RESUMEN
DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Replicación A/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Células CHO , Puntos de Control del Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cricetinae , Cricetulus , Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Mitosis , Mutación , Fosforilación , Proteínas Quinasas/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/genética , Serina/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
Type IIα DNA topoisomerase (TopoIIα) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIα activity and increase resistance to TopoIIα poisons. Using in vitro DNA decatenation assays we show that neoamphimedine potently inhibits TopoIIα-dependent DNA decatenation in the presence of Metnase. Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC(50) of 0.5 µM. Additionally, we find that the apparent K(m) of TopoIIα for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase.
Asunto(s)
Acridinas/farmacología , Adenosina Trifosfato/metabolismo , Antígenos de Neoplasias/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/metabolismo , Acridinas/administración & dosificación , Antígenos de Neoplasias/metabolismo , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Modelos MolecularesRESUMEN
Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.