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1.
J Allergy Clin Immunol ; 138(1): 84-96.e1, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27157131

RESUMO

BACKGROUND: Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. OBJECTIVE: We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) in vivo and in vitro. METHODS: We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. RESULTS: HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX) foci in bronchial epithelium. BEAS-2B cells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. CONCLUSION: This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Asma/etiologia , Asma/metabolismo , Quebras de DNA de Cadeia Dupla , Pulmão/imunologia , Pulmão/metabolismo , Estresse Oxidativo , Pyroglyphidae/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Caspase 3/metabolismo , Morte Celular/genética , Morte Celular/imunologia , Citocinas/metabolismo , Reparo do DNA , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/metabolismo , Eosinófilos/patologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/patologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
2.
DNA Repair (Amst) ; 106: 103176, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34365116

RESUMO

DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Reparo do DNA , Ensaios de Triagem em Larga Escala/métodos , Linhagem Celular , Linhagem Celular Tumoral , DNA/efeitos dos fármacos , DNA/metabolismo , DNA/efeitos da radiação , Reparo do DNA por Junção de Extremidades , Humanos , Mutagênicos/toxicidade
3.
SLAS Discov ; 25(8): 906-922, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32452708

RESUMO

Dysfunction of apoptosis and DNA damage response pathways often drive cancer, and so a better understanding of these pathways can contribute to new cancer therapeutic strategies. Diverse discovery approaches have identified many apoptosis regulators, DNA damage response, and DNA damage repair proteins; however, many of these approaches rely on indirect detection of DNA damage. Here, we describe a novel discovery platform based on the comet assay that leverages previous technical advances in assay precision by incorporating high-throughput robotics. The high-throughput screening (HTS) CometChip is the first high-throughput-compatible assay that can directly detect physical damage in DNA. We focused on DNA double-strand breaks (DSBs) and utilized our HTS CometChip technology to perform a first-of-its-kind screen using an shRNA library targeting 2564 cancer-relevant genes. Conditions of the assay enable detection of DNA fragmentation from both exogenous (ionizing radiation) and endogenous (apoptosis) sources. Using this approach, we identified LATS2 as a novel DNA repair factor as well as a modulator of apoptosis. We conclude that the HTS CometChip is an effective assay for HTS to identify modulators of physical DNA damage and repair.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Biblioteca Gênica , Testes Genéticos/tendências , Humanos , Proteínas de Neoplasias/genética , Neoplasias/genética , RNA Interferente Pequeno/genética , Robótica
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