RESUMO
Methylmercury (MeHg) is a potent neurotoxin, teratogen, and probable carcinogen, but the underlying mechanisms of its actions remain unclear. Although MeHg causes several types of DNA damage, the toxicological consequences of this macromolecular damage are unknown. MeHg enhances oxidative stress, which can cause various oxidative DNA lesions that are primarily repaired by oxoguanine glycosylase 1 (OGG1). Herein, we compared the response of wild-type and OGG1 null (Ogg1(-/-)) murine embryonic fibroblasts to environmentally relevant, low micromolar concentrations of MeHg by measuring clonogenic efficiency, cell cycle arrest, DNA double-strand breaks (DSBs), and activation of the DNA damage response pathway.Ogg1(-/-) cells exhibited greater sensitivity to MeHg than wild-type controls, as measured by the clonogenic assay, and showed a greater propensity for MeHg-initiated apoptosis. Both wild-type and Ogg1(-/-) cells underwent cell cycle arrest when exposed to micromolar concentrations of MeHg; however, the extent of DSBs was exacerbated in Ogg1(-/-) cells compared with that in wild-type controls. Pretreatment with the antioxidative enzyme catalase reduced levels of DSBs in both wild-type and Ogg1(-/-) cells but failed to block MeHg-initiated apoptosis at micromolar concentrations. Our findings implicate reactive oxygen species mediated DNA damage in the mechanism of MeHg toxicity; and demonstrate for the first time that impaired DNA repair capacity enhances cellular sensitivity to MeHg. Accordingly, the genotoxic properties of MeHg may contribute to its neurotoxic and teratogenic effects, and an individual's response to oxidative stress and DNA damage may constitute an important determinant of risk.
Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Compostos de Metilmercúrio/toxicidade , Animais , Citometria de Fluxo , Humanos , Camundongos , Camundongos KnockoutRESUMO
The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.
Assuntos
Proliferação de Células , Instabilidade Genômica , Camundongos/embriologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Apoptose , Linhagem da Célula , Centrossomo/fisiologia , Citocinese , Feminino , Fibroblastos/fisiologia , Amplificação de Genes , Genes Letais , Masculino , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Mitose , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático , Proteínas Supressoras de Tumor/genéticaRESUMO
Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus81-/- mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to gamma-irradiation. Remarkably, both homozygous Mus81-/- and heterozygous Mus81+/- mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Endonucleases , Genoma , Instabilidade Genômica , Neoplasias/genética , Alelos , Animais , Aberrações Cromossômicas , Dano ao DNA , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário e Fetal , Raios gama , Marcação de Genes , Predisposição Genética para Doença , Heterozigoto , Linfoma/etiologia , Linfoma/genética , Linfoma/patologia , Meiose , Camundongos , Mitomicina/farmacologia , Neoplasias/etiologia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Troca de Cromátide Irmã , Células-Tronco , Linfócitos T/fisiologiaRESUMO
Disruption of Brca1 results in cellular demise or tumorigenesis depending on cellular context. Inactivation of p53 contributes to Brca1-associated tumor susceptibility. However the activation of p53-dependent checkpoint/apoptotic signaling in the absence of Brca1 is poorly understood. Here, we show that Chk2 inactivation is partially equivalent to p53 inactivation, in that Chk2 deficiency facilitates the development, survival, and proliferation of Brca1-deficient T cells at the expense of genomic integrity. Brca1 deficiency was found to result in Chk2 phosphorylation and the Chk2-dependent accumulation and activation of p53. Furthermore, inactivation of Chk2 and Brca1 was cooperative in breast cancer. Our findings identify a critical role for Chk2 as a component of the DNA damage-signaling pathway activated in response to Brca1 deficiency.