RESUMEN
Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of female adults with poorly controlled atopic asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 13, 17 A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFα, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, we utilised a previously published assessment of the circulating "blood microbiome" performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in just the asthma group or just in the control group, and many more were observed to be expressed at significantly different levels in the asthma group compared to the control group. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthmatic state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.
Asunto(s)
Asma/inmunología , Hipersensibilidad/inmunología , Pyroglyphidae/inmunología , Adulto , Alérgenos/inmunología , Animales , Estudios de Casos y Controles , Femenino , Humanos , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , ARN Mensajero/genéticaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are short non-protein-coding RNA species that have a regulatory function in modulating protein translation and degradation of specific mRNAs. MicroRNAs are estimated to target approximately 60% of all human mRNAs and are associated with the regulation of all physiological processes. Similar to many messenger RNAs (mRNA), miRNAs exhibit marked tissue specificity, and appear to be dysregulated in response to specific pathological conditions. Perhaps, one of the most significant findings is that miRNAs are detectable in various biological fluids and are stable during routine clinical processing, paving the way for their use as novel biomarkers. Despite an increasing number of publications reporting individual miRNAs or miRNA signatures to be diagnostic of disease or indicative of response to therapy, there is still a paucity of baseline data necessary for their validation. To this end, we utilised state of the art sequencing technologies to determine the global expression of all circulating miRNAs within the plasma of 18 disease-free human subjects. RESULTS: In excess of 500 miRNAs were detected in our study population with expression levels across several orders of magnitude. Ten highly expressed miRNAs accounted for 90% of the total reads that mapped showing that despite the range of miRNAs present, the total miRNA load of the plasma was predominated by just these few species (50% of which are blood cell associated). Ranges of expression were determined for all miRNA detected (>500) and a set of highly stable miRNAs identified. Finally, the effects of gender, smoking status and body mass index on miRNA expression were determined. CONCLUSIONS: The data contained within will be of particular use to researchers performing miRNA-based biomarker screening in plasma and allow shortlisting of candidates a priori to expedite discovery or reduce costs as required.
Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , MicroARNs/genética , Transcriptoma , Adulto , Anciano , Análisis por Conglomerados , Biología Computacional/métodos , Femenino , Humanos , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Factores de Riesgo , Adulto JovenRESUMEN
Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Animales , Línea Celular , Estrés del Retículo Endoplásmico , Canales de Potasio Éter-A-Go-Go/metabolismo , Gosipol/análogos & derivados , Gosipol/farmacología , Células HeLa , Humanos , Células MCF-7 , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Nutrition during early mammalian development permanently influences health of the adult, including increasing the risk of type 2 diabetes and coronary heart disease. However, the molecular mechanisms underlying such programming are poorly defined. Here we demonstrate that programmed changes in miRNA expression link early-life nutrition to long-term health. Specifically, we show that miR-483-3p is upregulated in adipose tissue from low-birth-weight adult humans and prediabetic adult rats exposed to suboptimal nutrition in early life. We demonstrate that manipulation of miR-483-3p levels in vitro substantially modulates the capacity of adipocytes to differentiate and store lipids. We show that some of these effects are mediated by translational repression of growth/differentiation factor-3, a target of miR-483-3p. We propose that increased miR-483-3p expression in vivo, programmed by early-life nutrition, limits storage of lipids in adipose tissue, causing lipotoxicity and insulin resistance and thus increasing susceptibility to metabolic disease.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta , Factor 3 de Diferenciación de Crecimiento/metabolismo , MicroARNs/metabolismo , Regiones no Traducidas 3' , Adulto , Animales , Animales Recién Nacidos , Secuencia de Bases , Diferenciación Celular , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Factor 3 de Diferenciación de Crecimiento/antagonistas & inhibidores , Factor 3 de Diferenciación de Crecimiento/genética , Células HEK293 , Humanos , Metabolismo de los Lípidos , Masculino , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas WistarRESUMEN
Lung T lymphocytes are important in pulmonary immunity and inflammation. It has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5%+/-1.9% (n=11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8+/-2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.
Asunto(s)
Separación Celular/métodos , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Pulmón/citología , Receptores de Quimiocina/metabolismo , Receptores Virales/metabolismo , Linfocitos T/citología , Complejo CD3 , Citometría de Flujo , Humanos , Pulmón/inmunología , Microesferas , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Povidona , Receptores CXCR6 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dióxido de Silicio , Linfocitos T/metabolismoRESUMEN
Several histone deacetylase inhibitors (HDACi), which have recently entered early clinical trials, exert their anticancer activity in part through the induction of apoptosis although the precise mechanism of this induction is not known. Induction of apoptosis by structurally diverse HDACi in primary cells from patients with chronic lymphocytic leukemia (CLL) and different leukemic cell lines was mediated by the Bcl-2 regulated intrinsic pathway and demonstrated a requirement for de novo protein synthesis. A marked time-dependent induction of the pro-apoptotic BH3-only proteins, Bim, Noxa and Bmf was observed, which preceded the induction of apoptosis. A key role for both Bim and Noxa was proposed in HDACi-mediated apoptosis based on our findings that siRNA for Bim and Noxa but not Bmf largely prevented the HDACi-induced loss in mitochondrial membrane potential, caspase processing and phosphatidylserine externalization. Noxa, induced by HDACi, in CLL cells and tumor cell lines, bound extensively to Mcl-1, a major anti-apoptotic Bcl-2 family member present in CLL cells. Our data strongly suggests that HDACi induce apoptosis primarily through inactivation of anti-apoptotic Bcl-2 family members by increases in Bim and Noxa and highlights these increases as a potential clinical target for CLL/lymphoma therapy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Humanos , Inmunoprecipitación , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype. Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription. In this study the potential roles of PKC-alpha and PKC-theta in MDR1 gene transcriptional regulation were explored. Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-length PKC-alpha or PKC-theta genes driven by the ecdysone promoter. Stable transfectants were selected by use of the appropriate antibiotics. Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and down-regulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA). These cells were used to analyse PKC-mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to -8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC-alpha expressing cells TPA caused activation of all promoter fragments to -29 bp. This finding suggests that TPA-inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR-1) in this region of the promoter may be due to activation of PKC-alpha. In contrast, PKC-theta activated only two MDR1 fragments, -982 and -612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transcription can be regulated by PKC-alpha and PKC-theta. The results support the search for therapeutic strategies directed specifically against PKC-alpha to ameliorate resistance of tumours against cytotoxic agents.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Regulación Enzimológica de la Expresión Génica , Isoenzimas/metabolismo , Regiones Promotoras Genéticas , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Western Blotting , Catálisis , Cloranfenicol O-Acetiltransferasa/metabolismo , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Genes Reporteros , Humanos , Indoles/farmacología , Maleimidas/farmacología , Fenotipo , Isoformas de Proteínas , Proteína Quinasa C-alfa , Proteína Quinasa C-theta , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Pharmacogenomics links individual drug response variation to genetic differences, such as single nucleotide polymorphisms (SNPs). In particular, pharmacogenomics will allow clinicians to use genetic diagnostics to predict the response of a patient to a drug. We investigated whether SNPs in opioid receptors correlated with the development of morphine tolerance in mouse strains that showed either high or low tolerance to morphine. Sequencing identified five silent SNPs in the delta opioid receptor that varied from the published sequence in some strains, but which were found in both high and low tolerance strains. The mu and kappa opioid receptor sequences had no SNPs. Taken together, these data definitively demonstrate that morphine tolerance development in mice is independent of opioid receptor sequence.
Asunto(s)
Sistema Nervioso Central/efectos de los fármacos , Tolerancia a Medicamentos/genética , Dependencia de Morfina/genética , Polimorfismo de Nucleótido Simple/fisiología , Receptores Opioides/genética , Animales , Secuencia de Bases/fisiología , Sistema Nervioso Central/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Datos de Secuencia Molecular , Morfina/farmacología , Dependencia de Morfina/metabolismo , Dependencia de Morfina/fisiopatología , Narcóticos/farmacología , Receptores Opioides/metabolismoRESUMEN
The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure. Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance. A cluster of overexpressed genes related to DOX resistance was observed. Included in this cluster was ABCB1 the P-glycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target. Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity. In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identified which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway. Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK. The findings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Carcinoma/genética , Doxorrubicina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Resistencia a Medicamentos/genética , Femenino , Amplificación de Genes , Eliminación de Gen , Perfilación de la Expresión Génica , Humanos , Fenotipo , Receptores de Estrógenos/análisisRESUMEN
ATPase transporter proteins are commonly found in the hepatocyte canalicular membrane. Some of these, in particular the multidrug resistance (mdr1b) gene, have been previously demonstrated to be inducible genes. In this study, we found that tamoxifen induced expression of the mdr1b gene in the liver up to 40-fold after 14 days' exposure to tamoxifen in the diet at a concentration of 420 ppm. As tamoxifen and its metabolites are primarily excreted into the bile, we investigated if the increased expression of mdr1b in the liver following tamoxifen exposure had any effect on its excretion in rats. We found that the excretion of tamoxifen and its metabolites into bile was increased from 8 +/- 1% to 51 +/- 18% (mean +/- SD) of an administered dose of 180 nmol/kg over a collection period of 3 hr in rats that had received tamoxifen (35 mg/kg) orally for 12 days (plus a 3-day rest) prior to the experiment. These data suggest that prolonged treatment with tamoxifen may result in lower serum and tumour concentrations, due to a self-mediated enhancement of excretion via mdr1b gene-encoded P-glycoprotein. This may have implications for other drugs sharing the same route of excretion and co-administered with tamoxifen.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Canalículos Biliares/metabolismo , Tamoxifeno/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Antineoplásicos Hormonales/farmacocinética , Antineoplásicos Hormonales/farmacología , Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Tasa de Depuración Metabólica , Ratas , Ratas Endogámicas Lew , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND: Tamoxifen forms DNA adducts in rat liver and causes an increased mutation frequency at the lacI genes in the livers of lambda/lacI transgenic rats. Although an elevated occurrence of endometrial cancer is found in a small proportion of breast cancer patients treated with tamoxifen, there is conflicting evidence on whether or not low levels of DNA adducts are formed in humans. METHODS: Based on the finding that the progression of DNA/RNA polymerases on templates might be blocked by bulky DNA adducts, we successfully developed and used a polymerase stop assay to map the sites of adduct formation in the target lacI gene following its reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase/H2O2 (HRP/H2O2) activated 4-hydroxytamoxifen. RESULTS: Using a T4 DNA polymerase stop assay, adduct formation in the lacI gene of the plasmid constructs, after the reaction in vitro with alpha-acetoxytamoxifen and HRP/H2O2 activated 4-hydroxytamoxifen, was found to mainly occur with guanines. In particular, one site of adenosine adduction was found on a triplet of adenosines located between two runs of guanines. CONCLUSION: The success of our development of DNA polymerase stop assay to map the sites of tamoxifen-DNA adducts formation will be very useful for the investigation of the mutagenicity/carcinogenicity of tamoxifen. The mutagenic potential of the tamoxifen adducted bases shall be further examined by transfecting the adducted plasmids into suitable human cell lines. Also, further investigations of the sequence specificity in specific oncogenes and tumor suppressor genes may be useful to explore the relationship between the occurrence of human endometrial cancer and tamoxifen treatment.
Asunto(s)
Proteínas Bacterianas/genética , Aductos de ADN/análisis , ADN Polimerasa Dirigida por ADN/farmacología , Proteínas de Escherichia coli , Antagonistas de Estrógenos/metabolismo , Proteínas Represoras/genética , Tamoxifeno/metabolismo , Animales , Neoplasias Endometriales/inducido químicamente , Femenino , Humanos , Represoras Lac , Mutágenos/metabolismo , Ratas , Tamoxifeno/toxicidadRESUMEN
We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.
Asunto(s)
Aductos de ADN/metabolismo , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Mutágenos/metabolismo , Tamoxifeno/metabolismo , Bacteriófago T4/enzimología , Secuencia de Bases , Aductos de ADN/química , Aductos de ADN/genética , Análisis Mutacional de ADN/métodos , ADN Polimerasa I , ADN Polimerasa Dirigida por ADN , Operón Lac , Datos de Secuencia Molecular , Mutágenos/química , Radioisótopos de Fósforo/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Proteínas ViralesRESUMEN
Tamoxifen, a rat liver carcinogen, can induce mutations in the lacI gene in the livers of lambda/lacI transgenic rats. However, the presence of persistent tamoxifen adducts on the liver DNA raises the possibility that some contribution to the mutagenesis from ex vivo mutations during the in vitro lacI assay cannot be ruled out. To address this issue, mutagenesis at the cII gene of the transgenic shuttle vector was determined using a selection based assay which is unaffected by the presence of tamoxifen-DNA adducts. Female lambda/lacI transgenic rats were dosed orally with tamoxifen (20 mg/kg body wt) daily for 6 weeks, causing a 3.2-fold increase in the mutant frequency (MF) in the cII gene compared with that obtained with solvent treated animals. This was similar to the MF found previously at the lacI gene and confirms that tamoxifen is mutagenic in vivo. The major class of mutation induced by tamoxifen in the cII gene was G:C-->T:A transversions as was found previously in the lacI gene. However, in the one unreplicated study of mutations in the p53 gene of liver tumours induced by tamoxifen, no G:C-->T:A transversions were found; possible differences between mutagenesis in normal and tumour tissues are explored. The major proportion of the G:C-->T:A transversions occurred at 5'-CpG-3' dinucleotide (CpG) sites in the lacI gene, but not at such sites in the cII gene. The methylation of CpG sites greatly enhances the targeting of deoxyguanosine by carcinogens, thus this finding might be explained by differences in the methylation patterns at their respective CpG sites; however, nothing is known about the methylation status of either the lacI nor the cII gene in this transgenic rat. This study raises the important issue of which target genes (mammalian or transgenic) should be used as endpoints in mammalian mutagenesis assays.
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Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Hígado/efectos de los fármacos , Proteínas Represoras/genética , Tamoxifeno/toxicidad , Factores de Transcripción/genética , Transgenes/efectos de los fármacos , Aflatoxina B1/toxicidad , Animales , Animales Modificados Genéticamente , Islas de CpG , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Femenino , Vectores Genéticos/genética , Represoras Lac , Hígado/química , Pruebas de Mutagenicidad , Mutación Puntual , Ratas , Proteínas ViralesRESUMEN
Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2beta and gamma are integral membrane proteins, whereas alpha is intranuclear. When truncated recombinant human LAP2beta proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1-408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2. 5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2beta is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane-chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.
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Núcleo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Sistema Libre de Células , Cromatina/metabolismo , Clonación Molecular , Cartilla de ADN/genética , Replicación del ADN/efectos de los fármacos , ADN Complementario/genética , Femenino , Humanos , Técnicas In Vitro , Laminas , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Electrónica , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/ultraestructura , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.
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Neoplasias de la Mama/prevención & control , Genes fos/genética , Isoflavonas/uso terapéutico , Lignanos/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinógenos/metabolismo , Femenino , Humanos , Masoprocol/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The activity of glucose-6-phosphatase (G-6-Pase) in isolated rat microsomes was inhibited by a new selective inhibitor of the multi-subunit G-6-Pase system, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3,4-dihydroxy-5-(3-imid azo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (compound A) with a 50% inhibitory concentration (IC50) of approximately 10 nmol/l. Compound A (500 nmol/l) inhibited the uptake of [14C]glucose-6-phosphate (G-6-P) into intact isolated rat microsomes, confirming that this agent blocks G-6-P translocation, as suggested by previous studies using intact and permeabilized microsomes. The inhibition of microsomal G-6-P transport by compound A was associated with inhibition of the rate of glucose output from rat hepatocytes incubated in the presence of 25 nmol/l glucagon (IC50 approximately 320 nmol/l.) Compound A (1 micromol/l) also inhibited the basal rate of glucose production by rat hepatocytes by 47%. Intraperitoneal administration of compound A to fasted mice lowered circulating plasma glucose concentrations dose-dependently at doses as low as 1 mg/kg. This effect was comparatively short-lived; glucose lowering was maximal at 30 min after dosing with 100 mg/kg compound A (-71%) and declined thereafter, being reversed within 3 h. A similar time course of glycemic response was observed in fasted rats; glucose lowering was maximal 30 min after dosing with 100 mg/kg compound A (-36%) and declined until the effect was fully reversed by 3 h postdose. In rats subjected to compound A treatment, liver glycogen content was increased. G-6-P and lactate levels were maximally elevated 30 min after dosing and declined thereafter. Cumulatively, these results suggest that the mechanism of glucose lowering by compound A was via inhibition of G-6-Pase activity, mediated through inhibition of the T1 subunit of the microsomal G-6-Pase enzyme system. Drug levels measured over the same time course as that used to assess in vivo efficacy peaked within 30 min of administration, then declined, which is consistent with the transient changes in plasma glucose and liver metabolites.
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Glucemia/metabolismo , Ácidos Ciclohexanocarboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes , Fosfotransferasas/antagonistas & inhibidores , Animales , Antiportadores , Ácidos Ciclohexanocarboxílicos/química , Ácidos Ciclohexanocarboxílicos/farmacocinética , Glucosa/biosíntesis , Prueba de Tolerancia a la Glucosa , Glucosa-6-Fosfato/metabolismo , Glucógeno/metabolismo , Cinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Microsomas Hepáticos/enzimología , Estructura Molecular , Proteínas de Transporte de Monosacáridos , Obesidad/sangre , Ratas , Ratas Sprague-DawleyRESUMEN
Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. We have recently demonstrated that the MDR1 promoter is transcriptionally activated by the HTLV-I tax protein, providing an explanation for the development of drug resistance in HTLV-I infections. Here we report that HTLV-I mediated MDR1 activation is dependent on the presence of an NF-IL6-binding site located between base pairs -148 and -141 relative to the transcription start site. This finding opens up the possibility of moderating P-gp expression through interference with NF-IL6 binding to its trans recognition element and subsequent repression of MDR1 transcription.
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Productos del Gen tax/farmacología , Genes MDR/genética , Virus Linfotrópico T Tipo 1 Humano/fisiología , Activación Transcripcional , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Células COS , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Expresión Génica , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacosRESUMEN
At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.
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Membrana Nuclear/ultraestructura , Animales , Microscopía Electrónica/métodosRESUMEN
Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I-infected subjects was investigated. Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp-mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that the MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Regulación Neoplásica de la Expresión Génica , Genes MDR , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Leucemia de Células T/genética , Leucemia de Células T/virología , Adulto , Productos del Gen tax/genética , Humanos , PlásmidosRESUMEN
The multidrug resistance protein 2 (Mrp2) also called canalicular multidrug resistance protein (cMrp) or canalicular multispecific organic anion transporter (cMoat) is a transmembrane export pump located at the canalicular domain of hepatocytes. Mrp2 transports a broad spectrum of organic anions including glucuronides, glutathione conjugates, and organic sulphates into bile. Based on previous observations in rat hepatocytes, the inducibility of mrp2 gene expression in primate liver was investigated in rhesus monkeys treated with tamoxifen or rifampicin. It was found that treatment with tamoxifen (25 mg/kg per day; over 7 days) or rifampicin (15 mg/kg per day; over 7 days) leading to an induction of cytochrome P450 3A4, resulted in a strong increase in mrp2 mRNA in the liver of male and female rhesus monkeys. On the protein level, tamoxifen also was a highly effective inducer, while rifampicin showed some inducing effect in a female and was inactive in a male monkey. In sections of paraffin-embedded liver tissue, immunofluorescence staining confirmed the results of Western blot analysis. Induced Mrp2 was localized to the canalicular domain of the hepatocytes. In conclusion, our data show inducibility of mrp2 gene expression in the liver of primates which may represent an adaptive response aimed at the enhanced biliary elimination of the inducing drugs and/or their metabolites.