RESUMEN
Chemotherapeutics (e.g., aurora kinase inhibitors) designed to target proliferative cells are often nonspecific for tumor cells as normal cycling cells are also susceptible. Indeed, one of the major dose-limiting toxicities of aurora kinase inhibitors is a dangerous depletion of neutrophils in patients. In this study we proposed a strategy to selectively target p53 mutant cells while sparing normal ones. The strategy is based on the understanding that normal cells have an intact p53 pathway but not tumor cells carrying p53 mutations. Nongenotoxic activation of p53 using nutlin led to a reversible activation of G1 and G2 arrest in normal cells, which prevents them from entering mitosis, thus protecting them from the side effects of aurora kinase inhibition (VX-680), namely endoreduplication and apoptosis. Cells carrying mutant p53 are selectively killed by the nutlin/VX-680 combination, whereas p53 wild-type cells retain their proliferative capacity. The major implications drawn from these results are: (1) reversible nongenotoxic activation of p53 may be used as a strategy for the chemoprotection of normal tissues, and (2) aurora kinase inhibitors may have alleviated side effects when used in combination with nutlin-like inhibitors. We highlight the distinct roles of p53 and p73 in mediating the cellular responses to VX-680 and suggest that dual protection by p53 and p73 are needed to guard against endoreduplication and polyploidy.
Asunto(s)
Apoptosis/efectos de los fármacos , Imidazoles/farmacología , Mutación/fisiología , Piperazinas/farmacología , Proteína p53 Supresora de Tumor/genética , Apoptosis/genética , Aurora Quinasas , Cafeína/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Ciclina A2/genética , Ciclina A2/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Fase G2/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Imidazoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Piperazinas/uso terapéutico , Poliploidía , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Tetraploidía , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
The zebrafish has many advantages as a vertebrate model organism and has been extensively used in the studies of development. Its potential as a model in which to study tumour suppressor and oncogene function is now being realized. Whilst in situ hybridization of mRNA has been well developed in this species to study gene expression, antibody probes are in short supply. We have, therefore, generated a panel of anti-zebrafish p53 monoclonal antibodies and used these to study the p53 response in zebrafish embryos. By immunohistochemistry, we show that the exposure of zebrafish embryos to p53-activating agents such as R-roscovitine and gamma-irradiation results in the accumulation of p53 protein in the gut epithelium, liver and pancreas. A combination of R-roscovitine and gamma-irradiation results in massive p53 induction, not only in the pharyngeal arches, gut region and liver but also in brain tissues. Induction of apoptosis and expression of p53 response genes are seen in regions that correspond to sites of p53 protein accumulation. In contrast, although zebrafish tp53(M214K) mutant embryos showed a similar accumulation of p53 protein, a complete lack of a downstream p53-dependent response was observed. In this system the p53 gene is identified as a p53-responsive gene itself. Our results demonstrate that zebrafish p53 protein can readily be induced in embryos and detected using these new antibody tools, which will increase the usefulness of zebrafish as a model in compound-based screening for novel drugs in cancer research.