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1.
Nature ; 513(7516): 65-70, 2014 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-25079319

RESUMEN

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.


Asunto(s)
Regiones no Traducidas 5'/genética , Factor 4A Eucariótico de Iniciación/metabolismo , G-Cuádruplex , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Biosíntesis de Proteínas , Animales , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Secuencia de Bases , Línea Celular Tumoral , Epigénesis Genética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Motivos de Nucleótidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Triterpenos/farmacología
2.
Blood ; 125(1): 13-21, 2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25320243

RESUMEN

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.


Asunto(s)
Regulación Leucémica de la Expresión Génica , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Alelos , Animales , Línea Celular Tumoral , Supervivencia Celular , Estudios de Cohortes , Metilación de ADN , Epigénesis Genética , Femenino , Histonas/química , Humanos , Inmunofenotipificación , Interleucinas/metabolismo , Masculino , Ratones , Mutación , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores Sexuales , Linfocitos T/citología
3.
Int J Mol Sci ; 18(5)2017 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-28448471

RESUMEN

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and ß-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.


Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Animales , Enzimas Desubicuitinizantes/metabolismo , Humanos , Lisosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , beta-Arrestinas/metabolismo
4.
Biochem Soc Trans ; 44(2): 601-5, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-27068976

RESUMEN

Ubiquitination is a post-translational modification that targets proteins for degradation but can also regulate other cellular processes such as endocytosis, trafficking and DNA repair. We investigate ubiquitination of the dopamine D4receptor (D4R) which belongs to the superfamily of G protein-coupled receptors (GPCR). Several polymorphic variants of the D4R exist, which differ in the number of 16-amino acid repeats in the third intracellular loop (IC3) of the receptor. The functional role of this polymorphic region is not known but persons with the seven-repeat allele show a predisposition to develop attention deficit hyperactivity disorder (ADHD). We identified a protein, KLHL12, which specifically interacts with this polymorphic region and enhances ubiquitination of the D4R. We have tested the influence of KLHL12 on the ubiquitination of the most common D4R polymorphic variants and found that KLHL12 strongly promotes ubiquitination of the two- and four-repeat variant but has hardly any effect on ubiquitination of the seven-repeat D4R. This suggests that differential ubiquitination of the D4R may have functional implications. Moreover, we were able to demonstrate that KLHL12-mediated D4R ubiquitination does not lead to receptor degradation. Next, we aimed to identify specific residues in the sequence of D4R which undergo ubiquitination and observed that the lysine-less receptor mutant is still ubiquitinated. Subsequently, we have tested the hypothesis whether KLHL12 could promote ubiquitination on non-lysine residues of the D4R. The importance of the cysteine and serine/threonine residues in the ubiquitination process of the receptor was examined and the obtained results confirmed that D4R can be ubiquitinated on non-lysine residues. In this review we summarize our data on D4R ubiquitination and put this in the light of other GPCR ubiquitination studies.


Asunto(s)
Receptores de Dopamina D4/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Humanos , Lisina/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores de Dopamina D4/química , Ubiquitinación
5.
Haematologica ; 99(8): 1326-33, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24895337

RESUMEN

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia. In this context, our previous description of a cooperative microRNA regulatory network controlling several well-known T-cell acute lymphoblastic leukemia tumor suppressor genes, including PHF6, is of great importance. Given the high frequency of PHF6 lesions in T-cell acute lymphoblastic leukemia and the integration of PHF6 in this microRNA regulatory network, we aimed to identify novel oncogenic microRNAs in T-cell acute lymphoblastic leukemia which suppress PHF6. To this end, we performed an unbiased PHF6 3'UTR-microRNA library screen and combined the results with microRNA profiling data of samples from patients with T-cell acute lymphoblastic leukemia and normal thymocyte subsets. We selected miR-128-3p as a candidate PHF6-targeting, oncogenic microRNA and demonstrated regulation of PHF6 expression upon modulation of this microRNA in T-cell acute lymphoblastic leukemia cell lines. In vivo evidence of an oncogenic role of this microRNA in T-cell acute lymphoblastic leukemia was obtained through accelerated leukemia onset in a NOTCH1-induced T-cell acute lymphoblastic leukemia mouse model upon miR-128-3p over-expression. We conclude that miR-128-3p is a strong novel candidate oncogenic microRNA in T-cell acute lymphoblastic leukemia which targets the PHF6 tumor suppressor gene.


Asunto(s)
Proteínas Portadoras/genética , Marcación de Gen/métodos , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Animales , Células HEK293 , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Proteínas Represoras
6.
Haematologica ; 99(12): 1808-16, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25344525

RESUMEN

Genetic studies in T-cell acute lymphoblastic leukemia have uncovered a remarkable complexity of oncogenic and loss-of-function mutations. Amongst this plethora of genetic changes, NOTCH1 activating mutations stand out as the most frequently occurring genetic defect, identified in more than 50% of T-cell acute lymphoblastic leukemias, supporting a role as an essential driver for this gene in T-cell acute lymphoblastic leukemia oncogenesis. In this study, we aimed to establish a comprehensive compendium of the long non-coding RNA transcriptome under control of Notch signaling. For this purpose, we measured the transcriptional response of all protein coding genes and long non-coding RNAs upon pharmacological Notch inhibition in the human T-cell acute lymphoblastic leukemia cell line CUTLL1 using RNA-sequencing. Similar Notch dependent profiles were established for normal human CD34(+) thymic T-cell progenitors exposed to Notch signaling activity in vivo. In addition, we generated long non-coding RNA expression profiles (array data) from ex vivo isolated Notch active CD34(+) and Notch inactive CD4(+)CD8(+) thymocytes and from a primary cohort of 15 T-cell acute lymphoblastic leukemia patients with known NOTCH1 mutation status. Integration of these expression datasets with publicly available Notch1 ChIP-sequencing data resulted in the identification of long non-coding RNAs directly regulated by Notch activity in normal and malignant T cells. Given the central role of Notch in T-cell acute lymphoblastic leukemia oncogenesis, these data pave the way for the development of novel therapeutic strategies that target hyperactive Notch signaling in human T-cell acute lymphoblastic leukemia.


Asunto(s)
Biomarcadores de Tumor/genética , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , ARN Largo no Codificante/genética , Receptor Notch1/metabolismo , Timocitos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Biomarcadores de Tumor/metabolismo , Western Blotting , Estudios de Casos y Controles , Transformación Celular Neoplásica/patología , Células Cultivadas , Inmunoprecipitación de Cromatina , Estudios de Cohortes , Inhibidores Enzimáticos/farmacología , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Timocitos/citología , Timocitos/efectos de los fármacos
7.
Br J Haematol ; 154(3): 337-48, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21569010

RESUMEN

Chromosomal rearrangements involving the MECOM (MDS1 and EVI1 complex) locus are recurrent genetic events in myeloid leukaemia and are associated with poor prognosis. In this study, we assessed the role of MECOM locus protein EVI1 in the transcriptional regulation of microRNAs (miRNAs) involved in the leukaemic phenotype. For this, we profiled expression of 366 miRNAs in 38 MECOM-rearranged patient samples, normal bone marrow controls and MECOM (EVI1) knock down/re-expression models. Cross-comparison of these miRNA expression profiling data showed that MECOM rearranged leukaemias are characterized by down regulation of MIR449A. Reconstitution of MIR449A expression in MECOM-rearranged cell line models induced apoptosis resulting in a strong decrease in cell viability. These effects might be mediated in part by MIR449A regulation of NOTCH1 and BCL2, which are shown here to be bona fide MIR449A targets. Finally, we confirmed that MIR449A repression is mediated through direct promoter occupation of the EVI1 transcriptional repressor. In conclusion, this study reveals MIR449A as a crucial direct target of the MECOM locus protein EVI1 involved in the pathogenesis of MECOM-rearranged leukaemias and unravels NOTCH1 and BCL2 as important novel targets of MIR449A. This EVI1-MIR449A-NOTCH1/BCL2 regulatory axis might open new possibilities for the development of therapeutic strategies in this poor prognostic leukaemia subgroup.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo/fisiología , Leucemia/metabolismo , MicroARNs/biosíntesis , Proto-Oncogenes/fisiología , Factores de Transcripción/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Supervivencia Celular , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Lactante , Leucemia/genética , Leucemia/patología , Proteína del Locus del Complejo MDS1 y EV11 , MicroARNs/genética , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , ARN Neoplásico/genética , Receptor Notch1/biosíntesis , Receptor Notch1/fisiología , Elementos Reguladores de la Transcripción/fisiología , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas
8.
Cell Mol Life Sci ; 67(12): 1971-86, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20165900

RESUMEN

Dopamine is an important neurotransmitter that regulates several key functions in the brain, such as motor output, motivation and reward, learning and memory, and endocrine regulation. Dopamine does not mediate fast synaptic transmission, but rather modulates it by triggering slow-acting effects through the activation of dopamine receptors, which belong to the G-protein-coupled receptor superfamily. Besides activating different effectors through G-protein coupling, dopamine receptors also signal through interaction with a variety of proteins, collectively termed dopamine receptor-interacting proteins. We focus on the dopamine D4 receptor, which contains an important polymorphism in its third intracellular loop. This polymorphism has been the subject of numerous studies investigating links with several brain disorders, such as attention-deficit hyperactivity disorder and schizophrenia. We provide an overview of the structure, signalling properties and regulation of dopamine D4 receptors, and briefly discuss their physiological and pathophysiological role in the brain.


Asunto(s)
Encéfalo/metabolismo , Receptores de Dopamina D4/metabolismo , Recompensa , Esquizofrenia/metabolismo , Transmisión Sináptica , Animales , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Cricetinae , Dopamina/metabolismo , Proteínas de Unión al GTP/metabolismo , Memoria , Proteínas/metabolismo , Transducción de Señal
9.
PLoS One ; 13(3): e0194017, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29522551

RESUMEN

INTRODUCTION: Despite the established contribution of deregulated microRNA (miRNA) function to carcinogenesis, relatively few miRNA-cancer gene interactions have been validated, making it difficult to appreciate the true complexity of miRNA-cancer gene regulatory networks. RESULTS: In this effort, we identify miRNA interactomes of 17 well-established cancer genes, involved in various cancer types, through a miRNome-wide 3' UTR reporter screening. Using a novel and performant strategy for high-throughput screening data analysis, we identify 390 interactions, quadrupling the size of the known miRNA interactome for the cancer genes under investigation. Clear enrichments of established and predicted interactions underscore the validity of the interactome data set. Interactomes appear to be primarily driven by canonical binding site interactions. Nonetheless, non-canonical binding sites, such as offset 6mer and seed-mismatched or G:U wobble sites, also have regulatory activity, albeit clearly less pronounced. Furthermore, we observe enhanced regulation in the presence of 3' supplementary pairing for both canonical and non-canonical binding sites. CONCLUSIONS: Altogether, the cancer gene-miRNA interactome data set represents a unique resource that will aid in the unraveling of regulatory miRNA networks and the dynamic regulation of key protein-coding cancer genes. In addition, it uncovers aspects of the functional miRNA binding site's architecture and the relative contributions of different binding site types.


Asunto(s)
Regiones no Traducidas 3'/genética , Redes Reguladoras de Genes , Ensayos Analíticos de Alto Rendimiento/métodos , MicroARNs/genética , Oncogenes , Área Bajo la Curva , Sitios de Unión , Conjuntos de Datos como Asunto , Genes Reporteros , Humanos , Anotación de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Curva ROC , Reproducibilidad de los Resultados , Transfección
10.
Cell Signal ; 28(8): 1001-14, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27155323

RESUMEN

Dopamine receptors are G protein-coupled receptors involved in regulation of cognition, learning, movement and endocrine signaling. The action of G protein-coupled receptors is highly regulated by multifunctional proteins, such as ß-arrestins which can control receptor desensitization, ubiquitination and signaling. Previously, we have reported that ß-arrestin 2 interacts with KLHL12, a BTB-Kelch protein which functions as an adaptor in a Cullin3-based E3 ligase complex and promotes ubiquitination of the dopamine D4 receptor. Here, we have investigated the molecular basis of the interaction between KLHL12 and ß-arrestins and questioned its functional relevance. Our data demonstrate that ß-arrestin 1 and ß-arrestin 2 bind constitutively to the most common dopamine D4 receptor polymorphic variants and to KLHL12 and that all three proteins can interact within a single macromolecular complex. Surprisingly, stimulation of the receptor has no influence on the association between these proteins or their cellular distribution. We found that Cullin3 also interacts with both ß-arrestins but has no influence on their ubiquitination. Knockout of one of the two ß-arrestins hampers neither interaction between the dopamine D4 receptor and KLHL12, nor ubiquitination of the receptor. Finally, our results indicate that p44/42 MAPK phosphorylation, the signaling pathway which is often regulated by ß-arrestins is not influenced by KLHL12, but seems to be exclusively mediated by Gαi protein upon dopamine D4 receptor stimulation.


Asunto(s)
Proteínas de Microfilamentos/metabolismo , Receptores de Dopamina D4/metabolismo , beta-Arrestinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Cullin/metabolismo , Dopamina/farmacología , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Secuencia Kelch , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Biológicos , Proteínas Mutantes/metabolismo , Toxina del Pertussis/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Multimerización de Proteína/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos
11.
PLoS One ; 10(12): e0145654, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26717573

RESUMEN

DOPAMINE D4 RECEPTOR POLYMORPHISM: The dopamine D4 receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder. KLHL12 PROMOTES UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR ON NON-LYSINE RESIDUES: In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D4 receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D4 receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible. DIFFERENTIAL UBIQUITINATION OF THE DOPAMINE D4 RECEPTOR POLYMORPHIC VARIANTS: Additionally, we show that the dopamine D4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D4.2 and D4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process.


Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/genética , Lisina/genética , Proteínas de Microfilamentos/genética , Polimorfismo Genético/genética , Receptores de Dopamina D4/genética , Ubiquitinación/genética , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Genotipo , Células HEK293 , Humanos
12.
Exp Hematol ; 43(8): 625-39, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26123366

RESUMEN

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood cancer that accounts for about 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. It is considered as a paradigm for the multistep nature of cancer initiation and progression. Genetic and epigenetic reprogramming events, which transform T-cell precursors into malignant T-ALL lymphoblasts, have been extensively characterized over the past decade. Despite our comprehensive understanding of the genomic landscape of human T-ALL, leukemia patients are still treated by high-dose multiagent chemotherapy, potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable acute and long-term side effects, about 15% of pediatric and 40% of adult T-ALL patients still relapse, owing to acquired therapy resistance, and present with very dismal survival perspectives. Unfortunately, the molecular mechanisms by which residual T-ALL tumor cells survive chemotherapy and act as a reservoir for leukemic progression and hematologic relapse remain poorly understood. Nevertheless, it is expected that enhanced molecular understanding of T-ALL disease biology will ultimately facilitate a targeted therapy driven approach that can reduce chemotherapy-associated toxicities and improve survival of refractory T-ALL patients through personalized salvage therapy. In this review, we summarize recent biological insights into the molecular pathogenesis of T-ALL and speculate how the genetic landscape of T-ALL could trigger the development of novel therapeutic strategies for the treatment of human T-ALL.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Reprogramación Celular , Epigénesis Genética , Trasplante de Células Madre Hematopoyéticas , Células Precursoras de Linfocitos T , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adolescente , Adulto , Animales , Niño , Preescolar , Humanos , Lactante , Células Precursoras de Linfocitos T/metabolismo , Células Precursoras de Linfocitos T/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia
13.
Nat Commun ; 6: 5794, 2015 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-25565005

RESUMEN

Early T-cell precursor leukaemia (ETP-ALL) is a high-risk subtype of human leukaemia that is poorly understood at the molecular level. Here we report translocations targeting the zinc finger E-box-binding transcription factor ZEB2 as a recurrent genetic lesion in immature/ETP-ALL. Using a conditional gain-of-function mouse model, we demonstrate that sustained Zeb2 expression initiates T-cell leukaemia. Moreover, Zeb2-driven mouse leukaemia exhibit some features of the human immature/ETP-ALL gene expression signature, as well as an enhanced leukaemia-initiation potential and activated Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signalling through transcriptional activation of IL7R. This study reveals ZEB2 as an oncogene in the biology of immature/ETP-ALL and paves the way towards pre-clinical studies of novel compounds for the treatment of this aggressive subtype of human T-ALL using our Zeb2-driven mouse model.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Leucemia de Células T/fisiopatología , Proteínas Represoras/genética , Transducción de Señal/fisiología , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Técnicas Histológicas , Proteínas de Homeodominio/inmunología , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Quinasas Janus/metabolismo , Estimación de Kaplan-Meier , Cariotipificación , Luciferasas , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Interleucina-7/metabolismo , Proteínas Represoras/inmunología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc
14.
PLoS One ; 9(10): e109091, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25310091

RESUMEN

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species.


Asunto(s)
Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Pez Cebra/genética , Animales , Expresión Génica , Perfilación de la Expresión Génica/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia
15.
Sci Signal ; 7(352): ra111, 2014 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-25406379

RESUMEN

The posttranscriptional control of gene expression by microRNAs (miRNAs) is highly redundant, and compensatory effects limit the consequences of the inactivation of individual miRNAs. This implies that only a few miRNAs can function as effective tumor suppressors. It is also the basis of our strategy to define functionally relevant miRNA target genes that are not under redundant control by other miRNAs. We identified a functionally interconnected group of miRNAs that exhibited a reduced abundance in leukemia cells from patients with T cell acute lymphoblastic leukemia (T-ALL). To pinpoint relevant target genes, we applied a machine learning approach to eliminate genes that were subject to redundant miRNA-mediated control and to identify those genes that were exclusively targeted by tumor-suppressive miRNAs. This strategy revealed the convergence of a small group of tumor suppressor miRNAs on the Myb oncogene, as well as their effects on HBP1, which encodes a transcription factor. The expression of both genes was increased in T-ALL patient samples, and each gene promoted the progression of T-ALL in mice. Hence, our systematic analysis of tumor suppressor miRNA action identified a widespread mechanism of oncogene activation in T-ALL.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor , Genes myb/genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Traslado Adoptivo , Animales , Inteligencia Artificial , Trasplante de Células Madre Hematopoyéticas , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Modelos Genéticos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismo
16.
PLoS One ; 8(2): e56308, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23457546

RESUMEN

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs.


Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/patología , Células Mieloides/efectos de los fármacos , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Daunorrubicina/farmacología , Etopósido/farmacología , Femenino , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Células Mieloides/metabolismo , Células Mieloides/patología , Proto-Oncogenes/genética , Factores de Transcripción/genética
17.
PLoS One ; 8(8): e71776, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23977142

RESUMEN

BACKGROUND: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. RESULTS: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5). CONCLUSIONS: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.


Asunto(s)
Elementos Alu , Perfilación de la Expresión Génica/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Inhibidores de la Angiogénesis/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Secuencia Conservada , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , Estándares de Referencia , Proteínas Represoras , Transcriptoma/efectos de los fármacos , Tretinoina/farmacología , Witanólidos/farmacología
18.
Biochem Pharmacol ; 81(8): 1004-15, 2011 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-21300032

RESUMEN

Uncontrolled expression of IL-6 in the central nervous system is associated with neurodegenerative pathology and glioma development. Astrocytes are the predominant source of IL-6 in the central nervous system, and they are characteristically susceptible to synergistic IL-6 expression. Combined ß-adrenergic and TNF-receptor triggering induces synergistic IL-6 expression in 1321N1 cells via a transcriptional enhancer mechanism. Here, we have investigated the molecular basis of the very potent "super"-synergistic IL-6 expression that is apparent after combined treatment of astrocytes with a ß-adrenergic agonist, isoproterenol, and the inflammatory cytokines TNF-α and IL-1ß. We found that IL-1ß treatment strengthens the IL-6 synergy by inducing a distinct stabilization of IL-6 mRNA. Surprisingly, the mRNA-stabilizing effect seems to be dependent on protein kinase C (PKC), but not on the prototypical mRNA-stabilizing kinase p38. Moreover, although the mRNA-binding protein HuR basally stabilizes IL-6 mRNA, the mRNA-stabilizing effect of IL-1ß is independent of HuR. Our data using pharmacological inhibitors suggest PKC is an important modulator of IL-6 expression in the central nervous system and this might have therapeutic implications.


Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Astrocitos/efectos de los fármacos , Interleucina-1beta/farmacología , Interleucina-6/biosíntesis , Isoproterenol/farmacología , ARN Mensajero/biosíntesis , Astrocitos/inmunología , Astrocitos/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Genes Reporteros , Humanos , Interleucina-6/genética , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología
19.
FEBS J ; 278(8): 1333-44, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21320289

RESUMEN

Dopamine D(4) receptors (D(4) Rs) are G protein-coupled receptors that play a role in attention and cognition. In the present study, we investigated the dimerization properties of this receptor. Western blot analysis of the human D(4.2)R, D(4.4)R and D(4.7)R revealed the presence of higher molecular weight immunoreactive bands, which might indicate the formation of receptor dimers and multimers. Homo- and heterodimerization of the receptors was confirmed by co-immunoprecipitation and bioluminescence resonance energy transfer studies. Although dimerization of a large number of G protein-coupled receptors has been described, the functional importance often remains to be elucidated. Folding efficiency is rate-limiting for D(4)R biogenesis and quality control in the endoplasmic reticulum plays an important role for D(4)R maturation. Co-immunoprecipitation and immunofluorescence microscopy studies using wild-type and a nonfunctional D(4.4)R folding mutant show that oligomerization occurs in the endoplasmic reticulum and that this plays a role in the biogenesis and cell surface targeting of the D(4)R. The different polymorphic repeat variants of the D(4)R display differential sensitivity to the chaperone effect. In the present study, we show that this is also reflected by bioluminescence resonance energy transfer saturation assays, suggesting that the polymorphic repeat variants have different relative affinities to form homo- and heterodimers. In summary, we conclude that D(4)Rs form oligomers with different affinities and that dimerization plays a role in receptor biogenesis.


Asunto(s)
Receptores de Dopamina D4/metabolismo , Dimerización , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Mediciones Luminiscentes , Pliegue de Proteína , Receptores de Dopamina D4/química
20.
Nat Genet ; 43(7): 673-8, 2011 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-21642990

RESUMEN

The importance of individual microRNAs (miRNAs) has been established in specific cancers. However, a comprehensive analysis of the contribution of miRNAs to the pathogenesis of any specific cancer is lacking. Here we show that in T-cell acute lymphoblastic leukemia (T-ALL), a small set of miRNAs is responsible for the cooperative suppression of several tumor suppressor genes. Cross-comparison of miRNA expression profiles in human T-ALL with the results of an unbiased miRNA library screen allowed us to identify five miRNAs (miR-19b, miR-20a, miR-26a, miR-92 and miR-223) that are capable of promoting T-ALL development in a mouse model and which account for the majority of miRNA expression in human T-ALL. Moreover, these miRNAs produce overlapping and cooperative effects on tumor suppressor genes implicated in the pathogenesis of T-ALL, including IKAROS (also known as IKZF1), PTEN, BIM, PHF6, NF1 and FBXW7. Thus, a comprehensive and unbiased analysis of miRNA action in T-ALL reveals a striking pattern of miRNA-tumor suppressor gene interactions in this cancer.


Asunto(s)
Redes Reguladoras de Genes , Genes Supresores de Tumor , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Adulto , Animales , Apoptosis , Biomarcadores de Tumor/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Niño , Preescolar , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Lactante , Luciferasas/metabolismo , Masculino , Ratones , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Adulto Joven
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