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The expression of cytochrome P450 (CYP) enzymes is highly variable and associated with factors, such as age, genotype, sex, and disease states. In this study, quantification of metronidazole metabolizing CYP isoforms (CYP2A6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7) in human liver microsomes from 115 children and 35 adults was performed using a quantitative proteomics method. The data confirmed age-dependent increase in CYP2A6, CYP2E1, and CYP3A4 abundance, whereas, as expected, CYP3A7 abundance showed postnatal decrease with age. In particular, the fold difference (neonatal to adulthood levels) in the protein abundance of CYP2A6, CYP2E1, and CYP3A4 was 14, 11, and 20, respectively. In contrast, protein abundance of CYP3A7 was > 125-fold higher in the liver microsomes of neonates than of adults. The abundance of CYP2A6 and CYP3A5 was associated with genotypes, rs4803381 and rs776746, respectively. A proteomics-informed physiologically based pharmacokinetic (PBPK) model was developed to describe the pharmacokinetics of metronidazole and its primary metabolite, 2-hydroxymethylmetronidazole. The model revealed an increase in the metabolite-to-parent ratio with age and showed a strong correlation between CYP2A6 abundance and metabolite formation (r2 = 0.75). Notably, the estimated contribution of CYP3A7 was ~ 75% in metronidazole clearance in neonates. These data suggest that variability in CYP2A6 and CYP3A7 in younger children poses the risk of variable pharmacokinetics of metronidazole and its active metabolite with a potential impact on drug efficacy and safety. No sex-dependent difference was observed in the protein abundance of the studied CYPs. The successful integration of hepatic CYP ontogeny data derived from a large liver bank into the pediatric PBPK model of metronidazole can be extended to other drugs metabolized by the studied CYPs.
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BACKGROUND: Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. RESULTS: Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. CONCLUSION: These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.
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Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Melanocitos/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Melaninas/biosíntesis , Melaninas/genética , Melanocitos/citología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Neurodegenerative diseases require characterization based on underlying biology using biochemical biomarkers. Mixed pathology complicates discovery of biomarkers and characterization of cohorts, but inclusion of greater numbers of patients with different, related diseases with frequently co-occurring pathology could allow better accuracy. Combining cohorts collected from different studies would be a more efficient use of resources than recruiting subjects from each population of interest for each study. OBJECTIVE: To explore the possibility of combining existing datasets by controlling pre-analytic variables in the Alzheimer's Disease Neuroimaging Initiative (ADNI) and Parkinson's Progression Markers Initiative (PPMI) studies. METHODS: Cerebrospinal fluid (CSF) was collected and processed from 30 subjects according to both the ADNI and PPMI protocols. Relationships between reported levels of Alzheimer's disease (AD) and Parkinson's disease (PD) biomarkers in the same subject under each protocol were examined. RESULTS: Protocol-related differences were observed for Aß, but not t-tau or α-syn, and trended different for p-tau and pS129. Values of α-syn differed by platform. Conversion of α-syn values between ADNI and PPMI platforms did not completely eliminate differences in distribution. DISCUSSION: Factors not captured in the pre-analytical sample handling influence reported biomarker values. Assay standardization and better harmonized characterization of cohorts should be included in future studies of CSF biomarkers.
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Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Enfermedad de Parkinson/diagnóstico , Proteínas tau/líquido cefalorraquídeo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeo , FosforilaciónRESUMEN
The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by â¼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine.
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Genotipo , Glucuronosiltransferasa/metabolismo , Microsomas Hepáticos/enzimología , Adolescente , Factores de Edad , Analgésicos Opioides/farmacología , Antimetabolitos/farmacología , Niño , Preescolar , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Lactante , Recién Nacido , Microsomas Hepáticos/efectos de los fármacos , Morfina/farmacología , Adulto Joven , Zidovudina/farmacologíaRESUMEN
Association of Alzheimer's disease (AD) with cerebral glucose hypometabolism, likely due to impairments of insulin signaling, has been reported recently, with encouraging results when additional insulin is provided to AD patients. Here, we tested the potential effects of the anti-diabetic vanadium, vanadyl (IV) acetylacetonate (VAC), on AD in vitro and in vivo models. The experimental results showed that VAC at sub-micromolar concentrations improved the viability of neural cells with or without increased ß-amyloid (Aß) burden; and in APP/PS1 transgenic mice, VAC treatment (0.1 mmol kg-1 d-1) preserved cognitive function and attenuated neuron loss, but did not reduce brain Aß plaques. Further studies revealed that VAC attenuated Aß pathogenesis by (i) activation of the PPARγ-AMPK signal transduction pathway, leading to improved glucose and energy metabolism; (ii) up-regulation of the expression of glucose-regulated protein 75 (Grp75), thus suppressing p53-mediated neuronal apoptosis under Aß-related stresses; and (iii) decreasing toxic soluble Aß peptides. Overall, our work suggested that vanadyl complexes may have great potential for effective therapeutic treatment of AD.
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Enfermedad de Alzheimer/prevención & control , Encéfalo/efectos de los fármacos , Hipoglucemiantes/farmacología , Compuestos Organometálicos/farmacología , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Hipoglucemiantes/química , Ratones , Compuestos Organometálicos/química , Vanadatos/química , Vanadio/químicaRESUMEN
The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (â¼10-fold) with â¼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.
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Andrógenos/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hígado/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Variaciones en el Número de Copia de ADN/genética , Femenino , Genotipo , Humanos , Inactivación Metabólica/genética , Lactante , Recién Nacido , Masculino , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Testosterona/metabolismo , Adulto JovenRESUMEN
Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.
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Diferenciación Celular , ADN Helicasas/metabolismo , Melanocitos/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Elementos de Facilitación Genéticos , Expresión Génica , Regulación de la Expresión Génica , Melaninas/biosíntesis , Glicoproteínas de Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidorreductasas/genéticaRESUMEN
The age-dependent absolute protein abundance of carboxylesterase (CES) 1 and CES2 in human liver was investigated and applied to predict infant pharmacokinetics (PK) of oseltamivir. The CES absolute protein abundance was determined by liquid chromatography-tandem mass spectrometry proteomics in human liver microsomal and cytosolic fractions prepared from tissue samples obtained from 136 pediatric donors and 35 adult donors. Two surrogate peptides per protein were selected for the quantification of CES1 and CES2 protein abundance. Purified CES1 and CES2 protein standards were used as calibrators, and the heavy labeled peptides were used as the internal standards. In hepatic microsomes, CES1 and CES2 abundance (in picomoles per milligram total protein) increased approximately 5-fold (315.2 vs. 1664.4) and approximately 3-fold (59.8 vs. 174.1) from neonates to adults, respectively. CES1 protein abundance in liver cytosol also showed age-dependent maturation. Oseltamivir carboxylase activity was correlated with protein abundance in pediatric and adult liver microsomes. The protein abundance data were then used to model in vivo PK of oseltamivir in infants using pediatric physiologically based PK modeling and incorporating the protein abundance-based ontogeny function into the existing pediatric Simcyp model. The predicted pediatric area under the curve, maximal plasma concentration, and time for maximal plasma concentration values were below 2.1-fold of the clinically observed values, respectively.
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Envejecimiento/metabolismo , Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Hígado/enzimología , Modelos Biológicos , Oseltamivir/farmacocinética , Adulto , Cromatografía Liquida , Citosol/efectos de los fármacos , Citosol/enzimología , Humanos , Técnicas In Vitro , Lactante , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Oseltamivir/sangre , Proteómica , Espectrometría de Masas en TándemRESUMEN
Precision medicine approach has a potential to ensure optimum efficacy and safety of drugs at individual patient level. Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models could play a significant role in precision medicine by predicting interindividual variability in drug disposition and response. In order to develop robust PBPK/PD models, it is imperative that the critical physiological parameters affecting drug disposition and response and their variability are precisely characterized. Currently used PBPK/PD modeling software, for example, Simcyp and Gastroplus, encompass information such as organ volumes, blood flows to organs, body fat composition, glomerular filtration rate, etc. However, the information on the interindividual variability of the majority of the proteins associated with PK and PD, for example, drug metabolizing enzymes, transporters, and receptors, are not fully incorporated into these PBPK modeling platforms. Such information is significant because the population factors such as age, genotype, disease, and gender can affect abundance or activity of these proteins. To fill this critical knowledge gap, mass spectrometry-based quantitative proteomics has emerged as an important technique to characterize interindividual variability in the protein abundance of drug metabolizing enzymes, transporters, and receptors. Integration of these quantitative proteomics data into in silico PBPK/PD modeling tools will be crucial toward precision medicine.
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Modelos Biológicos , Preparaciones Farmacéuticas/metabolismo , Medicina de Precisión/métodos , Proteómica/métodos , Animales , Proteínas Portadoras/metabolismo , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Polimorfismo Genético/fisiología , Medicina de Precisión/tendencias , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteómica/tendencias , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.
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Sistema de Señalización de MAP Quinasas , Melanoma/genética , Melanoma/metabolismo , Factores de Transcripción/genética , Sustitución de Aminoácidos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células Cultivadas , ADN Helicasas/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Melanoma/patología , Mutación , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to directly activate genes that encode components of peripheral myelin.
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Vaina de Mielina/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Citometría de Flujo , Immunoblotting , Ratones , Vaina de Mielina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción SOXE/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Pathological cardiac hypertrophy is characterized by a sustained increase in cardiomyocyte size and re-activation of the fetal cardiac gene program. Previous studies implicated SWI/SNF chromatin remodeling enzymes as regulators of the fetal cardiac gene program in surgical models of cardiac hypertrophy. Although hypertension is a common risk factor for developing cardiac hypertrophy, there has not yet been any investigation into the role of SWI/SNF enzymes in cardiac hypertrophy using genetic models of hypertension. In this study, we tested the hypothesis that components of the SWI/SNF complex are activated and recruited to promoters that regulate the fetal cardiac gene program in hearts that become hypertrophic as a result of salt induced hypertension. Utilizing the Dahl salt-sensitive (S) rat model, we found that the protein levels of several SWI/SNF subunits required for heart development, Brg1, Baf180, and Baf60c, are elevated in hypertrophic hearts from S rats fed a high salt diet compared with normotensive hearts from Dahl salt-resistant (R) rats fed the same diet. Furthermore, we detected significantly higher levels of SWI/SNF subunit enrichment as well as evidence of more accessible chromatin structure on two fetal cardiac gene promoters in hearts from S rats compared with R rats. Our data implicate SWI/SNF chromatin remodeling enzymes as regulators of gene expression in cardiac hypertrophy resulting from salt induced hypertension. Thus we provide novel insights into the epigenetic mechanisms by which salt induced hypertension leads to cardiac hypertrophy.
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Cardiomegalia/fisiopatología , Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Hipertensión/fisiopatología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Cardiomegalia/genética , Ensamble y Desensamble de Cromatina/genética , Modelos Animales de Enfermedad , Expresión Génica , Histonas/genética , Histonas/metabolismo , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas Dahl/genética , Ratas Endogámicas Dahl/metabolismo , Ratas Endogámicas Dahl/fisiología , Cloruro de Sodio Dietético/metabolismo , Activación TranscripcionalRESUMEN
There exists a general recognition of the fact that LXR-α, being a member of the nuclear receptor family, plays a crucial role in the biological process that connects inflammation, cholesterol homeostasis, and cellular decisions. In this context the present study was addressed to understand the role of LXR-α gene in the selective and specific reprogramming of cancer cells into a state of apoptosis leaving the normal cells unaffected. The results of this study revealed that LXR-α gene when activated in cancerous cells of diverse origin results in the regulation of genes coding for Bcl-2, AATF, and Par-4 in a fashion, forcing these cells to enter into the state of apoptosis leaving the normal cells unaffected. On the basis of this study we propose that in near future LXR-α agonist (Withaferin A) may definitely find its use in the therapeutic interventions directed towards the treatment of cancer.
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Apoptosis/efectos de los fármacos , Neoplasias/patología , Receptores Nucleares Huérfanos/metabolismo , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Factor de Transcripción E2F1/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Reporteros , Humanos , Hidroxicolesteroles/farmacología , Ligandos , Receptores X del Hígado , Neoplasias/metabolismo , Receptores Nucleares Huérfanos/genética , Unión Proteica , Interferencia de ARN , Elementos de Respuesta , Transducción de Señal , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/ultraestructura , Transcripción Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Witanólidos/farmacologíaRESUMEN
The discovery of cooperativity between pRB and E2F greatly prompted various investigators to find how E2F biology contributes to oncogenesis. Although E2F family of transcription factors have been linked to proliferation, apoptosis and differentiation, yet no heed has been paid to understand the role of E2F biology in cellular quiescence. To understand the functional RNomics (regulation of gene transcription through RNA interference) of E2F-1 gene, 2 cancer cell lines, such as Jurkat exhibiting E2F-1 gene overamplification and Hela-229 exhibiting intrinsic downregulation of E2F-1 gene expression, were used in our study. E2F-1 gene knockdown via siRNA within Jurkat cells resulted in upregulation of genes characteristic of quiescence both translationally and transcriptionally, which was accompanied by downregulation of genes at both translational and transcriptional level involved in cell cycle progression and apoptosis. This genomic phenomenon also translated into ultrastructural and phenotypic features typical of quiescent state. These observed results in Jurkat cells were simulated by upregulation of E2F-1 gene in Hela-229 cells through the downregulation of miR 17-5p. This E2F-1-regulated pathway explained as to how Jurkat cells entered exclusively into quiescent state when E2F-1 was downregulated in these cells and how Hela-229 cells proliferate vigorously when E2F-1 was upregulated in such cells. Here, we propose a gene-regulatory pathway which the cell might be using in its entry into either quiescent or proliferative states. Furthermore, this pathway may be useful in designing strategies for the treatment of cancer in general and acute lymphoblastic leukemia in particular.
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Biomarcadores de Tumor/genética , Proliferación Celular , Factor de Transcripción E2F1/genética , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/genética , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/metabolismo , Perfilación de la Expresión Génica , Células HeLa , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismoRESUMEN
The study, addressed to explore the transcriptional expression and regulation of Apoptosis-antagonizing transcription factor (AATF) gene within various types of human leukemic cell lines, revealed that AATF gene was overexpressed ubiquitously in all the leukemic cell lines studied and this upregulation was accompanied by c-myc gene overamplification in these cells. Downregulation of AATF gene transcription within leukemic cells not only resulted in the downregulation of c-myc gene and vice-versa but also contributed to apoptosis leading to cell death. Further, the link between AATF expression and leukemic cellular apoptosis involved PI3K/Akt pathway. Based on these results we propose that AATF gene may be of crucial importance in maintaining the leukemic state of a cell compartment through its ability to initiate cell proliferation coupled with repression of cellular apoptosis.