RESUMO
The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Óxidos/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Trióxido de Arsênio , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Methylglyoxal (MG) is a reactive endogenous metabolite that is produced from the process of degradation of triose-phosphates. Under hyperglycemic conditions the rate of MG formation increases as a result of elevated concentrations of precursors. It has been established that MG elicits oxidative stress signaling, leading to the activation of MAP kinases, p38 MAPK and JNK, yet it remains largely unknown about a role of cell-cycle checkpoint regulation in MG-induced signaling. Here, we show that checkpoint kinases, Chk1 and Chk2, as well as their upstream ATM kinase are phosphorylated and activated following MG treatment of cultured cells. This MG-induced activation of Chk1 and Chk2 were inhibited by either aminoguanidine (AG), an inhibitor of production of advanced glycation end products (AGEs) or N-acetyl-l-cysteine (NAC), an anti-oxidant in dose dependent manners, indicating that oxidative stress via AGEs is involved critically in the activation of Chk1 and Chk2 by MG. Furthermore, it was found that cell-cycle synchronized cells exhibited G(2)/M checkpoint arrest following MG treatment, and that siRNA-mediated knock-down of Chk2, but not Chk1, results in a failure of MG-induced G(2)/M arrest. Thus, the results indicate a critical role for Chk2 in MG-induced G(2)/M cell-cycle checkpoint arrest.
