RESUMO
The checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related) transduce genomic stress signals to halt cell cycle progression and promote DNA repair. We report the identification of an ATR-interacting protein (ATRIP) that is phosphorylated by ATR, regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway. ATR and ATRIP both localize to intranuclear foci after DNA damage or inhibition of replication. Deletion of ATR mediated by the Cre recombinase caused the loss of ATR and ATRIP expression, loss of DNA damage checkpoint responses, and cell death. Therefore, ATR is essential for the viability of human somatic cells. Small interfering RNA directed against ATRIP caused the loss of both ATRIP and ATR expression and the loss of checkpoint responses to DNA damage. Thus, ATRIP and ATR are mutually dependent partners in cell cycle checkpoint signaling pathways.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular , Exodesoxirribonucleases , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Morte Celular , Linhagem Celular , Sobrevivência Celular , Sequência Conservada , Dano ao DNA , Proteínas de Ligação a DNA , Éxons/genética , Deleção de Genes , Genes Essenciais/genética , Células HeLa , Humanos , Integrases/genética , Integrases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/genética , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Alinhamento de Sequência , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The objective of this research was to determine the amount and timing of nitric oxide (NO, nitrogen monoxide) gas produced by the lungs, intestinal mucosa, and organ surfaces facing the peritoneal cavity after iv injection of a bacterial toxin, lipopolysaccharide (LPS). Some of the deleterious effects of LPS on organ function have been attributed to NO or strong oxidants formed locally from NO. Medical-grade air was used as an inspiratory air source (50 strokes/min x 3 ml/stroke) or was pumped through the ileal lumen or peritoneal cavity (20 strokes/min x 3 ml/stroke). The air was collected at intervals of 15-30 min for 3 h after LPS and analyzed for authentic NO gas by chemiluminescence. LPS (5 mg/kg) or saline was injected iv. Sodium nitroprusside (SNP) was injected to determine the appearance of its NO released into the perfused compartments. Blood pressure, plasma nitrate plus nitrite (NO(x)), and total plasma leukocytes were measured as other manifestations of LPS effects. NO began to increase in the pulmonary expired air 90 min after LPS and continued to increase for the remainder of the experiment. The final pulmonary post-LPS [NO] was about 20-fold greater than the [NO] before LPS. LPS had no effect on intraluminal or intraperitoneal [NO]. The saline injection had no effect on [NO] in any compartment. SNP injection increased NO entry into all three air-perfused compartments. Thus, NO from an exogenous tissue source was not prevented from being detected. Blood pressure was decreased by LPS only during the pulmonary perfusion. There were no significant effects of LPS on leukocytes or plasma NO(x). LPS decreased blood pressure and leukocytes and increased plasma NO(x) when air perfusion was not done. It was concluded that different organs can produce LPS-induced NO at markedly different rates and times. However, some aspect of the experimental technique of air perfusion could alter the effects of LPS.
Assuntos
Mucosa Intestinal/fisiologia , Lipopolissacarídeos/farmacologia , Pulmão/fisiologia , Óxido Nítrico/sangue , Nitroprussiato/farmacologia , Mucosa Respiratória/fisiologia , Anestesia Geral , Animais , Pressão Sanguínea/efeitos dos fármacos , Feminino , Hipotensão/fisiopatologia , Íleo , Mucosa Intestinal/efeitos dos fármacos , Cinética , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Medições Luminescentes , Pulmão/efeitos dos fármacos , Nitratos/sangue , Nitritos/sangue , Nitroprussiato/administração & dosagem , Perfusão , Cavidade Peritoneal , Ratos , Ratos Sprague-Dawley , Mucosa Respiratória/efeitos dos fármacos , Fatores de TempoRESUMO
Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.
