RESUMEN
Alternative splicing as a mean to control gene expression and diversify function is suspected to considerably influence drug response and clearance. We report the quantitative expression profiles of the human UGT genes including alternatively spliced variants not previously annotated established by deep RNA-sequencing in tissues of pharmacological importance. We reveal a comprehensive quantification of the alternative UGT transcriptome that differ across tissues and among individuals. Alternative transcripts that comprise novel in-frame sequences associated or not with truncations of the 5'- and/or 3'- termini, significantly contribute to the total expression levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential tissue expression patterns and remodeling in favor of alternative variants upon tumorigenesis. These complex alternative splicing programs have the strong potential to contribute to interindividual variability in drug metabolism in addition to diversify the UGT proteome.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Preparaciones Farmacéuticas/metabolismo , Variantes Farmacogenómicas/genética , Transcriptoma/fisiología , Humanos , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Preparaciones Farmacéuticas/administración & dosificación , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiología , Transcriptoma/efectos de los fármacosRESUMEN
A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.
Asunto(s)
Glucuronosiltransferasa/genética , Fase II de la Desintoxicación Metabólica/genética , Transcriptoma , Mama/enzimología , Neoplasias de la Mama/enzimología , Endometrio/enzimología , Femenino , Glucuronosiltransferasa/metabolismo , Humanos , Neoplasias Intestinales/enzimología , Intestinos/enzimología , Riñón/enzimología , Neoplasias Renales/enzimología , Hígado/enzimología , Neoplasias Hepáticas/enzimología , Masculino , Especificidad de Órganos , Próstata/enzimología , Neoplasias de la Próstata/enzimología , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Neoplasias Uterinas/enzimologíaRESUMEN
Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.
Asunto(s)
Células del Cúmulo/fisiología , AMP Cíclico/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/métodos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Colforsina/farmacología , Células del Cúmulo/citología , Células del Cúmulo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dipiridamol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Understanding the mechanisms regulating oocyte developmental competence is essential to enhance the clinical efficiency of assisted reproduction. FSH orchestrates the acquisition of oocyte competence, both in vivo and in vitro. Multiple pathways are implicated in FSH signalling; however, their precise coordination remains unresolved. A robust system to investigate FSH signalling is oocyte in vitro maturation (IVM) and we have previously demonstrated better bovine embryo development after FSH addition for the first 6 h during IVM. Using this model, we investigated FSH signalling in cumulus through transcriptomic and pharmacological tools. We demonstrate modulation of cumulus transcriptome by FSH mainly through protein kinase A (PKA) and epidermal growth factor (EGF) pathways. Differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism and oocyte competence. FSH required rouse-sarcoma oncogene (SRC) for EGF receptor transactivation. PKA and EGF pathway crosstalk was investigated using extracellular signal-regulated kinases (ERK1/2) phosphorylation as the functional end-point. FSH enhanced ERK1/2 activation by the EGF pathway with a simultaneous diminution through PKA. More specifically, FSH increased dual specific phosphatase (DUSP1) transcripts via PKA although DUSP1 protein did not change since EGF was required to prevent degradation. Our findings implicate FSH in PKA and EGF pathway activation, which interact to maintain appropriate levels of ERK1/2 phosphorylation and eventually cumulus expansion, metabolism and steroidogenesis. Moreover, considering the implication of the EGF pathway in GDF9 and BMP15 actions, our findings suggest that FSH may have a role in modulation of the cumulus response to oocyte-secreted factors. This information has implications for improvement of IVM and hence oocyte developmental competence.
Asunto(s)
Células del Cúmulo/efectos de los fármacos , Fármacos para la Fertilidad/farmacología , Hormona Folículo Estimulante/farmacología , Oocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Bovinos , Biología Computacional , Células del Cúmulo/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/genética , Factores de Tiempo , TranscriptomaRESUMEN
The risk of severe irinotecan-induced neutropenia has been shown to be related to the UGT1 variant UGT1A1*28, which increases exposure to the potent metabolite SN-38. Our goal was to identify a novel UGT1 marker(s) using 28 haplotype-tagged single nucleotide polymorphisms genotyped by mass spectrometry. By characterizing the UGT1 sequence from a cohort of 167 Canadian metastatic colorectal cancer (mCRC) patients and a validation cohort of 250 Italian mCRC patients, we found rs11563250G, located in the intergenic region downstream of UGT1, to be significantly associated with reduced risk of severe neutropenia (odds ratio (OR)=0.21; P=0.043 and OR=0.27; P=0.036, respectively, and OR=0.31 when combined; P=0.001), which remained significant upon correction for multiple testing in the combined cohort (P=0.041). For the two-marker haplotype rs11563250G and UGT1A1*1 (rs8175347 TA6), the OR was of 0.17 (P=0.0004). Genetic testing of this marker may identify patients who might benefit from increased irinotecan dosing.
Asunto(s)
Camptotecina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Glucuronosiltransferasa/genética , Neutropenia/inducido químicamente , Neutropenia/genética , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/uso terapéutico , Biomarcadores de Tumor/genética , Camptotecina/efectos adversos , Camptotecina/uso terapéutico , Canadá , Femenino , Pruebas Genéticas/métodos , Haplotipos/genética , Humanos , Irinotecán , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P<0.01) for all substrates tested. Functional data support a dominant-negative function for endogenous exon 5b-spliced forms of UGT1A, hence potentially affecting in vivo glucuronidation capacity. This new regulatory strategy may ensure an additional mean to modulate cellular response to endo/xeno stimulus.
Asunto(s)
Empalme Alternativo , Glucurónidos/biosíntesis , Glucuronosiltransferasa/genética , Western Blotting , Línea Celular Tumoral , Colon/enzimología , Colon/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Humanos , Inmunohistoquímica , Fase II de la Desintoxicación Metabólica , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por SustratoRESUMEN
Previous studies have shown that using nonspecific phosphodiesterase (PDE) inhibitors such as caffeine improved milk production, supporting the premise that modulation of intracellular concentration of cyclic nucleotides (cyclic AMP, cyclic guanosine 3'-5'-monophosphate) is involved. Intracellular cyclic nucleotides are degraded by the PDE enzyme family. The contribution of type IV PDE (PDE4) in the secretion of casein has been reported in rat mammary gland. The objective of this study was to demonstrate the functional presence of the PDE4 family in the bovine mammary gland. To understand the enzymatic expression pattern in the mammary gland, tissue samples were taken randomly from udders obtained from a local slaughterhouse. Reverse transcription PCR revealed that the PDE4D transcript was amplified, and the expected size fragment was obtained in a 1% agarose gel. Sequence analysis of the amplicon resulted in 99% homology to PDE4D. Moreover, Western blotting using a specific PDE4D antibody has confirmed that the protein of the isoenzyme PDE4D1 is present. A clear immunoreactive signal was also observed within the acini where epithelial cells are located. Assaying cyclic AMP PDE activity reported a total activity of 38.71 +/- 3.22 fmol/min per microg of total protein. Rolipram, a specific PDE4 inhibitor, showed a sensitive activity of 8.48 +/- 1.28 fmol/min per microg of total protein, indicating that PDE4 is responsible for one-fifth of the total enzymatic activity of PDE in the mammary gland. To further validate the presence of PDE4D in the bovine mammary epithelial cells, protein extracts from bovine mammary epithelial cells were separated on SDS-PAGE gels, and PDE4D protein was detected. The PDE assays reported a total activity of 30.16 +/- 4.82 fmol/min per microg of total protein. Rolipram showed a sensible activity of 11.91 +/- 5.93 fmol/min per microg of total protein. In conclusion, these results not only demonstrate the presence of PDE4D transcript and protein, but also show an active enzyme, suggesting a functional role of PDE4D in bovine mammary gland.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Glándulas Mamarias Animales/enzimología , Animales , Bovinos , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Mycophenolate mofetil (MMF) is an immunosuppressive prodrug approved for use in transplantation. Its active metabolite, mycophenolic acid, is mainly metabolized by UDP-glucuronosyltransferase (UGT) enzymes. In this study, we retrospectively analyzed 74 kidney transplant patients who had been prescribed MMF as part of their immunosuppression regimen. Polymorphisms in UGT1A8 (-999C > T, codon 255A > G, codon 277G > A) were correlated with the occurrence of side effects, such as diarrhea, blood disorders, and infections. The infectious episodes were more frequently observed among individuals receiving MMF (2 g/d) who carryied the variant UGT1A8 codon 277A (P = .031), the haplotype UGT1A8H5 (-999C/codon 55A/codon 277A; P = .02), and the diplotype UGT1A8H2/H5 (-999CC/codon 255AA/codon 277GA; P = .015). The molecular data from this study suggest that UGT polymorphisms may be a factor influencing clinical outcomes among patients receiving MMF for transplant therapy; however, larger studies are warranted.
Asunto(s)
Glucuronosiltransferasa/genética , Trasplante de Riñón/inmunología , Ácido Micofenólico/análogos & derivados , Polimorfismo de Nucleótido Simple , Codón/genética , Diarrea/inducido químicamente , Enfermedades Hematológicas/inducido químicamente , Humanos , Infecciones/epidemiología , Ácido Micofenólico/efectos adversos , Estudios RetrospectivosRESUMEN
We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 -275/-2152 selected among 305 individuals genetically screened for UDP-glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype -1248A>G, -1241T>C, -1054T>C, -842G>A, -268A>G, -102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high-performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 -275/-2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration-time curve (AUC6-12 h/AUC0-12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 -275/-2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA C(max) values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Glucuronosiltransferasa/genética , Ácido Micofenólico/farmacocinética , Polimorfismo Genético/fisiología , Adolescente , Adulto , Área Bajo la Curva , Disponibilidad Biológica , Biotransformación , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Haplotipos , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , UDP Glucuronosiltransferasa 1A9RESUMEN
We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.
Asunto(s)
Haplotipos , Microscopía de Fuerza Atómica/métodos , Análisis de Secuencia de ADN/métodos , Alelos , Bacteriófago M13/genética , Marcadores Genéticos , Genotipo , Glucuronosiltransferasa/genética , Heterocigoto , Humanos , Microscopía de Fuerza Atómica/instrumentación , Hibridación de Ácido Nucleico , Mapeo Físico de Cromosoma , Plásmidos/genética , Polimorfismo de Nucleótido Simple , Estreptavidina/genéticaRESUMEN
Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2 ± 49.5 and 1252.6 ± 86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400 nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.
Asunto(s)
Papaverina/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Espermatozoides/enzimología , Reacción Acrosómica/efectos de los fármacos , Animales , Calcio/metabolismo , Bovinos , Epidídimo/citología , Epidídimo/metabolismo , Masculino , Fosforilación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Espermatozoides/efectos de los fármacosRESUMEN
Previously, we observed strong positive associations between circulating concentrations of free testosterone and free dihydrotestosterone (DHT) in relation to Barrett's esophagus in a US male military population. To replicate these findings, we conducted a second study of sex steroid hormones and Barrett's esophagus in the Factors Influencing the Barrett/Adenocarcinoma Relationship (FINBAR) Study based in Northern Ireland and Ireland. We used mass spectrometry to quantitate EDTA plasma concentrations of nine sex steroid hormones and ELISA to quantitate sex hormone-binding globulin in 177 male Barrett's esophagus cases and 185 male general population controls within the FINBAR Study. Free testosterone, free DHT, and free estradiol were estimated using standard formulas. Multivariable logistic regression estimated odds ratios (OR) and 95% confidence intervals (95%CI) of associations between exposures and Barrett's esophagus. While plasma hormone and sex hormone-binding globulin concentrations were not associated with all cases of Barrett's esophagus, we did observe positive associations with estrogens in younger men (e.g. estrone + estradiol ORcontinuous per ½IQR = 2.92, 95%CI:1.08, 7.89), and free androgens in men with higher waist-to-hip ratios (e.g. free testosterone ORcontinuous per ½IQR = 2.71, 95%CI:1.06, 6.92). Stratification by body mass index, antireflux medications, and geographic location did not materially affect the results. This study found evidence for associations between circulating sex steroid hormones and Barrett's esophagus in younger men and men with higher waist-to-hip ratios. Further studies are necessary to elucidate whether sex steroid hormones are consistently associated with esophageal adenocarcinogenesis.
Asunto(s)
Esófago de Barrett/sangre , Dihidrotestosterona/sangre , Estradiol/sangre , Globulina de Unión a Hormona Sexual/metabolismo , Testosterona/sangre , Factores de Edad , Anciano , Índice de Masa Corporal , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Factores de RiesgoRESUMEN
BACKGROUND: UDP-glucuronosyltransferase 1A7 (UGT1A7) detoxifies several tobacco carcinogens. We determined whether UGT1A7 expression is observed in normal orolaryngeal tissue and whether UGT1A7 allelic variations are associated with the risk for orolaryngeal cancer. METHODS: UGT1A7 expression in normal orolaryngeal tissue was determined by semiquantitative reverse transcription-polymerase chain reaction (PCR). Buccal cell DNA isolated from 194 case subjects with orolaryngeal cancer and from 388 control subjects who were matched by sex, age, and race was subjected to UGT1A7 genotyping with the use of combined PCR-restriction fragment length polymorphism and allelic discrimination analysis. All statistical tests were two-sided. RESULTS: UGT1A7 messenger RNA was expressed at similar levels in the esophagus, tongue, tonsil, floor of the mouth, and larynx. Genotyping revealed the presence of three variant reduced-activity UGT1A7 alleles in both Caucasians and African-Americans. Individuals with any of the predicted low-activity UGT1A7 genotypes had an increased risk of orolaryngeal cancer (odds ratio [OR] = 3.7; 95% confidence interval [CI] = 1.7 to 8.7) relative to subjects with the wild-type genotype. Both Caucasians and African-Americans with the low-activity genotypes had statistically significantly increased orolaryngeal cancer risk compared with Caucasians and African-Americans with the wild-type genotype (OR = 2.8 [95% CI = 1.1 to 7.6] and OR = 6.2 [95% CI = 1.2 to 31], respectively). For subjects with the predicted low-activity genotypes, the risks of oral cavity cancer (OR = 4.2; 95% CI = 1.7 to 10) and laryngeal cancer (OR = 3.7; 95% CI = 0.99 to 14) were similar. There was no association between UGT1A7 genotype and orolaryngeal cancer risk in never smokers, whereas subjects with predicted low-activity UGT1A7 genotypes who were light smokers (OR = 3.7; 95% CI = 1.1 to 12) or heavy smokers (OR = 6.1; 95% CI = 1.5 to 25) had an increased risk. CONCLUSIONS: The tissue expression of UGT1A7 is consistent with the possibility of a physiologic role in orolaryngeal cancer. Variations in the UGT1A7 gene that reduce UGT1A7 activity may affect the risk of smoking-related orolaryngeal cancer.
Asunto(s)
Carcinógenos/farmacocinética , Carcinoma de Células Escamosas/enzimología , Glucuronosiltransferasa/fisiología , Neoplasias Laríngeas/enzimología , Neoplasias de la Boca/enzimología , Nicotiana , Humo/análisis , Alelos , Sustitución de Aminoácidos , Población Negra/genética , Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/etiología , Codón , Estudios de Cohortes , Esófago/enzimología , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Glucuronosiltransferasa/análisis , Glucuronosiltransferasa/genética , Humanos , Inactivación Metabólica/genética , Neoplasias Laríngeas/epidemiología , Neoplasias Laríngeas/etiología , Laringe/enzimología , Estilo de Vida , Hígado/enzimología , Masculino , Persona de Mediana Edad , Boca/enzimología , Neoplasias de la Boca/epidemiología , Neoplasias de la Boca/etiología , New York/epidemiología , Especificidad de Órganos , Tonsila Palatina/enzimología , Philadelphia/epidemiología , Polimorfismo Genético , Riesgo , Fumar/efectos adversos , Encuestas y Cuestionarios , Lengua/enzimología , Población Blanca/genéticaRESUMEN
We examined the role of constitutional genetic variation at the UDP-glucuronosyltransferase (UGT) 1A1 locus in breast cancer susceptibility. The UGT1A1 enzyme is a major UGT involved in estradiol glucuronidation. To date, four UGT1A1 variant alleles characterized by a variation in the number of TA from five through eight repeats in the atypical TATA box region have been described in the African-American population. Functional analyses of the transcriptional activity in breast and liver cells revealed that the transcription activation of a reporter gene is inversely correlated with the number of repeats. Reverse transcription-PCR analysis confirmed the expression of UGT1A1 in human liver in the hepatocarcinoma cell line HepG2 and provided evidence of the expression of UGT1A1 in breast cancer tissue, where a positive signal was observed in 11 of 12 breast cancer cell lines tested. The population-based case-control study involved 200 women with breast cancer and 200 female controls of African ancestry. We postulated that breast cancer cases might have a higher prevalence of low activity allele-containing genotypes than controls (alleles presenting seven and eight repeats in the A(TA)nTAA motif of the TATA box). The age-adjusted odds ratio (OR) for breast cancer comparing women with seven and eight allele-containing genotypes versus 5/5, 5/6, and 6/6 genotypes was 1.8 [95% confidence interval (CI), 1.0-3.1; P = 0.06] in premenopausal women and 1.0 (95% CI, 0.5-1.7; P = 0.9) in postmenopausal women. The observed 1.8-fold elevated risk in premenopausal women with invasive breast cancer is highly suggestive of a possible interaction between UGT genotype and hormones. Additional analyses suggested a stronger association of UGT1A1 genotype with estrogen receptor (ER)-negative breast cancer. Among premenopausal women, the association was stronger for ER- breast cancer (OR, 2.1; 95% CI, 1.0-4.2; P = 0.04) than ER+ breast cancer (OR, 1.3; 95% CI, 0.6-3.0; P = 0.5). The OR was slightly stronger among women who used oral contraceptives, and the association remained null in postmenopausal women, regardless of whether they took hormone replacement therapy. Our current findings suggest that further investigations are warranted to elucidate the role of UGT1A1 in breast cancer risk.
Asunto(s)
Población Negra/genética , Neoplasias de la Mama/etnología , Glucuronosiltransferasa/genética , Adulto , Anciano , Neoplasias de la Mama/genética , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Polimorfismo Genético , Regiones Promotoras Genéticas , Receptores de Estrógenos/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5'AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1 ± 1.5 fmoles/min/µg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2 ± 14.8 fmoles/min/µg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.
Asunto(s)
AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Semen/metabolismo , Animales , Bovinos , Masculino , Inhibidores de Fosfodiesterasa/farmacología , Semen/efectos de los fármacos , Espermatozoides/metabolismoRESUMEN
The antiretroviral protease inhibitor atazanavir inhibits hepatic uridine diphosphate glucuronosyltransferase (UGT) 1A1, thereby preventing the glucuronidation and elimination of bilirubin. Resultant indirect hyperbilirubinemia with jaundice can cause premature discontinuation of atazanavir. Risk for bilirubin-related discontinuation is highest among individuals who carry two UGT1A1 decreased function alleles (UGT1A1*28 or *37). We summarize published literature that supports this association and provide recommendations for atazanavir prescribing when UGT1A1 genotype is known (updates at www.pharmgkb.org).
Asunto(s)
Sulfato de Atazanavir/efectos adversos , Glucuronosiltransferasa/antagonistas & inhibidores , Inhibidores de la Proteasa del VIH/efectos adversos , Hiperbilirrubinemia/inducido químicamente , Ictericia/inducido químicamente , Hígado/efectos de los fármacos , Farmacogenética/normas , Predisposición Genética a la Enfermedad , Genotipo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Hiperbilirrubinemia/enzimología , Hiperbilirrubinemia/genética , Ictericia/enzimología , Ictericia/genética , Hígado/enzimología , Fenotipo , Medición de Riesgo , Factores de RiesgoRESUMEN
Although much attention has been focused on the synthesis of dihydrotestosterone (DHT), the inactivation and elimination of active androgens can also be key points in regulating androgen levels in tissues such as the prostate. Recent data suggest that 5alpha-reduced C19 steroids can be converted to glucuronide derivatives in the human prostate, leading to complete inactivation of these steroids. These results are supported by the recent finding of at least two steroid uridine diphosphoglucuronosyltransferase (UGT) enzymes in the prostate as well as in the human prostatic cancer LNCaP cell line. To ascertain the role of UGTs in regulating active steroid levels, we investigated the modulation of UGT levels in response to steroid treatments in LNCaP cells. Results demonstrate the down-regulation of UGT activities specific for 3-hydroxysteroids and 17-hydroxy-steroids after treatment with androgens and estrogens. Treating the cells with DHT or R1881 for 7 days inhibited UGT activity by 60%; however, 80% of the total activity was recovered after 5 days in the absence of the androgens. The inhibition of UGT activities by DHT and R1881 increases with the time of incubation and with increasing concentrations of the androgens used. The decrease in UGT enzyme activity occurred in parallel with a diminution in UGT transcript levels, as observed in Northern blot analyses. A correlation between the effect of steroids on the androgen-dependent growth response of LNCaP cells, the secretion of prostate-specific antigen, and the inhibition of UGT activities was clearly demonstrated, which implicates the androgen signaling pathway. Treating cells with Casodex, an androgen antagonist that binds the mutated androgen receptor expressed in LNCaP cells, partially blocked the androgen- and estrogen-induced decrease in UGT activity, suggesting that the regulation of UGT levels involves the androgen receptor. In addition to the formation of DHT, the inactivation of steroids by glucuronidation, which is regulated by steroids themselves, is an important mechanism controlling the level of androgens in the prostate.
Asunto(s)
Andrógenos/farmacología , Dihidrotestosterona/farmacología , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucuronosiltransferasa/metabolismo , Transcripción Genética/efectos de los fármacos , Antagonistas de Andrógenos/farmacología , División Celular/efectos de los fármacos , Línea Celular , Glucuronatos/metabolismo , Glucuronosiltransferasa/biosíntesis , Humanos , Cinética , Análisis de los Mínimos Cuadrados , Masculino , Metribolona/farmacología , Neoplasias de la Próstata , Especificidad por Sustrato , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1alpha (IL-1alpha), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1alpha led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1alpha decreased both UGT activity and LNCaP cell proliferation in the absence and presence of DHT (0.5 nM); a maximal inhibition of 70% was observed. IL-6 inhibited LNCaP cell proliferation as well as the DHT-induced proliferation of these cells. However, neither IL-4 nor IL-6 significantly affected the formation of DHT glucuronide. Ribonuclease protection and Western blot analyses demonstrated a specific reduction of UGT2B17 transcript and protein levels in IL-1alpha-treated LNCaP cells. The level of UGT2B15 was not affected by cytokine treatments, indicating a differential regulation between these two UGT enzymes. Transfection experiments performed with the UGT2B17 gene promoter region indicates that the regulation occurs at the transcription level via putative cis-acting elements. This study indicates that cell proliferation and UGT expression in steroid-responsive cancer cells are differentially regulated depending on the cytokines present in the cell microenvironment.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Interleucinas/farmacología , División Celular , Dihidrotestosterona/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucuronatos/metabolismo , Glucuronosiltransferasa/genética , Humanos , Interleucina-1/farmacología , Interleucina-4/farmacología , Interleucina-6/farmacología , Masculino , Regiones Promotoras Genéticas , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Although androgens are important regulators in the prostate, other effectors such as growth factors may also act to maintain normal function of the gland. Human prostate and human prostate cancer LNCaP cells express steroid conjugating uridine diphospho-glucuronosyltransferase (UGT) enzymes, and it was shown that the level of UGT activities and transcripts is down-regulated by androgens, especially dihydrotestosterone (DHT). In the present study, we examined the interaction between androgen, epidermal growth factor (EGF), and steroid UGT enzymes. The formation of DHT glucuronide (DHT-G) was inhibited by 47% when LNCaP cells were treated for 6 days with 10 ng/ml of EGF. Northern blot analysis also demonstrated a decrease in the steady-state level of UGT2B transcripts. Treatment with both DHT (0.5 nM) and EGF (10 ng/ml) caused a greater decrease of DHT glucuronidation and UGT2B messenger RNA levels than when the cells were treated with either compound alone. RNase protection assays showed that treatment with DHT and EGF caused a specific decrease of UGT2B17 transcript in LNCaP cells treated; however, the level of UGT2B15 messenger RNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. UGT2B17 was shown to be more labile than UGT2B15, indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids.
Asunto(s)
Glucuronosiltransferasa/metabolismo , Próstata/enzimología , División Celular , Línea Celular , Deshidroepiandrosterona/metabolismo , Dihidrotestosterona/metabolismo , Dihidrotestosterona/farmacología , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas , Factor de Crecimiento Epidérmico/administración & dosificación , Factor de Crecimiento Epidérmico/farmacología , Glucuronosiltransferasa/genética , Humanos , Masculino , Próstata/citología , Transcripción GenéticaRESUMEN
One of the most important mechanisms involved in host defense against xenobiotic chemicals and endogenous toxins is the glucuronidation catalysed by UDP-glucuronosyltransferase enzymes (UGT). The role of genetic factors in determining variable rates of glucuronidation is not well understood, but phenotypic evidence in support of such variation has been reported. In the present study, six single nucleotide polymorphisms were discovered in the first exon of the UGT1A7 gene, which codes for the putative substrate-binding domain, revealing a high structural heterogeneity at the UGT1 gene locus. The new UGT1A7 proteins differ in their primary structure at amino acid positions 129, 131 and 208, creating four distinct UGT1A7 allelic variants in the human population: UGT1A7*1 (N129 R131 W208), *2 (K129 K131 W208), *3 (K129 K131 R208), and *4 (N129 R131 R208). In functional studies, HEK cells stably transfected to express the four allelic UGT1A7 variants exhibited significant differences in catalytic activity towards 3-, 7-, and 9-hydroxy-benzo(a)pyrene. UGT1A7*3 exhibited a 5.8-fold lower relative Vmax compared to wild-type *1, whereas *2 and *4 had a 2.6- and 2.8-fold lower relative Vmax than *1, respectively, suggesting that these mutations confer slow glucuronidation phenotype. Kinetic characterization suggested that these differences were primarily attributable to altered Vmax. Additionally, it suggested that each amino acid substitutions can independently affect the UGT1A7 catalytic activity, and that their effects are additive. The expression pattern of UGT1A7 studied herein and its catalytic activity profile suggest a possible role of UGT1A7 in the detoxification and elimination of carcinogenic products in lung. A population study demonstrated that a considerable proportion of the population (15.3%) was found homozygous for the low activity allele containing all three missense mutations, UGT1A7*3. These findings suggest that further studies are needed to investigate the impact of the low UGT1A7 conjugator genotype on individual susceptibility to chemical-induced diseases and responses to therapeutic drugs.