RESUMEN
Inhibiting the bromodomain and extra-terminal (BET) domain family of epigenetic reader proteins has been shown to have potent anti-tumoral activity, which is commonly attributed to suppression of transcription. In this study, we show that two structurally distinct BET inhibitors (BETi) interfere with replication and cell cycle progression of murine Myc-induced lymphoma cells at sub-lethal concentrations when the transcriptome remains largely unaltered. This inhibition of replication coincides with a DNA-damage response and enhanced sensitivity to inhibitors of the upstream replication stress sensor ATR in vitro and in mouse models of B-cell lymphoma. Mechanistically, ATR and BETi combination therapy cause robust transcriptional changes of genes involved in cell death, senescence-associated secretory pathway, NFkB signaling and ER stress. Our data reveal that BETi can potentiate the cell stress and death caused by ATR inhibitors. This suggests that ATRi can be used in combination therapies of lymphomas without the use of genotoxic drugs.
Asunto(s)
Linfoma/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Acetiltransferasas , Chaperonas de Histonas , Humanos , Linfoma/genética , Linfoma/patología , Ratones , Proteínas Nucleares/genética , Transducción de SeñalRESUMEN
The p53 tumor suppressor protein is a dimer of dimers that binds its consensus DNA sequence (containing two half-sites) as a pair of clamps. We show here that after one wild-type dimer of a tetramer binds to a half-site on the DNA, the other (unbound) dimer can be in either the wild-type or the mutant conformation. An equilibrium state between these two conformations exists and can be modulated by two types of regulators. One type modifies p53 biochemically and determines the intrinsic balance of the equilibrium. The other type of regulator binds directly to one or both dimers in a p53 tetramer, trapping each dimer in one or the other conformation. In the wild-type conformation, the second dimer can bind to the second DNA half-site, resulting in drastically enhanced stability of the p53-DNA complex. Importantly, a genotypically mutant p53 can also be in equilibrium with the wild-type conformation, and when trapped in this conformation can bind DNA.
Asunto(s)
ADN/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , ADN/genética , Dimerización , Estabilidad de Medicamentos , Genotipo , Humanos , Técnicas In Vitro , Ratones , Mutación , Conformación Proteica , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The tumour suppressor p53 becomes activated as a transcription factor in response to DNA damage, but the mechanism for this activation is unclear. A good candidate for an upstream activator of p53 is the DNA-dependent protein kinase (DNA-PK) that depends on the presence of DNA breaks for its activity. Here we investigate the link between DNA damage and the activation of DNA-PK and of p53. To determine whether DNA-PK is an upstream mediator of the p53 DNA-damage response, we analysed a severe combined-immunodeficiency (SCID) mouse cell line, SCGR11, and the human glioma cell line M059J . Both cell lines lack any detectable DNA-PK activity. We find that p53 is incapable of binding to DNA in the absence of DNA-PK, that DNA-PK is necessary but not sufficient for activation of p53 sequence-specific DNA binding, and that this activation occurs in response to DNA damage. Our results establish DNA-PK as a link between DNA damage and p53 activation, and reveal the existence of a mammalian DNA-damage-response pathway.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , ADN/metabolismo , Proteína Quinasa Activada por ADN , Activación Enzimática , Inhibidores Enzimáticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Datos de Secuencia Molecular , Proteínas Nucleares , Unión Proteica , Transcripción Genética , Activación Transcripcional , Transfección , Células Tumorales CultivadasRESUMEN
The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (--><-- --><--). Each p53 subunit binds to one quarter-site, resulting in all four DNA quarter-sites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps.
Asunto(s)
ADN/metabolismo , Polímeros/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Supresores de Tumor/fisiología , Humanos , Polímeros/química , Proteína p53 Supresora de Tumor/químicaRESUMEN
It is generally accepted that wild type (growth suppressing) p53 is capable of binding to a consensus DNA sequence and is in a conformation recognizable by antibody PAb246 (for murine p53), but not by antibody PAb240. Conversely, mutant forms of p53 incapable of DNA binding often assume conformations that display the PAb240, but not the PAb246 epitope. Exposure of these two epitopes on p53 is therefore believed to be mutually exclusive. We show that wild type p53 translated in vitro in rabbit reticulocyte lysate (RRL) has a PAb240 epitope that is not always cryptic, even on p53 that is bound sequence-specifically to DNA (presumably as a tetramer). All of the DNA-bound, PAb240+ p53 concurrently displays the PAb246 epitope, and both epitopes can be occupied by antibody while p53 is bound to DNA. This novel 'dual positive' conformation also exists in the absence of DNA and suggests that p53 is not necessarily inactive when the PAb240 epitope is displayed. When the C-terminal 58 amino acids of p53 containing the dimer/tetramerization domains are replaced with a heterologous dimerization domain, the resultant dimeric p53 manifests only the PAb246+/PAb240- conformation while bound to DNA. Thus, the C-terminal 58 amino acids of p53 are required for the PAb246+/PAb240+ phenotype, possibly due to tetramerization. This novel 'dual positive' p53 conformation exists in an excess of wild type p53 that has the PAb246-/PAb240+ 'mutant' conformation, suggesting that the 'mutant' conformation is not dominant negative in and of itself.