RESUMEN
Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.
RESUMEN
Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.
Asunto(s)
Melanoma/genética , Mutación , Fosfohidrolasa PTEN/fisiología , Proteínas Proto-Oncogénicas B-raf/genética , Proteína de Retinoblastoma/fisiología , Proteínas Supresoras de Tumor/fisiología , Quinasas raf/fisiología , Animales , Quinasa 4 Dependiente de la Ciclina/genética , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas B-raf/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiologíaAsunto(s)
Crisis Blástica/genética , Proteínas de Unión al ADN/genética , Leucemia Mielomonocítica Crónica/genética , Trastornos Mieloproliferativos/genética , Proteínas de Neoplasias/genética , Mielofibrosis Primaria/genética , Factores de Transcripción/genética , Anciano , Sustitución de Aminoácidos , Crisis Blástica/mortalidad , Estudios de Cohortes , ADN de Neoplasias/genética , Dioxigenasas , Proteína Potenciadora del Homólogo Zeste 2 , Exones/genética , Femenino , Genotipo , Mutación de Línea Germinal , Humanos , Leucemia Mielomonocítica Crónica/mortalidad , Masculino , Mutación Missense , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Complejo Represivo Polycomb 2 , Polimorfismo de Nucleótido Simple , Mielofibrosis Primaria/mortalidad , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Análisis de Secuencia de ADNRESUMEN
The temporary disruption of reproductive activities due to equine coital exanthema (ECE), caused by equid herpesvirus 3 (EHV-3), at thoroughbred breeding facilities and embryo transfer centres, has an appreciable economic impact. The aim of the present study was to estimate the prevalence of excretion of EHV-3 in mares without clinical symptoms under field conditions and the re-excretion patterns of the virus in two seropositive (presumably latently infected) mares maintained in isolation for 11 mo. The EHV-3 virus was detected in perineal-vaginal swabs by real time PCR in 14 (6%) of 220 thoroughbred mares without clinical symptoms at the time of breeding. In the two isolated mares, re-excretion of EHV-3 was demonstrated on two occasions, 3 mo apart (each for a 3 d interval) in one mare, and on only 1 d in the other mare. Antibodies against EHV-3 were identified by seroneutralization in 105 (48%) of the thoroughbred mares, and during the entire period in the two isolated mares. Therefore, the present study provided evidence of EHV-3 shedders in a healthy mare population under both field and isolation conditions. Furthermore, at least two periods of spontaneous EHV-3 reactivation and re-excretion in the presence of serum antibodies occurred in one mare in an 11 mo interval. These findings could assist in the design and implementation of measures to minimize the spread of EHV-3 and control ECE outbreaks.
Asunto(s)
Infecciones por Herpesviridae/veterinaria , Herpesvirus Équido 3/aislamiento & purificación , Enfermedades de los Caballos/virología , Esparcimiento de Virus , Animales , Infecciones por Herpesviridae/virología , Herpesvirus Équido 3/fisiología , Enfermedades de los Caballos/inmunología , Caballos , Periodicidad , Activación ViralRESUMEN
The cytokine tumour necrosis factor alpha (TNF-alpha) has been shown to play a role in human immunodeficiency virus (HIV) replication by activating transcription of the provirus in both T cells and macrophages. Therefore, agents that block TNF-alpha-induced HIV expression could have therapeutic value in the treatment of AIDS. We have sought to identify antiviral agents that block TNF-alpha induction of HIV LTR-directed transcription, using a cell-based, virus-free assay system in automated high-throughput screening. HeLa cells were transfected with an HIV LTR-luciferase reporter plasmid and a stable line was isolated in which TNF-alpha increased luciferase production by two- to threefold. This cell line was used to screen approximately 15,000 fungal extracts. An inhibitory activity specific for TNF-alpha-induced HIV LTR transcription was observed in culture OS-F67406. The active component was isolated and identified as a known metabolite, 3-O-methylviridicatin, by NMR and mass spectrometry. No biological activity has been associated with this compound previously. This compound blocks TNF-alpha activation of the HIV LTR in the HeLa-based system, with an IC50 of 5 microM, and inhibited virus production in the OM-10.1 cell line, a model of chronic infection responsive to induction by TNF-alpha, with an IC50 of 2.5 microM.
Asunto(s)
Fármacos Anti-VIH/farmacología , Hongos/química , VIH/efectos de los fármacos , Hidroxiquinolinas/aislamiento & purificación , Quinolonas/aislamiento & purificación , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/efectos de los fármacos , VIH/fisiología , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Hidroxiquinolinas/química , Hidroxiquinolinas/farmacología , Pruebas de Sensibilidad Microbiana , Quinolonas/química , Quinolonas/farmacologíaRESUMEN
The viral transactivator Rev is essential for HIV replication, since it allows the nuclear export of unspliced and partially spliced viral mRNAs that encode the structural proteins. Rev is an RNA binding protein that interacts with a highly structured RNA element, the RRE, found within the envelope sequences. This viral protein also interacts with cellular proteins, termed nucleoporins, and acts as an adaptor between the viral mRNAs and the cellular nuclear export machinery. Both interactions are specific, and required for Rev function. Because of its crucial role in the HIV replication cycle, and its novel mechanism of action, Rev represents an ideal target for therapeutic intervention. This review describes the efforts towards Rev inhibition. Gene therapy approaches, including the expression of trans-dominant mutants and RNA decoys, as well as antisense therapies and small molecule inhibitors of Rev-RRE binding or Rev interaction with the cellular machinery will be discussed
Asunto(s)
Productos del Gen rev/genética , Productos del Gen rev/fisiología , Terapia Genética , Infecciones por VIH/genética , Infecciones por VIH/terapia , Humanos , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
We have described a protein (Hep27) [Donadel, G., Garzelli, C., Frank, R. & Gabrielli, F. (1991) Eur. J. Biochem. 195, 723-729] which is synthesized and accumulated in the nucleus of human hepatoblastoma (HepG2) cells, following growth arrest induced by butyrate treatment. The synthesis of Hep27 is inhibited in cells that, released from the butyrate block, have resumed DNA synthesis. This report describes the cloning and the characterization of the cDNA coding for the Hep27 protein. The translation of the Hep27 cDNA predicts an amino acid sequence that can be aligned with those of the known short-chain alcohol dehydrogenase enzymes (SCAD) family. Both the recognition of enzymic functional domains and the similarity with the SCAD family of proteins of several amino acid blocks throughout the molecule, strongly suggest that this protein is a new member of the SCAD family. In agreement with its nuclear localization Hep27 has a region similar to the bipartite nuclear-targeting sequence. The study of Hep27 mRNA expression and protein synthesis suggests the existence of a regulation at the post-transcriptional level. The possible nuclear role of the Hep27 protein is discussed.
Asunto(s)
Alcohol Deshidrogenasa/biosíntesis , Proteínas Nucleares/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasas , Alcohol Deshidrogenasa/genética , Oxidorreductasas de Alcohol , Secuencia de Aminoácidos , Secuencia de Bases , Carbonil Reductasa (NADPH) , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Clonación Molecular , ADN Complementario/genética , Fase G1 , Humanos , Hidroxiesteroide Deshidrogenasas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Over the last 7 years we have carried out a major research effort focused on gene transcription as a novel approach to drug discovery. The goal is to identify small molecular weight compounds that modulate the expression of a target gene in a specific manner, thereby either increasing or decreasing the concentration of the corresponding protein product. Transcriptional modulation not only provides a potential means to replace recombinant proteins as drugs, but also provides a novel approach to manipulate key gene targets in many therapeutic areas. This article describes some of the features and advantages of transcription-based pharmaceuticals and illustrates how this approach can be applied to drug discovery with a program we are pursuing to identify new treatments for sickle cell disease and beta-thalassemia.
Asunto(s)
Diseño de Fármacos , Expresión Génica/efectos de los fármacos , Anemia de Células Falciformes/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica , Globinas/genética , HumanosRESUMEN
T-cell antigen receptor triggering results in activation of protein kinase C and mobilization of calcium. These two signals are necessary and sufficient to activate the T-cell specific transcription factor NF-AT, which cooperates with other transcription factors activated by accessory signals to initiate expression of interleukin 2 and its receptor. The protein kinase C mediated pathway involves activation of ras proteins. In a Jurkat cell model of T-cell activation, treatment with antigen receptor agonists results in induction of expression of a reporter gene under the control of a NF-AT dependent promoter. Overexpression of the ras GTPase activating protein p120GAP in these cells caused a significant inhibition of T-cell antigen receptor mediated induction, suggesting a role for p120GAP in regulation of ras. The inhibition was overcome by expression of a valine-12 mutant ras which lacks GTPase activity.
Asunto(s)
GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica/genética , Genes ras/genética , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Humanos , Plásmidos , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/genética , Células Tumorales CultivadasRESUMEN
A reporter gene under the control of a T-cell antigen receptor element was activated in Jurkat cells by antigen receptor triggering or by a combination of phorbol myristate acetate, which activates protein kinase C, and a calcium ionophore. Both these signals were necessary for expression of the reporter gene. When co-transfected with a construct capable of overexpressing the tyrosine kinase p59fyn, the reporter gene was activated by PMA alone. Thus p59fyn could replace the calcium ionophore but not activation of protein kinase C. The activation by p59fyn plus PMA was blocked by EGTA and by the immunosuppressant drug cyclosporin A.
Asunto(s)
Calcio/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Calcimicina/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Ácido Egtácico/farmacología , Humanos , Factores de Transcripción NFATC , Plásmidos , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes/metabolismo , Linfocitos T/fisiología , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/genética , Transfección , Células Tumorales CultivadasRESUMEN
The type I interleukin-1 receptor (IL-1R) is capable of transducing a signal resulting in promoter activation in T cells. This signal transduction is dependent on the cytoplasmic domain, which consists of 213 amino acids. In contrast to the type I receptor, the type II IL-1R has a small cytoplasmic tail, and it is not clear whether this receptor is a signal-transducing or a regulatory molecule. Here we report that the type II IL-1R does not mediate gene activation in Jurkat cells. However, a hybrid receptor composed of the extracellular and transmembrane regions of the human type II interleukin-1 fused to the cytoplasmic domain of the human type I IL-1R was capable of transducing a signal across the membrane resulting in a pattern of gene activation identical to that mediated by the type I IL-1R. Our results indicated that the extracellular domain of the type II IL-1R was capable of functionally interacting with interleukin-1 and transmitting the resulting signal to a heterologous cytoplasmic domain.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Receptores de Interleucina-1/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Expresión Génica/efectos de los fármacos , Humanos , Cinética , Datos de Secuencia Molecular , FN-kappa B/genética , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/genética , Receptores de Interleucina-1/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Virus 40 de los Simios/genética , Linfocitos T , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
T-cell antigen receptor engagement results in suboptimal activation of protein kinase C and a prolonged increase in intracellular free calcium concentration. These signals, in combination with stimulation via accessory molecules usually supplied by the antigen presenting cell, activate expression of interleukin-2 (IL-2) and initiate autocrine growth. The lymphocyte-specific tyrosine kinase p56lck is physically associated with CD4 and is brought into close proximity of the intracellular domain of the antigen receptor by CD4 recognition of the major histocompatibility complex during antigen presentation. p56lck activation enhances and may be essential for antigen receptor signaling. We report that a constitutively active form of p56lck delivers a signal which contributes to IL-2 promoter activation. The signal substituted for a calcium-mobilizing signal in a Jurkat cell model of T-cell activation. The activation was sensitive to EGTA and cyclosporin A, indicating that p56lck functions at an early stage of the calcium-mediated pathway. The transcription factor NF-AT mediated, at least in part, the p56lck activation of IL-2 expression. In addition, activated p56lck synergized with constitutively active p21Ha-ras, which can replace protein kinase C activation, resulting in activation of NF-AT in the absence of external signals.
Asunto(s)
Calcio/metabolismo , Ciclosporina/metabolismo , Interleucina-2/genética , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Interleucina-2/metabolismo , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Mutación , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , TransfecciónRESUMEN
In a Jurkat cell model of T-cell activation an interleukin-2 promoter/reporter gene construct was activated by antigen receptor agonism in combination with the lymphokine interleukin-1. Antigen receptor signals could be mimicked by suboptimal activation of protein kinase C (PKC) with phorbol esters in combination with calcium mobilization by an ionophore. In cotransfection experiments, oncogenic rats obviated the need for PKC stimulation but did not replace either the calcium signal or interleukin-1. Activated ras expression also replaced the requirement for PKC stimulation in activation of the T-cell transcription factor NF-AT. A dominant inhibitory ras mutant specifically blocked antigen receptor agonism, indicating that ras activity is required for antigen receptor signaling. In addition, an inhibitor of PKC blocked both activated ras and phorbol ester stimulation, suggesting a role for ras upstream of PKC.
Asunto(s)
Activación de Linfocitos , Proteínas Nucleares , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Transducción de Señal , Linfocitos T/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Proteínas de Unión al ADN/fisiología , Expresión Génica , Genes Dominantes , Humanos , Interleucina-2/genética , Isoquinolinas/farmacología , Factores de Transcripción NFATC , Piperazinas/farmacología , Proteína Quinasa C/fisiología , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.
Asunto(s)
Aminoácidos/metabolismo , Proteínas de Drosophila , Hormonas de Insectos/metabolismo , Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular , Receptores Inmunológicos/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Cromatografía en Capa Delgada , Drosophila , Escherichia coli/metabolismo , Humanos , Hormonas de Insectos/genética , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores Inmunológicos/genética , Receptores de Interleucina-1 , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Receptores Toll-Like , Células Tumorales CultivadasRESUMEN
The evolutionary conservation of a sequence or part of it can help to identify the essential functional and structural domains within a protein. We have cloned and characterised a cDNA coding for the type-I interleukin-1 receptor (IL-1R) of chick (ch) embryo fibroblasts. The comparison of the amino acid (aa) sequences of the avian with that of murine (m) and human (h) IL-1Rs shows a 60% homology. The intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to the murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila melanogaster. The alignment between the chicken and D. melanogaster proteins shows the presence of four aa blocks with more than 80% homology. The possible functional significance of this homology is discussed. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain.
Asunto(s)
Proteínas de Drosophila , Interleucina-1 , Receptores de Antígenos de Linfocitos T/genética , Receptores de Superficie Celular , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Hormonas de Insectos/química , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/química , Receptores Inmunológicos/química , Receptores de Interleucina-1 , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Receptores Toll-LikeRESUMEN
We have used an interleukin-2 (IL-2) promoter-CAT fusion gene to study activation of IL-2 gene expression by IL-1, phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and calcium ionophore in the murine thymoma line EL4 and the human lymphoma line Jurkat. The two cell lines respond differently to combinations of these stimuli. IL-1 in combination with suboptimal concentration of PMA induced chloramphenicol acetyltransferase (CAT) activity in EL4. In Jurkat cells, IL-1 failed to synergize with PMA or PHA. Cotransfection with the IL-2/CAT gene and a construct capable of expressing murine T-cell type IL-1 receptors converted Jurkat cells to IL-1 responsiveness. IL-1 in combination with PHA but not with PMA resulted in induction of CAT activity in these cells. Induction of IL-2/CAT activity by all stimuli in both cell lines was blocked by the presence of EGTA in the culture medium. EGTA did not inhibit IL-1/PMA activation of an SV40 early promoter-CAT fusion gene in either EL4 or Jurkat cells; therefore, calcium was not required for IL-1 or PMA signal transduction. Jurkat cells were shown to differ from EL4 in their requirement for calcium mobilization. Two different calcium-dependent pathways of gene activation were distinguished, both of which were blocked by the immunosuppressive drug cyclosporin A.
Asunto(s)
Calcio/fisiología , Ciclosporina/farmacología , Regulación de la Expresión Génica , Interleucina-2/genética , Linfocitos T/metabolismo , Ácido Egtácico , Humanos , Interleucina-1/fisiología , Fitohemaglutininas , Regiones Promotoras Genéticas , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-1 , Transducción de Señal , Virus 40 de los Simios/genética , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.
Asunto(s)
Receptores Inmunológicos/fisiología , Transducción de Señal/genética , Autorradiografía , Sitios de Unión , Línea Celular/fisiología , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferasa , Clonación Molecular , Citoplasma/metabolismo , Análisis Mutacional de ADN , Regulación de la Expresión Génica/genética , Humanos , Interleucina-1/genética , Interleucina-1/fisiología , Receptores Inmunológicos/química , Receptores de Interleucina-1 , Proteínas Recombinantes de Fusión , Transducción de Señal/fisiología , Virus 40 de los SimiosRESUMEN
Several distinct octamer-binding transcription factors (OTFs) interact with the sequence ATTTGCAT (the octamer motif), which acts as a transcription regulatory element for a variety of differentially controlled genes. The ubiquitous OTF-1 plays a role in expression of the cell cycle-regulated histone H2b gene as well as several other genes, while the tissue-specific OTF-2 has been implicated in the tissue-specific expression of immunoglobulin genes. In an attempt to understand the apparent transcriptional selectivity of these factors, we have investigated the physical and functional characteristics of OTF-1 purified from HeLa cells and both OTF-1 and OTF-2 purified from B cells. High-resolution footprinting and mobility shift-competition assays indicated that these factors were virtually indistinguishable in binding affinities and DNA-protein contacts on either the H2b or an immunoglobulin light-chain (kappa) promoter. In addition, each of the purified factors showed an equivalent intrinsic capacity to activate transcription from either immunoglobulin promoters (kappa and heavy chain) or the H2b promoter in OTF-depleted HeLa and B-cell extracts. However, with OTF-depleted HeLa extracts, neither factor could restore immunoglobulin gene transcription to the relatively high level observed in unfractionated B-cell extracts. Restoration of full immunoglobulin gene activity appears to require an additional B-cell regulatory component which interacts with the OTFs. The additional B-cell factor could act either by facilitating interaction of OTF activation domains with components of the general transcriptional machinery or by contributing a novel activation domain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Genes de Inmunoglobulinas , Cadenas Ligeras de Inmunoglobulina/genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Bases , Clonación Molecular , Desoxirribonucleasa I , Células HeLa/inmunología , Células HeLa/metabolismo , Histonas/genética , Factor C1 de la Célula Huésped , Humanos , Cinética , Datos de Secuencia Molecular , Mapeo Nucleótido , Factor 1 de Transcripción de Unión a Octámeros , Factor 2 de Transcripción de Unión a Octámeros , Sondas de Oligonucleótidos , Plásmidos , Mapeo RestrictivoRESUMEN
The human class II gene, HLA-DR alpha, contains an octanucleotide sequence ATTTGCAT located approximately 40 base pairs upstream of the transcription initiation site. We have investigated the transcriptional function of the DR alpha octamer in human B-lymphoblastoid cells and non-B cells. Deletion and substitution mutagenesis of the octamer sequence greatly reduced the activity of the DR alpha promoter in both in vivo and in vitro cell-free transcription systems of B-cell origin. Conversely, these mutations did not affect promoter activity in several non-B-cell lines that express the DR alpha gene. Removal of octamer-binding proteins by in vivo titration with an octamer-containing competitor plasmid reduced DR alpha promoter activity in B-lymphoblastoid cells. These results suggest that a protein-octamer interaction, most likely involving the B-cell-specific octamer binding protein (OTF-2), is required for DR alpha promoter function in B-lymphoblastoid cells but not in non-B cells.