RESUMO
Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Variantes Farmacogenômicos/genética , Pravastatina/metabolismo , Transporte Biológico , Regulação da Expressão Gênica , Células HEK293 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Heterozigoto , Humanos , Cinética , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Fenótipo , Transfecção , População Branca/genéticaRESUMO
The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8- and 2.8-fold, respectively), and 2.9-fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time- and concentration-dependent induction by metamizole of CYP2B6 (7.8- and 3.1-fold for mRNA and protein, respectively, at 100 muM) and CYP3A4 (2.4- and 2.9-fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital-like mechanism of induction. These data warrant further studies to elucidate the drug-interaction potential of metamizole, especially in patients with long-term treatment.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Hidrocarboneto de Aril Hidroxilases/biossíntese , Sistema Enzimático do Citocromo P-450/biossíntese , Dipirona/farmacologia , Oxirredutases N-Desmetilantes/biossíntese , Idoso , Hidrocarboneto de Aril Hidroxilases/genética , Western Blotting , Catálise , Células Cultivadas , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , DNA/biossíntese , DNA/genética , Dipirona/análogos & derivados , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Feminino , Genótipo , Hepatócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Oxirredutases N-Desmetilantes/genética , Plasmídeos , Receptor de Pregnano X , RNA/biossíntese , RNA/genética , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Esteroides/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/efeitos dos fármacosRESUMO
The retroviral oncogene v-myb is a mutated and truncated version of the c-myb proto-oncogene and encodes a transcription factor (v-Myb) that specifically transforms myelomonocytic cells. v-Myb is thought to transform myelomonocytic cells by affecting the expression of specific target genes, most of which as yet remain unknown. To identify novel v-Myb regulated genes we have employed 'differential display', using a myelomonocytic chicken cell line that expresses a conditional version of v-Myb. Here we describe the identification of the gene encoding the A2b adenosine receptor, a member of the seven transmembrane receptor superfamily, as a v-Myb target gene. Our results provide the first evidence that v-Myb directly regulates a gene encoding a membrane receptor and establish a link between Myb function and adenosine receptor signaling.
Assuntos
Regulação da Expressão Gênica , Receptores Purinérgicos P1/genética , Proteínas Oncogênicas de Retroviridae/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Dados de Sequência Molecular , Proteínas Oncogênicas v-myb , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-myb , Transativadores/fisiologiaRESUMO
The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the ability of avian myeloblastosis virus (AMV) to transform myelomonocytic cells. v-Myb is thought to disrupt the differentiation of myelomonocytic cells by affecting the expression of specific target genes. To identify such genes we have analysed the gene expression in a myelomonocytic chicken cell line that carries an estrogen inducible version of v-Myb by differential display. Here we describe the identification of the chicken homolog of the mouse Pdcd4 gene as a novel v-Myb target gene. Pdcd4 is also known as MA-3, TIS and H731 and has recently been shown to suppress the transformation of epidermal cells by tumor promoters. Our results provide the first evidence that v-Myb directly regulates the expression of a potential tumor suppressor gene.
Assuntos
Galinhas/genética , Proteínas Oncogênicas v-myb/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA , Alpharetrovirus/genética , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Vírus da Mieloblastose Aviária/genética , Sequência de Bases , Núcleo Celular/metabolismo , Células Cultivadas , Citarabina/farmacologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Células Mieloides/fisiologia , Células Mieloides/efeitos da radiação , Células Mieloides/virologia , Proteínas Oncogênicas v-myb/genética , Homologia de Sequência de Aminoácidos , Timo/metabolismo , Raios UltravioletaRESUMO
Transformation of myeloid cells by the retroviral oncogene v-myb is thought to be caused by deregulated expression of specific cellular genes that act as targets of v-Myb in myeloid cells. Recently, we have identified the chicken tom-1 gene as a direct target for v-Myb. tom-1 has two promoters, only one of which (the tom-1A promoter) is activated by v-Myb. Here, we show that v-Myb activates the tom-1A promoter by cooperating with Ets-2, a member of the Ets transcription factor family. Interestingly, we find that the ability of v-Myb to cooperate with Ets proteins differs from that of its non-oncogenic cellular counterpart c-Myb. c-Myb cooperates with Ets-1 and Ets-2, whereas v-Myb only cooperates with Ets-2. Truncation of the N-terminus of c-Myb, which is known to activate the oncogenic potential of c-Myb, specifically abrogates the ability of the protein to cooperate with Ets-1. Our findings, therefore, reveal a novel function for the N-terminus of c-Myb and raise the possibility that oncogenic activation of c-Myb is linked to the loss of cooperation between Myb and c-Ets-1.
Assuntos
Proteínas de Ligação a DNA , Genes myb/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Transformação Celular Viral , Galinhas , Regulação da Expressão Gênica , Genes Reporter , Dados de Sequência Molecular , Plasmídeos , Mutação Puntual , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas c-ets , Proteínas Oncogênicas de Retroviridae/genética , Transativadores/genética , TransfecçãoRESUMO
Previous studies have implicated the CCAAT box/enhancer binding protein beta (C/EBPbeta) in the regulation of cell-type specific gene expression in myelomonocytic cells and in the activation of target genes by the transcription factor v-Myb. To better understand the role of C/EBPbeta in myelomonocytic cells we have cloned the chicken C/EBPbeta gene and studied its regulation. The chicken C/EBPbeta promoter contains a number of C/EBP binding sites and is activated by C/EBPbeta, suggesting that the C/EBPbeta gene is autoregulated by its own protein product. Interestingly, the C/EBPbeta promoter is not activated by C/EBPalpha, another C/EBP family member highly expressed in myelomonocytic cells, indicating that the autoregulation is specific for C/EBPbeta. Comparison of different C/EBP inducible promoters shows that the relative transactivation potential of C/EBPalpha and beta is extremely dependent on the promoter context. By using the promoters of the mim-1 and C/EBPbeta genes and by exchanging the DNA-binding domains between C/EBPalpha and beta we show that the observed promoter preferences of C/EBPalpha and beta are not due to differential DNA-binding but instead depend on the transactivation domains of these proteins. The C/EBPbeta promoter also contains several Myb binding motifs, suggesting that the C/EBPbeta gene is also myb-inducible. We show that the C/EBPbeta promoter is activated synergistically by v-Myb and C/EBPbeta and that transcription of the endogenous C/EBPbeta gene is increased by v-Myb. Thus, our results identify the C/EBPbeta gene as a novel v-Myb target gene. Taken together, our data suggest a model for the regulation of C/EBPbeta expression in which v-Myb stimulates the synthesis of C/EBPbeta by enhancing an autoregulatory loop acting on the C/EBPbeta promoter.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes myb , Monócitos/fisiologia , Proteínas Nucleares/genética , Animais , Comunicação Autócrina/genética , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Galinhas , Clonagem Molecular , Coturnix , Elementos Facilitadores Genéticos/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. OBJECTIVES: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. METHODS AND RESULTS: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. CONCLUSIONS: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.
Assuntos
Plaquetas/enzimologia , Lesões das Artérias Carótidas/enzimologia , Grânulos Citoplasmáticos/enzimologia , Proteínas Imediatamente Precoces/sangue , Ativação Plaquetária , Proteínas Serina-Treonina Quinases/sangue , Vesículas Secretórias/enzimologia , Trombose/enzimologia , Trifosfato de Adenosina/sangue , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Lesões das Artérias Carótidas/sangue , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Modelos Animais de Doenças , Feminino , Genótipo , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Masculino , Megacariócitos/enzimologia , Megacariócitos/metabolismo , Camundongos Knockout , Fenótipo , Agregação Plaquetária , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Serotonina/sangue , Serotonina/metabolismo , Transdução de Sinais , Tetraspanina 30/sangue , Tetraspanina 30/metabolismo , Trombose/sangue , Trombose/genética , Trombose/patologia , Fatores de Tempo , Transfecção , Regulação para Cima , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica , Oxirredutases N-Desmetilantes/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Citocromo P-450 CYP3A , Primers do DNA , DNA Complementar , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rifampina/farmacologia , Ativação TranscricionalRESUMO
CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Polimorfismo de Nucleotídeo Único , Processamento Alternativo , Western Blotting , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Mutação da Fase de Leitura , Expressão Gênica , Frequência do Gene , Marcadores Genéticos , Alemanha , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos Hepáticos/enzimologia , Fenótipo , Alinhamento de Sequência , Análise de Sequência de DNA , Suíça , Transcrição Gênica , População Branca/genéticaRESUMO
BACKGROUND AND PURPOSE: Widespread resistance to antimalarial drugs requires combination therapies with increasing risk of pharmacokinetic drug-drug interactions. Here, we explore the capacity of antimalarial drugs to induce drug metabolism via activation of constitutive androstane receptors (CAR) by ligand binding. EXPERIMENTAL APPROACH: A total of 21 selected antimalarials and 11 major metabolites were screened for binding to CAR isoforms using cellular and in vitro CAR-coactivator interaction assays, combined with in silico molecular docking. Identified ligands were further characterized by cell-based assays and primary human hepatocytes were used to elucidate induction of gene expression. KEY RESULTS: Only two artemisinin derivatives arteether and artemether, the metabolite deoxyartemisinin and artemisinin itself demonstrated agonist binding to the major isoforms CAR1 and CAR3, while arteether and artemether were also inverse agonists of CAR2. Dihydroartemisinin and artesunate acted as weak inverse agonists of CAR1. While arteether showed the highest activities in vitro, it was less active than artemisinin in inducing hepatic CYP3A4 gene expression in hepatocytes. CONCLUSIONS AND IMPLICATIONS: Artemisinin derivatives and metabolites differentially affect the activities of CAR isoforms and of the pregnane X receptor (PXR). This negates a common effect of these drugs on CAR/PXR-dependent induction of drug metabolism and further provides an explanation for artemisinin consistently inducing cytochrome P450 genes in vivo, whereas arteether and artemether do not. All these drugs are metabolized very rapidly, but only artemisinin is converted to an enzyme-inducing metabolite. For better understanding of pharmacokinetic drug-drug interaction possibilities, the inducing properties of artemisinin metabolites should be considered.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Animais , Antimaláricos/metabolismo , Artemisininas/metabolismo , Células COS , Células CACO-2 , Chlorocebus aethiops , Receptor Constitutivo de Androstano , Citocromo P-450 CYP3A/genética , Interações Medicamentosas , Resistência a Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Isoformas de Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
BACKGROUND AND PURPOSE: Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACH: Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTS: All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONS: Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Ácidos Heptanoicos/farmacologia , Proteínas de Membrana Transportadoras/biossíntese , Pirróis/farmacologia , Receptores de Esteroides/biossíntese , Receptores de Esteroides/genética , Hidrocarboneto de Aril Hidroxilases/biossíntese , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Atorvastatina , Linhagem Celular Tumoral , Proteínas Correpressoras/metabolismo , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Interações Medicamentosas , Genes Reporter/efeitos dos fármacos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Ácidos Heptanoicos/metabolismo , Humanos , Ligantes , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxirredutases N-Desmetilantes/biossíntese , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Receptor de Pregnano X , Regiões Promotoras Genéticas , Pirróis/metabolismo , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismoRESUMO
Delayed graft function (DGF) is an important complication in renal transplantation, contributing significantly to decrease in long-term allograft survival. In addition to donor- and recipient-related risk factors such as immunosuppression, altered renal excretion of xenobiotics by membrane transporters may influence DGF. Using DNA samples from recipients and donors, we assessed the impact on DGF of genetic variants in P-glycoprotein (ABCB1), multidrug resistance protein 2 (ABCC2), and the nuclear pregnane X receptor (PXR/NR1I2), which regulates the transcription of enzymes and transporters. In our local cohort of renal transplant recipients (n = 178), DGF occurred in 27.5%. The PXR 8055TT genotype of the donor only (not of the recipient) was significantly associated with an increased risk for DGF. This finding emerged from univariate as well as multivariate logistic regression analysis including 16 nongenetic factors and held true after correction for multiple testing. Our findings provide the first evidence that PXR may be associated with risk of DGF, independent of previously identified risk factors.
Assuntos
Função Retardada do Enxerto/etiologia , Transplante de Rim/efeitos adversos , Receptores de Esteroides/genética , Doadores de Tecidos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adolescente , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Análise Multivariada , Receptor de Pregnano X , Fatores de RiscoRESUMO
The impact of thyroid dysfunction on the regulation, expression, and function of ABCB1 remains unclear. We therefore investigated ABCB1 mRNA expression and function in patients with thyroid dysfunction and studied the disposition of the ABCB1 substrate digoxin before and after treatment for thyroid disease. In patients with hypothyroidism, normalization of thyroid function was associated with a 1.8-fold increase in mRNA expression and a 26% increase in rhodamine efflux from CD56(+) cells. In hypothyroidism, digoxin clearance was significantly decreased, whereas bioavailability, volume of distribution, half-life time, and protein binding were unaltered. In hyperthyroidism, ABCB1 mRNA expression, rhodamine efflux, and disposition of digoxin were not significantly affected other than in relation to renal clearance. Experiments using the LS174T cell line indicated that the gene is a direct target of thyroid hormone receptors. In conclusion, thyroid abnormalities can exert significant effects on the expression of P-glycoprotein, thereby altering the disposition and action of drugs that are substrates of P-glycoprotein.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Cardiotônicos/farmacocinética , Digoxina/farmacocinética , Hipertireoidismo/metabolismo , Hipotireoidismo/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Administração Oral , Adulto , Idoso , Células CACO-2 , Cardiotônicos/administração & dosagem , Digoxina/administração & dosagem , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica , Humanos , Hipertireoidismo/tratamento farmacológico , Hipertireoidismo/genética , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/genética , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Rodaminas/metabolismo , Hormônios Tireóideos/metabolismo , Transfecção , Adulto JovemRESUMO
The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA binding phosphoproteins that are thought to regulate the expression of myb-responsive genes during myeloid differentiation. To identify such myb-regulated genes, and to explore the mechanisms by which v-myb affects their expression, we have established a conditional expression system for v-myb. We have converted the v-myb protein to an estrogen-inducible transactivator by fusing the protein to the hormone binding domain of the human estrogen receptor. Expression of the chimeric protein in a chicken macrophage cell-line causes estrogen-dependent, reversible changes in the differentiation state as well as alterations in the gene expression program of the cells. We have used this estrogen-dependent v-myb expression system to identify a novel v-myb regulated gene.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Transformada , Galinhas , Coturnix , DNA/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Proteínas Oncogênicas v-myb , RNA/análise , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , TransativadoresRESUMO
Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1 are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5'-upstream region of human MDR1 was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about -8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about -8 kilobase pairs, to which PXR binds.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores de Esteroides/fisiologia , Rifampina/farmacologia , Sequência de Bases , DNA , Primers do DNA , Dimerização , Regulação da Expressão Gênica/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Dados de Sequência Molecular , Receptor de Pregnano X , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/fisiologia , Receptores X de Retinoides , Fatores de Transcrição/fisiologiaRESUMO
The retroviral oncogene v-myb is a mutated and truncated version of the c-myb proto-oncogene and encodes a transcription factor (v-Myb) that specifically transforms myelomonocytic cells. Two different variants of v-myb, transduced independently by the oncogenic chicken retroviruses AMV and E26, have been characterized. It is believed that both variants of v-Myb transform myelomonocytic cells by affecting the expression of specific genes; however, no target genes common to both oncogenic viruses have been identified. Here, we describe the identification of a novel v-Myb target gene, designated as tom-1 (target of myb 1). The tom-1 gene has two promoters, one of which is Myb-inducible. tom-1 is expressed at elevated levels in AMV-transformed as well as in E26-transformed myeloid cells. We show that tom-1 activation by v-Myb does not require de novo protein synthesis and that the Myb-inducible tom-1 promoter contains a functional Myb binding site. Thus, tom-1 is the first example of a direct target gene for both oncogenic forms of the v-myb gene. Further analysis of the Myb-inducible tom-1 promoter shows that a C/EBP binding site is juxtaposed to the Myb binding site and that C/EBP is required for the Myb-dependent activation of the promoter. Together with previous work our results suggest that C/EBP may be a general cooperation partner for v-Myb in myelomonocytic cells.
Assuntos
Vírus da Leucose Aviária/genética , Vírus da Mieloblastose Aviária/genética , Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Transformação Celular Neoplásica , Transformação Celular Viral , Galinhas , Clonagem Molecular , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Genes Virais , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas v-myb , Regiões Promotoras Genéticas , Proteínas Oncogênicas de Retroviridae/genéticaRESUMO
The retroviral oncogene v-myb encodes a transcriptional activator which is responsible for the activation of the mim-1 gene in myelomonocytic cells transformed by v-myb. The mim-1 promoter contains several myb consensus binding sites and has previously been shown to be regulated directly by v-myb. Here we report that the mim-1 gene is activated synergistically by v-myb and different C/EBP transcription factors. We have cloned a chicken C/EBP-related gene that is highly expressed in myeloid cells and identified it as the chicken homolog of C/EBP beta. A dominant-negative variant of chicken C/EBP beta interferes with the v-myb induced activation of the mim-1 gene in these cells, suggesting that C/EBP beta or another C/EBP transcription factor is required for the activation of mim-1 by v-myb. We found that C/EBP beta and other C/EBP transcription factors confer to fibroblasts the ability to induce the mim-1 gene in the presence of v-myb. Finally we show that, in contrast to v-myb, c-myb synergizes with C/EBP transcription factors only at low concentrations of c-myb protein. Our results suggest a role for C/EBP beta, and possibly for other C/EBP transcription factors, in v-myb function and in myeloid-specific gene activation.
Assuntos
Acetiltransferases , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Vírus da Mieloblastose Aviária , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Linhagem Celular , Galinhas , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Muramidase/genética , Oligodesoxirribonucleotídeos , Proteínas Oncogênicas v-myb , Regiões Promotoras Genéticas , Proteínas/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Ativação TranscricionalRESUMO
A novel approach to reducing organ toxicity of anticancer agents is the application of nontoxic glucuronide prodrugs from which the active drug is released by human beta-glucuronidase, an enzyme present at high levels in many tumors. In view of high interindividual variability in beta-glucuronidase expression, regulation of this enzyme is an essential factor modulating bioactivation of glucuronide prodrugs. However, data on regulation of human beta-glucuronidase expression are not available. Preliminary evidence from animal experiments points to a role of intracellular calcium in regulation of beta-glucuronidase activity. Therefore, we investigated regulation of beta-glucuronidase by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin in the human hepatoma cell line HepG2. The enzyme was characterized on activity, protein, and mRNA levels by cleavage of 4-methylumbelliferyl-beta-D-glucuronide, Western blotting, Northern blotting, and nuclear run-on transcription. Incubation of HepG2 cells with A23187 and thapsigargin, respectively, revealed a time and concentration dependent down-regulation of beta-glucuronidase activity to about 50% of the control level. This effect could also be demonstrated in several other cell lines (e.g., HL-60, ECV 304, 32M1, Caco-2/TC7). Effects on protein and mRNA levels paralleled those obtained on enzymatic activity. In line with these data, A23187 and thapsigargin decreased beta-glucuronidase transcriptional rate. Our data demonstrate regulation of human beta-glucuronidase by xenobiotics. Down-regulation of beta-glucuronidase by A23187 and thapsigargin is at least partly mediated by a transcriptional mechanism. Based on our findings, we speculate that beta-glucuronidase activity and hence bioactivation of glucuronide prodrugs in humans can be modulated by exogenous factors.
Assuntos
Calcimicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Tapsigargina/farmacologia , Northern Blotting , Western Blotting , Carcinoma Hepatocelular , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Glucuronidase/efeitos dos fármacos , Glucuronidase/metabolismo , Humanos , Ionóforos/farmacologia , Neoplasias Pulmonares , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Verapamil is subject to extensive oxidative metabolism mediated by cytochrome P450 enzymes with less than 5% of an oral dose being excreted unchanged in urine. Furthermore, verapamil is known to be a potent inhibitor of P-glycoprotein function. There is evidence from in vivo investigations that some verapamil metabolites might be actively transported. The aim of the present study was to investigate P-glycoprotein-mediated transport and inhibition properties of verapamil and its metabolites norverapamil, D-620, D-617, and D-703. Polarized transport of these compounds was assessed in P-glycoprotein-expressing Caco-2 and L-MDR1 cells (LLC-PK1 cells stably transfected with human MDR1-P-glycoprotein). Inhibition of P-glycoprotein-mediated transport by these compounds was determined using digoxin as P-glycoprotein substrate. At concentrations of 5 microM, significant differences between basal-to-apical and apical-to-basal apparent permeability coefficients were observed for D-617 and D-620 in all P-glycoprotein-expressing cell monolayers, indicating that both are P-glycoprotein substrates. In contrast, no P-glycoprotein-dependent transport was found for verapamil, norverapamil, and D-703 in Caco-2 cells and for D-703 in L-MDR1 cells. Moreover, verapamil, norverapamil, and D-703 inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 1.1, 0.3, and 1.6 microM, respectively, whereas D-617 and D-620 did not (at concentrations up to 100 microM). We conclude that verapamil phase I metabolites exhibit different P-glycoprotein substrate and inhibition characteristics, with the N-dealkylated metabolites D-617 and D-620 being P-glycoprotein substrates and norverapamil and D-703 being inhibitors of P-glycoprotein function, which may influence P-glycoprotein-dependent drug disposition and elimination.