RESUMO
Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.
Assuntos
Antígenos Nucleares/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/genética , Western Blotting , Linhagem Celular Tumoral , Ensaio Cometa , Reparo do DNA , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Autoantígeno Ku , Espectrometria de Massas , Fosforilação , Isoformas de Proteínas/genética , RNA Interferente Pequeno , TransfecçãoRESUMO
Hydrogenated nanodiamonds (H-NDs) exhibit a negative electron affinity that confers a high reactivity with oxygen species and a positive charge in aqueous solutions. It allows electron emission from H-NDs following irradiation by photons and in consequence may enhance the effects of radiation on cancer cells. By using three human radioresistant cancer cell lines, we showed a potentialization of cytotoxicity after a co-exposure to H-NDs and irradiation; an event occurring through the induction of DNA damage and reactive oxygen species. This occurred together with a decrease in cell impedance, the activation of G1/S, an unlocking of G2 cell cycle check-points and early low cell death rate. At later stage of exposure, persistent increases in heterochromatinization, large γ-H2AX foci and ß-galactosidase activity were detected providing evidence of cells' entrance into senescence. Similar potentialization was observed with neocarzinostatin (NCS), a radiomimetic drug. This original finding underlines a wide clinical potential of H-NDs to intensify radiation effects on radio-resistant cancer cells.
