RESUMEN
Aberrant MYC gene expression by the Wnt/beta-catenin pathway is implicated in colorectal carcinogenesis. Wnt/beta-catenin signaling stimulates association of the beta-catenin coactivator complex with two Wnt responsive enhancers (WREs) located in close proximity to MYC gene boundaries. Each enhancer directly binds members of the TCF/Lef family of transcription factors that, in turn, recruit beta-catenin. In a previous report, we showed that the downstream MYC enhancer (MYC 3' WRE) cooperated with the upstream enhancer (MYC 5' WRE) to activate expression of a heterologous reporter gene in response to Wnt/beta-catenin and mitogen signaling. Here we use chromatin conformation capture (3C) to show that the MYC 5' and 3' WREs are juxtaposed at the genomic MYC locus during active transcription. This MYC 5'3' chromatin loop is present in HCT116 human colorectal cancer cells that contain high levels of nuclear beta-catenin and is absent in HEK293 cells that contain trace amounts of nuclear beta-catenin. Depletion of functional beta-catenin/TCF complexes blocks formation of the MYC 5'3 chromatin loop. Furthermore, we find that the chromatin loop is absent in quiescent cells, but is rapidly and transiently induced by serum mitogens in a beta-catenin-dependent manner. Thus, we propose that a distinct chromatin architecture coordinated by beta-catenin/TCF-bound WREs accompanies transcriptional activation of MYC gene expression.
Asunto(s)
Cromatina/química , Elementos de Facilitación Genéticos , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Línea Celular , Cromatina/genética , Expresión Génica , Células HCT116 , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Conformación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción TCF/genética , Proteínas Wnt/genética , beta Catenina/genéticaRESUMEN
In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph(+) metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34(+) cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph(+) metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated beta-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.
Asunto(s)
Antígenos CD34/metabolismo , Antineoplásicos/administración & dosificación , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Proteínas de Neoplasias/biosíntesis , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Adulto , Anciano , Benzamidas , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Cromosoma Filadelfia/efectos de los fármacosRESUMEN
Post-translational modification of the p53 family members is key to their regulation. Here we report the phosphorylation of TAp63gamma, but not DeltaNp63gamma, by IkappaB kinase beta (IKKbeta). Activation of IKKbeta by gamma radiation or tumor necrosis factor-alpha led to increased TAp63gamma protein levels in cells. IKKbeta, but not its kinase-defective mutant IKKbeta-K44A, led to this observed stabilization of TAp63gamma. This stabilization of TAp63gamma in response to gamma radiation was significantly decreased in the absence of IKKbeta. Phosphorylation of TAp63gamma blocks ubiquitylation and possible degradation of this protein. We postulate that phosphorylation of TAp63gamma by IKKbeta stabilizes the TAp63gamma protein by blocking ubiquitylation-dependent degradation of this protein.
Asunto(s)
Quinasa I-kappa B/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación/fisiología , Sustitución de Aminoácidos , Línea Celular Tumoral , Activación Enzimática/genética , Activación Enzimática/efectos de la radiación , Rayos gamma , Humanos , Quinasa I-kappa B/genética , Mutación Missense , Fosforilación/efectos de la radiación , Transactivadores/genética , Factores de Transcripción , Proteínas Supresoras de Tumor/genética , Ubiquitinación/efectos de la radiaciónRESUMEN
Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Mutación/genética , Fosfotransferasas/metabolismo , Piperazinas/farmacología , Pirimidinas/farmacología , Secuencia de Aminoácidos , Animales , Benzamidas , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Modelos Animales de Enfermedad , Femenino , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Células Progenitoras Mieloides/citología , Fosfotirosina/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Especificidad por SustratoRESUMEN
The transcriptional co-activator p300 has been reported to regulate the tumor suppressor p53 and its ortholog p73. Here we describe a study showing that this coactivator also regulates the transcriptional function of p63. p300 bound to the N-terminal domain of p63gamma, and p63gamma bound to the N terminus of p300 in vitro and in cells. p300, but not its acetylase-defective mutant AT2, stimulated p63gamma-dependent transcription and induction of p21 in cells, consequently leading to G1 arrest. Inversely, the deltaN-p63gamma isoform as well as p300AT2 inhibited the induction of p21 by p63gamma. These results suggest that p300 regulates p63-dependent transcription of p21.