas Tiroideas/metabolismo , Tiroxina/metabolismo , Xenobióticos/toxicidad , Animales , Animales Modificados Genéticamente , Sistema Biliar/patología , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Glucurónidos/metabolismo , Homeostasis/fisiología , Hígado/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Tirotropina/metabolismo , Triyodotironina/metabolismo
9.
Toxicol Sci ; 106(2): 319-28, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18757529

RESUMEN

Perfluorooctanoic acid and perfluorodecanoic acid (PFDA) are commonly used as emulsifiers and surfactants in fluoropolymer manufacturing and are known peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists. PPAR alpha activation induces beta- and omega-oxidation enzymes such as Cyp4a14 and acyl-CoA oxidase, which are a likely cause of subsequent oxidative stress and peroxisome proliferation. Conversely, NF-E2-related factor-2 (Nrf2) is a transcription factor that protects against oxidative stress and inflammation by regulating several detoxification and xenobiotic transporter genes. Because PFDA markedly induces hepatic metabolism and oxidative stress, we hypothesized that PFDA exposure would increase expression of hepatic efflux multidrug resistance-associated protein (Mrp) transporters. A single PFDA dose (80 mg/kg) administered to mice increased hepatic Mrp3 (fourfold) and Mrp4 (31-fold) mRNA expression. Upregulation of Mrp3 and Mrp4 correlated with elevated serum-conjugated bilirubin and bile acids, respectively. To determine the mechanism of Mrp3 and Mrp4 induction, PFDA was administered to Nrf2-null mice, PPAR alpha-null mice, and mice pretreated with gadolinium chloride, a Kupffer cell-depleting chemical capable of inhibiting the inflammatory cytokine response. In both PPAR alpha- and Nrf2-null mice, maximal induction of Mrp3 and Mrp4 mRNA after PFDA administration was attenuated. Gadolinium chloride pretreatment reduced serum and hepatic tumor necrosis factor-alpha levels after PFDA treatment, as well as Mrp4 mRNA expression by 30%, suggesting that Kupffer cell-derived mediators may contribute to Mrp induction. Thus, after PFDA administration, the liver mounts a compensatory hepatoprotective response via PPAR alpha and Nrf2, markedly increasing Mrp3 and Mrp4 expression, with corresponding increases in serum of known Mrp3 and Mrp4 substrates.


Asunto(s)
Caprilatos/toxicidad , Ácidos Decanoicos/toxicidad , Fluorocarburos/toxicidad , Hígado/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor 2 Relacionado con NF-E2/fisiología , PPAR alfa/fisiología , Animales , Ácidos y Sales Biliares/sangre , Bilirrubina/sangre , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética
10.
Mol Pharmacol ; 71(4): 1159-64, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17244698

RESUMEN

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.


Asunto(s)
AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Transportadores de Anión Orgánico Sodio-Independiente/genética , Sistemas de Mensajero Secundario , Animales , Proteína de Unión a CREB , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ayuno , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hígado/metabolismo , Ratones , Mutación , Elementos de Respuesta , Transcripción Genética , Transfección
11.
Drug Metab Dispos ; 33(7): 956-62, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15833929

RESUMEN

The multidrug resistance-associated proteins (Mrp) are ATP-dependent transporters that export a variety of conjugated and unconjugated compounds out of cells. There are nine identified Mrp transporters in humans, with murine orthologs for all except Mrp8. Because nuclear receptors mediate induction of phase I enzymes, Mrp transporter expression might be similarly regulated by these receptors to coordinate metabolism and export of chemicals from liver. To test the hypothesis that Mrp expression may be coordinately regulated with phase I enzyme expression in liver, 15 different compounds were given representing known transcriptionally mediated pathways: aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor-E2-related factor 2 (Nrf2). Each of these compounds induced expression of their respective target enzyme in liver, demonstrating that the chemical regimens were effective. The AhR ligands [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl 126 (PCB126), and beta-naphthoflavone] induced Mrp2, -3, -5, and -6 mRNA expression. The CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induced Mrp2, -3, -4, -6, and -7 mRNA expression. Mrp3 was also induced by two other CAR activators phenobarbital and diallyl sulfide, two PXR ligands, pregnenalone-16alpha-carbonitrile and spironolactone, and the PPARalpha ligands clofibrate, ciprofibrate, and diethylhexylphthalate. The Nrf2 activators (butylated hydroxyanisole, oltipraz, and ethoxyquin) induced Mrp2-6. In conclusion, a variety of mechanisms are suggested for Mrp3 induction, including AhR, CAR, PXR, PPARalpha, and Nrf2, whereas on a whole, a predominant role for AhR and Nrf2 in hepatic induction of the Mrp family was observed. Thus, these specific transcription factors are implicated in regulation of both drug metabolism and efflux transport.


Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Receptores Citoplasmáticos y Nucleares/agonistas , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Inducción Enzimática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
12.
Drug Metab Dispos ; 33(9): 1276-82, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15919853

RESUMEN

Drug-metabolizing enzymes and transporters are key factors that affect disposition of xenobiotics. Phase I enzyme induction by classes of microsomal enzyme inducers occurs via activation of transcription factors such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor erythroid 2-related factor 2 (Nrf2). However, regulation of organic anion-transporting polypeptide (Oatp) uptake transporters by these factors is poorly understood. Hepatic Oatp uptake of some chemicals must occur prior to biotransformation; thus, we hypothesize that expression of Oatps and biotransformation enzymes is coordinately regulated in liver. In the present study, the effects of known chemical activators of AhR, CAR, PXR, PPARalpha, and Nrf2 on the hepatic mRNA expression of mouse Oatps and drug-metabolizing enzymes were quantified by the branched DNA assay. All chemicals increased the expression of their well characterized target drug-metabolizing enzymes: AhR ligands increased Cyp1A1, CAR activators increased Cyp2B10, PXR ligands increased Cyp3A11, PPARalpha ligands increased Cyp4A14, and Nrf2 activators induced NAD(P)H:quinone oxidoreductase 1. AhR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated biphenyl 126, and beta-naphthoflavone) increased Oatp2b1 and 3a1 mRNA expression in liver. CAR activators [phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and diallyl sulfide] decreased Oatp1a1 mRNA expression. Two PXR ligands [pregnenolone-16alpha-carbonitrile (PCN) and spironolactone] increased Oatp1a4 mRNA expression in liver, whereas PXR ligands (PCN, spironolactone, and dexamethasone) and PPARalpha ligands (clofibrate, ciprofibrate, and diethylhexylphthalate) decreased Oatp1a1, 1b2, 2a1, and 2b1 mRNA expression in liver. Nrf2 activators (oltipraz, ethoxyquin, and butylated hydroxyanisole) down-regulated Oatp1a1 but up-regulated Oatp2b1 mRNA expression. Therefore, only a few transcription factor activators increased Oatp expression, and, surprisingly, many decreased Oatp expression.


Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Activadores de Enzimas/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/enzimología , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , Transportadores de Anión Orgánico/metabolismo , Animales , Receptor de Androstano Constitutivo , Inducción Enzimática , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico/genética , PPAR alfa/metabolismo , Receptor X de Pregnano , ARN Mensajero/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo
13.
Comp Biochem Physiol C Toxicol Pharmacol ; 139(4): 209-18, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15683829

RESUMEN

The ABCG5 and ABCG8 genes encode half-transporter proteins that heterodimerize to form a transporter of plant sterols and cholesterol. The purpose of this study was to examine the expression and regulation of ABCG5 and ABCG8 at the mRNA level in Sprague-Dawley rats. Both ABCG5 and ABCG8 mRNA were expressed primarily in rat small intestine and liver, and gender-specific differences in expression were observed. The effects of treatment with a battery of microsomal enzyme inducers on ABCG5 and ABCG8 mRNA were examined; most treatments had no effect, but of three PXR ligands, PCN was an effective inducer, spironolactone was repressive, and dexamethasone was ineffective. The effects of a 1% cholesterol diet on the regulation of rat ABCG5 and ABCG8 were also examined, and compared with those in C57BL/6 mice. Cholesterol caused a suppression of ABCG5 and ABCG8 mRNA in rat liver, but the same treatment increased the expression of these genes in mouse liver. ABCG5 and ABCG8 mRNA was also induced by cholesterol in rat ileum, but not mouse ileum. These results suggest variation between rats and mice in regulatory mechanisms controlling ABCG5 and ABCG8 expression, and may explain some differences in lipid metabolism observed between these two species.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Xenobióticos/farmacología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Animales , Duodeno/metabolismo , Inducción Enzimática , Femenino , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores Sexuales
14.
J Biol Chem ; 277(51): 49446-52, 2002 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-12384496

RESUMEN

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.


Asunto(s)
Glutamato-Cisteína Ligasa/química , Ratones Noqueados , Estrés Oxidativo , Factores de Edad , Alelos , Animales , Northern Blotting , Southern Blotting , Peso Corporal , Muerte Celular , Cromatografía en Gel , Cisteína/metabolismo , Relación Dosis-Respuesta a Droga , Fibroblastos/metabolismo , Genotipo , Glutamato-Cisteína Ligasa/genética , Ácido Glutámico/química , Glutatión/metabolismo , Homocigoto , Peróxido de Hidrógeno/farmacología , Immunoblotting , Riñón/enzimología , Cinética , Hígado/enzimología , Ratones , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Factores de Tiempo , Distribución Tisular
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