RESUMO
The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21(WAF1/CIP1)) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.
Assuntos
Embrião de Mamíferos/efeitos da radiação , Proteínas Nucleares , Proteína Supressora de Tumor p53/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Cisteína Endopeptidases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Humanos , Cinética , Complexos Multienzimáticos/efeitos dos fármacos , Fosforilação , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-mdm2 , Raios XRESUMO
DNA-damaging agents induce phosphorylation of the p53 protein, resulting in its accumulation in the nucleus. To clarify the signal transduction pathway(s) involved in p53 protein accumulation in normal human embryo cells following X-irradiation, the effects of three protein kinase inhibitors were examined. Quercetin, an inhibitor of heat-shock response, dose dependently suppressed the p53 accumulation induced by X-rays at more than 100 microM. No suppression, however, was observed with calphostin-C, a specific inhibitor of protein kinase C, in the range of 0.05 to 0.25 microM. Wortmannin was the most potent inhibitor of p53 accumulation. Its suppressive effect appears within a few minutes of pretreatment with a dose of 25 microM, but posttreatment was less effective. Our findings suggest that PKC is not involved in X-ray-induced p53 accumulation in normal human embryo cells and that a wortmannin-sensitive pathway acts as a sensor of DNA damage.
