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1.
PLoS One ; 9(12): e115327, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25514749

RESUMEN

Small-interfering RNAs and microRNAs are small ∼21-22 nucleotide long RNAs capable of posttranscriptional suppression of gene expression. The synthetic siRNAs are especially designed to target pre-specified genes and are common molecular biology tools. The miRNAs are endogenous regulators of gene expression found in a wide variety of eukaryotes. miRNAs are currently utilized for diagnostics applications. Therapeutically, various miRNA-antagonizing tools are being explored and miRNAs are also utilized for cell-specific inhibition of the expression of gene therapy vectors harboring target sites for specific miRNAs. Here we show, for the first time, that siRNAs and miRNAs can be harnessed to induce gene expression. We designed special expression vectors in which target sites for artificial siRNAs or endogenous miRNAs are located between the transgene and an Upstream Inhibitory Region (UIR). We hypothesized that cleavage of the mRNA by siRNAs or miRNAs will separate the transgene from the UIR and the resulting uncapped mRNA will be capable of being translated. A UIR composed of seven open reading frames was found to be the most efficient inhibitor of the translation of the downstream transgene. We show that under such a configuration both artificial siRNAs and endogenous miRNAs were capable of inducing transgene expression. We show that using the diphtheria toxin A-chain gene, in combination with target sites for highly expressed miRNAs, specific induction of cell-death can be achieved, setting the stage for application to cancer therapy.


Asunto(s)
Regulación de la Expresión Génica/genética , Terapia Genética/métodos , Vectores Genéticos/genética , MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Toxina Diftérica , Perfilación de la Expresión Génica , Células HEK293 , Humanos , MicroARNs/genética , Sistemas de Lectura Abierta/genética , Fragmentos de Péptidos , ARN Interferente Pequeño/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transgenes/genética
2.
Proc Natl Acad Sci U S A ; 111(21): 7849-54, 2014 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-24825887

RESUMEN

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Adipocitos/metabolismo , Adipocitos/fisiología , Animales , Compuestos Azo , Ácidos Grasos no Esterificados/sangre , Humanos , Immunoblotting , Ratones , Ratones Transgénicos , Peroxidasas , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína Desacopladora 1
3.
J Endocrinol ; 197(2): 325-33, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18434362

RESUMEN

We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.


Asunto(s)
Leptina/sangre , Receptores de Leptina/genética , Secuencia de Aminoácidos , Animales , Bioensayo , Bovinos , Línea Celular , Pollos , Humanos , Datos de Secuencia Molecular , Receptores de Leptina/química , Xenopus
4.
Methods Mol Biol ; 342: 139-57, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16957373

RESUMEN

MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression controlling central biological processes. These small, approx 22-nucleotide (nt)-long RNA molecules induce translational suppression when they are imperfectly matched to their target messenger RNA (mRNA) or direct mRNA cleavage when perfectly, or nearly perfectly, matched to their target. Direct roles in developmental processes have been described in a variety of species, and involvement in human diseases, such as cancer and diabetes, has been implied. These studies highlight the need to obtain detailed expression profiles of miRNAs in tissues, during development, and in disease. Their small size and the existence of miRNA families of related sequences pose critical problems in approaching expression analysis of miRNAs, especially using high-throughput approaches. All methodologies presented here address the special requirements for the analysis of miRNA expression using a variety of platforms, including cloning, microarrays, and microbeads. The different variables, as well as the different approaches, used by various laboratories are detailed and general recommendations are provided.


Asunto(s)
Perfilación de la Expresión Génica/métodos , MicroARNs/genética , MicroARNs/metabolismo , Animales , Humanos , MicroARNs/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Nat Genet ; 37(7): 766-70, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15965474

RESUMEN

MicroRNAs are noncoding RNAs of approximately 22 nucleotides that suppress translation of target genes by binding to their mRNA and thus have a central role in gene regulation in health and disease. To date, 222 human microRNAs have been identified, 86 by random cloning and sequencing, 43 by computational approaches and the rest as putative microRNAs homologous to microRNAs in other species. To prove our hypothesis that the total number of microRNAs may be much larger and that several have emerged only in primates, we developed an integrative approach combining bioinformatic predictions with microarray analysis and sequence-directed cloning. Here we report the use of this approach to clone and sequence 89 new human microRNAs (nearly doubling the current number of sequenced human microRNAs), 53 of which are not conserved beyond primates. These findings suggest that the total number of human microRNAs is at least 800.


Asunto(s)
Genoma Humano , MicroARNs/análisis , Secuencia de Bases , Secuencia Conservada , Humanos , Análisis por Micromatrices , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Alineación de Secuencia , Análisis de Secuencia de ADN
6.
Genome Res ; 14(12): 2486-94, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15574827

RESUMEN

MicroRNAs (MIRs) are a novel group of conserved short approximately 22 nucleotide-long RNAs with important roles in regulating gene expression. We have established a MIR-specific oligonucleotide microarray system that enables efficient analysis of the expression of the human MIRs identified so far. We show that the 60-mer oligonucleotide probes on the microarrays hybridize with labeled cRNA of MIRs, but not with their precursor hairpin RNAs, derived from amplified, size-fractionated, total RNA of human origin. Signal intensity is related to the location of the MIR sequences within the 60-mer probes, with location at the 5' region giving the highest signals, and at the 3' end, giving the lowest signals. Accordingly, 60-mer probes harboring one MIR copy at the 5' end gave signals of similar intensity to probes containing two or three MIR copies. Mismatch analysis shows that mutations within the MIR sequence significantly reduce or eliminate the signal, suggesting that the observed signals faithfully reflect the abundance of matching MIRs in the labeled cRNA. Expression profiling of 150 MIRs in five human tissues and in HeLa cells revealed a good overall concordance with previously published results, but also with some differences. We present novel data on MIR expression in thymus, testes, and placenta, and have identified MIRs highly enriched in these tissues. Taken together, these results highlight the increased sensitivity of the DNA microarray over other methods for the detection and study of MIRs, and the immense potential in applying such microarrays for the study of MIRs in health and disease.


Asunto(s)
Sondas de ADN/genética , Regulación de la Expresión Génica , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencia de Bases , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Células HeLa , Humanos , Masculino , MicroARNs/genética , Hibridación de Ácido Nucleico , Placenta/metabolismo , Alineación de Secuencia , Testículo/metabolismo , Timo/metabolismo
7.
Invest Ophthalmol Vis Sci ; 45(10): 3796-805, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15452091

RESUMEN

PURPOSE: Ischemic proliferative retinopathy, which occurs as a complication of diabetes mellitus, prematurity, or retinal vein occlusion, is a major cause of blindness worldwide. In addition to retinal neovascularization, it involves retinal degeneration, of which apoptosis is the main cause. A prior report has described the cloning of a novel HIF-1-responsive gene, RTP801, which displays strong hypoxia-dependent upregulation in ischemic cells of neuronal origin, both in vitro and in vivo. Moreover, inducible overexpression of RTP801 promotes the apoptotic death of differentiated neuron-like PC12 cells and increases their sensitivity to ischemic injury and oxidative stress. The purpose of the study was to examine the potential role of RTP801 in the pathogenesis of retinopathy, using RTP801-deficient mice. METHODS: Wild-type and RTP801-knockout mice were used in a model of retinopathy of prematurity (ROP). Their retinas were collected at postnatal day (P)14 and P17. They were examined by fluorescein angiography and by analysis of VEGF expression, neovascularization, and apoptosis. RESULTS: The expression of RTP801 was induced in the wild-type retina after hypoxia treatment. The retinal expression of VEGF after transfer to normoxic conditions was similarly upregulated in both wild-type and knockout mice. Nevertheless, the retinas of the RTP801-knockout mice in an ROP model showed a significant reduction in retinal neovascularization (P < 0.0001) and in the number of apoptotic cells in the inner nuclear layer (P < 0.0001). CONCLUSIONS: In the absence of RTP801 expression, development of retinopathy in the mouse model of ROP was significantly attenuated, thus implying an important role of RTP801 in the pathogenesis of ROP.


Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/fisiología , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/prevención & control , Factores de Transcripción/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Humanos , Hiperoxia/complicaciones , Hibridación in Situ , Recién Nacido , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno/toxicidad , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Retinopatía de la Prematuridad/etiología , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patología , Factores de Transcripción/deficiencia , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismo
8.
Bone ; 34(2): 246-60, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14962803

RESUMEN

Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.


Asunto(s)
Huesos/fisiología , Condrocitos/fisiología , Osteocitos/fisiología , Biosíntesis de Proteínas , Células Madre/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Fracturas Óseas/genética , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Cráneo/fisiología , Estrés Mecánico , Regulación hacia Arriba
9.
Oncogene ; 22(6): 797-806, 2003 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-12584558

RESUMEN

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.


Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Transducción de Señal/genética , Transactivadores/metabolismo , Receptor fas/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Factor 2 Relacionado con NF-E2 , Transactivadores/biosíntesis , Transactivadores/genética
10.
Clin Cancer Res ; 8(12): 3813-9, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12473594

RESUMEN

PURPOSE: In this study, we first sought to evaluate whether individuals heterozygous for ATM mutations may have an increased susceptibility to radiation-induced breast cancer (BC) after treatment for Hodgkin's disease (HD). We next sought to determine the frequency of ATM variants in patients with Hodgkin's lymphoma, regardless of coexisting BC, compared with healthy volunteers. EXPERIMENTAL DESIGN: Full sequence analysis of ATM was performed on cDNA from peripheral blood lymphocytes from 37 cases of BC after therapeutic radiation therapy for HD and 27 comparison cases with HD and no BC treated during the same time period. The frequency of ATM variants was analyzed in the total group of 64 cases of HD and compared to allele frequencies in 128 ethnically matched controls from the same geographical region. RESULTS: No protein-truncating ATM mutations were observed in cases with HD with or without BC. Missense mutations were more frequent in the cohort with HD compared with patients with BC following HD (P = 0.02). The median time from HD to the development of BC was 18 years in patients with ATM variants compared with 16 years in those with no ATM variants (P = 0.04). Multiple ATM variants, including one homozygous mutation, were observed in 9 HD cases. CONCLUSIONS: Heterozygous protein-truncating or missense mutations of ATM were not associated with increased radiation-associated risk of BC after HD. The observation of multiple germ-line mutations and a homozygote suggests that rare ATM variants may constitute cancer-susceptibility alleles in a subset of cases.


Asunto(s)
Neoplasias de la Mama/etiología , Frecuencia de los Genes/genética , Enfermedad de Hodgkin/radioterapia , Neoplasias Inducidas por Radiación/etiología , Proteínas Serina-Treonina Quinasas/genética , Adolescente , Adulto , Anciano , Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Proteínas de Ciclo Celular , Células Cultivadas , Niño , Estudios de Cohortes , ADN Complementario/análisis , Proteínas de Unión al ADN , Exones/genética , Femenino , Humanos , Linfocitos/sangre , Linfocitos/metabolismo , Masculino , Mutación , Neoplasias Inducidas por Radiación/genética , ARN Neoplásico/sangre , Proteínas Supresoras de Tumor
11.
Oncogene ; 21(39): 6017-31, 2002 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-12203114

RESUMEN

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (gamma- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H(2)O(2). Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.


Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Northern Blotting , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , División Celular , Supervivencia Celular , Clonación Molecular , Cartilla de ADN/química , Doxorrubicina/farmacología , Glioma/metabolismo , Glioma/patología , Humanos , Peróxido de Hidrógeno/farmacología , Hipoxia/metabolismo , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
12.
Genes Dev ; 16(16): 2058-72, 2002 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-12183361

RESUMEN

beta-catenin and plakoglobin (gamma-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. beta-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by beta-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either beta-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulated cell growth, enhanced motility, induced transformation, and produced rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression was elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreased Nr-CAM expression and antibodies to Nr-CAM inhibited the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by beta-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility.


Asunto(s)
Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Neoplasias del Colon/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Melanoma/metabolismo , Transactivadores/biosíntesis , Factores de Transcripción/biosíntesis , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Western Blotting , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Desmoplaquinas , Genes Dominantes , Humanos , Luciferasas/metabolismo , Factor de Unión 1 al Potenciador Linfoide , Masculino , Ratones , Ratones Desnudos , Microscopía Fluorescente , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , ARN/metabolismo , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transactivadores/metabolismo , Activación Transcripcional , Transducción Genética , Transfección , Células Tumorales Cultivadas , Cicatrización de Heridas , beta Catenina , gamma Catenina
13.
Mol Cell Biol ; 22(7): 2283-93, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11884613

RESUMEN

Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.


Asunto(s)
Apoptosis/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Diferenciación Celular , Clonación Molecular , Proteínas de Unión al ADN/química , Humanos , Peróxido de Hidrógeno/farmacología , Hipoxia/genética , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hibridación in Situ , Liposomas/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Datos de Secuencia Molecular , Células PC12 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras , Homología de Secuencia de Aminoácido , Accidente Cerebrovascular/genética , Factores de Transcripción/química , Células Tumorales Cultivadas , Regulación hacia Arriba
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