RESUMEN
The p53 tumor suppressor is a universal sensor of genotoxic stress that regulates the transcription of genes required for cell-cycle arrest and apoptosis. In response to DNA damage, the p53 protein is phosphorylated at its amino-terminus and becomes stabilized upon disruption of an interaction with its negative regulator, MDM2. Subsequent phosphorylation and acetylation of p53 promote different interactions with other proteins and with target gene regulatory elements to facilitate cell-cycle arrest, apoptosis, or adaptation in response to DNA damage. Downstream of p53, p21 is responsible for growth arrest in G1, but other p53 target genes are responsible for G2 cell-cycle arrest. In response to genotoxic insult, p53-induced apoptosis results from overlapping downstream pathways that both suppress mitogenic and survival signaling and promote pro-apoptotic signaling. Adaptation to DNA damage is manifested by p53-mediated expression of its negative regulator, MDM2. The frequency of observed mutations in p53 predicts that its inactivation is a requisite step in tumorigenesis, as p53 is mutated in approximately 50% of human tumors. Thus, it is likely that in the remaining tumors, genetic aberrations will occur in pathways that regulate p53 or in pathways directly downstream of p53. The advances in the understanding of p53 signaling over the past few years point to many potential overlapping signaling pathways, where mutations may occur as alternative modes to p53 mutation.
Asunto(s)
Mutágenos/farmacología , Proteínas Nucleares , Proteína p53 Supresora de Tumor/efectos de los fármacos , Daño del ADN , Regulación de la Expresión Génica , Humanos , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Células 3T3 , Animales , Elementos sin Sentido (Genética) , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , Clonación Molecular , Cósmidos , Cartilla de ADN , Embrión de Mamíferos , Exones , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Expresión Génica , Biblioteca Genómica , Hibridación in Situ , Intrones , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-mdm2 , Mapeo Restrictivo , Dedos de ZincRESUMEN
The colony stimulating factor-1 receptor (CSF-1R) affects mitogenic growth and gene expression in NIH 3T3 cells through signaling pathways that require the products of the c-ras and c-myc proto-oncogenes. In this work we tested the hypothesis that there is direct communication between the Ras and Myc pathways. In transient transfection assays Ras increased by 5-fold transcriptional transactivation by chimeric c-Myc-Gal4 proteins. A constitutive active form of the CSF-1R also stimulated this activity and co-expression of a dominant negative ras gene ablated receptor stimulation. Deletion analysis of the c-Myc N-terminal region demonstrated that amino acid residues between positions 92 and 143 are the targets for Ras action. Transactivation by chimeric Myc proteins that were stably expressed could be transiently enhanced by either CSF-1 or serum, with peak activity occurring 2 h after mitogen stimulation. The steady-state levels of the chimeric c-Myc transactivators were increased following stimulation with CSF-1 or serum, but this increase in steady-state protein level did not strictly correlate with the increase in transactivation activity. Thus, Ras signaling may directly affect the activity of the c-Myc N-terminal region.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Fragmentos de Péptidos/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas de Saccharomyces cerevisiae , Transducción de Señal , Factores de Transcripción , Activación Transcripcional , Proteínas ras/metabolismo , Células 3T3 , Animales , Sangre , Proteínas de Unión al ADN , Éteres Cíclicos/farmacología , Proteínas Fúngicas/genética , Expresión Génica , Genes myc , Genes ras , Humanos , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Mutagénesis , Ácido Ocadaico , Proteínas Recombinantes/farmacología , Transfección , Proteínas ras/farmacologíaRESUMEN
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Proto-Oncogénicas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Elementos de Facilitación Genéticos , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteína Proto-Oncogénica c-ets-1 , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas c-ets , Transducción de Señal , Treonina/genéticaRESUMEN
The mouse urokinase-type plasminogen activator (uPA) gene was used as a model macrophage colony-stimulating factor 1 (CSF-1)-inducible gene to investigate CSF-1 signalling pathways. Nuclear run-on analysis showed that induction of uPA mRNA by CSF-1 and phorbol myristate acetate (PMA) was at the transcriptional level in bone marrow-derived macrophages. CSF-1 and PMA synergized strongly in the induction of uPA mRNA, showing that at least some components of CSF-1 action are mediated independently of protein kinase C. Promoter targets of CSF-1 signalling were investigated with NIH 3T3 cells expressing the human CSF-1 receptor (c-fms). uPA mRNA was induced in these cells by treatment with CSF-1, and a PEA3/AP-1 element at -2.4 kb in the uPA promoter was involved in this response. Ets transcription factors can act through PEA3 sequences, and the involvement of Ets factors in the induction of uPA was confirmed by use of a dominant negative Ets-2 factor. Expression of the DNA binding domain of Ets-2 fused to the lacZ gene product prevented CSF-1-mediated induction of uPA mRNA in NIH 3T3 cells expressing the CSF-1 receptor. Examination of ets-2 mRNA expression in macrophages showed that it was also induced synergistically by CSF-1 and PMA. In the macrophage cell line RAW264, the uPA PEA3/AP-1 element mediated a response to both PMA and cotransfected Ets-2. uPA promoter constructs were induced 60- to 130-fold by Ets-2 expression, and the recombinant Ets-2 DNA binding domain was able to bind to the uPA PEA3/AP-1 element. This work is consistent with a proposed pathway for CSF-1 signalling involving sequential activation of fms, ras, and Ets factors.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/farmacología , Transcripción Genética/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/genética , Células 3T3 , Animales , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ésteres del Forbol/farmacología , ARN Mensajero/biosíntesis , Transducción de Señal , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Proteína Oncogénica gp140(v-fms)/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Secuencia de Bases , Sitios de Unión , Elementos Transponibles de ADN/genética , Genes fms , Genes ras , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Proto-Oncogénicas c-jun/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismoRESUMEN
Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC 286193), is a synthetic nucleoside inhibitor of inosine monophosphate dehydrogenase and blocks guanine nucleotide biosynthesis. In the present study, we examined the effects of tiazofurin on mouse erythroleukemia (MEL) cell differentiation and protooncogene expression. Tiazofurin induced hemoglobin production in MEL cells in a concentration-dependent manner, as measured by an increase in benzidine staining. Northern blot analysis of MEL cells treated with 7 mumol/L tiazofurin demonstrated accumulation of both alpha- and beta-globin RNA transcripts. This induction of differentiation was blocked by the presence of exogenous guanosine (100 mumol/L). In contrast to the down-regulation of c-myc and c-myb RNA in MEL cells induced by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), there was no detectable change in levels of these transcripts after tiazofurin treatment. Furthermore, MEL cells induced by tiazofurin did not commit to terminal differentiation. These results suggest a role for guanine nucleotides, at least in part, in the regulation of MEL cell differentiation.