RESUMEN
The gene Trp53 is among the most frequently mutated and studied genes in human cancer, but the mechanisms by which it suppresses tumour formation remain unclear. We generated mice with an allele encoding changes at Leu25 and Trp26, known to be essential for transcriptional transactivation and Mdm2 binding, to enable analyses of Trp53 structure and function in vivo. The mutant Trp53 was abundant, its level was not affected by DNA damage and it bound DNA constitutively; however, it showed defects in cell-cycle regulation and apoptosis. Both mutant and Trp53-null mouse embryonic fibroblasts (MEFs) were readily transformed by oncogenes, and the corresponding mice were prone to tumours. We conclude that the determining pathway for Trp53 tumour-suppressor function in mice requires the transactivation domain.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes p53 , Activación Transcripcional , Proteína p53 Supresora de Tumor , Alelos , Animales , Apoptosis/genética , Daño del ADN/efectos de los fármacos , Dactinomicina/farmacología , Femenino , Ratones , Ratones Transgénicos , Modelos Animales , Trasplante de Neoplasias , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The mammalian UV response results in rapid and dramatic induction of c-jun. Induction of a protooncogene, normally involved in mitogenic responses, by a genotoxic agent that causes growth arrest seems paradoxical. We now provide an explanation for the role of c-Jun in the UV response of mouse fibroblasts. c-Jun is necessary for cell-cycle reentry of UV-irradiated cells, but does not participate in the response to ionizing radiation. Cells lacking c-Jun undergo prolonged cell-cycle arrest, but resist apoptosis, whereas cells that express c-Jun constitutively do not arrest and undergo apoptosis. This function of c-Jun is exerted through negative regulation of p53 association with the p21 promoter. Cells lacking c-Jun exhibit prolonged p21 induction, whereas constitutive c-Jun inhibits UV-mediated p21 induction.
Asunto(s)
Ciclo Celular , Ciclinas/metabolismo , Regulación de la Expresión Génica , Genes jun/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Animales , Línea Celular , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/antagonistas & inhibidores , Ciclinas/genética , ADN/biosíntesis , ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Fibroblastos , Genes Reporteros , Humanos , Ratones , Transducción de Señal , Transcripción Genética/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Cricetinae , ADN/metabolismo , Reparación del ADN , Proteína Quinasa Activada por ADN , Ratones , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.