RESUMO
Human oxidation resistance 1 (OXR1) was identified as a protein that decreases genomic mutations in Escherichia coli caused by oxidative DNA damage. However, the mechanism by which OXR1 defends against genome instability has not been elucidated. To clarify how OXR1 maintains genome stability, the effects of OXR1-depletion on genome stability were investigated in OXR1-depleted HeLa cells using gamma-rays (γ-rays). The OXR1-depleted cells had higher levels of superoxide and micronucleus (MN) formation than control cells after irradiation. OXR1-overexpression alleviated the increases in reactive oxygen species (ROS) level and MN formation after irradiation. The increased MN formation in irradiated OXR1-depleted cells was partially attenuated by the ROS inhibitor N-acetyl-L-cysteine, suggesting that OXR1-depeletion increases ROS-dependent genome instability. We also found that OXR1-depletion shortened the duration of γ-ray-induced G2/M arrest. In the presence of the cell cycle checkpoint inhibitor caffeine, the level of MN formed after irradiation was similar between control and OXR1-depleted cells, demonstrating that OXR1-depletion accelerates MN formation through abrogation of G2/M arrest. In OXR1-depleted cells, the level of cyclin D1 protein expression was increased. Here we report that OXR1 prevents genome instability by cell cycle regulation as well as oxidative stress defense.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Raios gama , Instabilidade Genômica/efeitos da radiação , Proteínas Mitocondriais/metabolismo , Mitose/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Micronúcleo Germinativo/efeitos dos fármacos , Micronúcleo Germinativo/metabolismo , Micronúcleo Germinativo/efeitos da radiação , Proteínas Mitocondriais/deficiência , Mitose/efeitos dos fármacos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Superóxidos/metabolismoRESUMO
Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.
Assuntos
Apoptose/efeitos dos fármacos , Argônio/farmacologia , Dano ao DNA , Histonas/metabolismo , Gases em Plasma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , Peróxido de Hidrogênio/toxicidade , Ácido Hipocloroso/toxicidade , Micronúcleo Germinativo/efeitos dos fármacos , Micronúcleo Germinativo/metabolismo , Micronúcleo Germinativo/efeitos da radiação , Oxirredução , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Raios UltravioletaRESUMO
We describe here an automated imaging system developed at the Center for High Throughput Minimally Invasive Radiation Biodosimetry. The imaging system is built around a fast, sensitive sCMOS camera and rapid switchable LED light source. It features complete automation of all the steps of the imaging process and contains built-in feedback loops to ensure proper operation. The imaging system is intended as a back end to the RABiT-a robotic platform for radiation biodosimetry. It is intended to automate image acquisition and analysis for four biodosimetry assays for which we have developed automated protocols: The Cytokinesis Blocked Micronucleus assay, the γ-H2AX assay, the Dicentric assay (using PNA or FISH probes) and the RABiT-BAND assay.
Assuntos
Automação/métodos , Processamento de Imagem Assistida por Computador/métodos , Micronúcleo Germinativo/química , Citocinese , Histonas/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Testes para Micronúcleos , Micronúcleo Germinativo/efeitos da radiação , RadiometriaRESUMO
In the ciliate Tetrahymena, meiotic micronuclei (MICs) undergo extreme elongation, and meiotic pairing and recombination take place within these elongated nuclei (the "crescents"). We have previously shown that elongation does not occur in the absence of Spo11p-induced DNA double-strand breaks (DSBs). Here we show that elongation is restored in spo11Delta mutants by various DNA-damaging agents including ones that may not cause DSBs to a notable extent. MIC elongation following Spo11p-induced DSBs or artificially induced DNA lesions is probably a DNA-damage response mediated by a phosphokinase signal transduction pathway, since it is suppressed by the ATM/ATR kinase inhibitors caffeine and wortmannin and by knocking out Tetrahymena's ATR orthologue. MIC elongation occurs concomitantly with the movement of centromeres away from the telomeric pole of the MIC. This DNA damage-dependent reorganization of the MIC helps to arrange homologous chromosomes alongside each other but is not sufficient for exact pairing. Thus, Spo11p contributes to bivalent formation in two ways: by creating a favorable spatial disposition of homologues and by stabilizing pairing by crossovers. The polarized chromosome orientation inside the crescent resembles the conserved meiotic bouquet, and crescent and bouquet also share the putative function of aiding meiotic pairing. However, they are regulated differently because in Tetrahymena, DSBs are required for entering rather than exiting this stage.
Assuntos
Núcleo Celular/enzimologia , Dano ao DNA , Meiose , Proteínas Quinases/metabolismo , Tetrahymena/citologia , Tetrahymena/enzimologia , Androstadienos/farmacologia , Animais , Cafeína/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Centrômero/efeitos dos fármacos , Centrômero/metabolismo , Centrômero/efeitos da radiação , Pareamento Cromossômico/efeitos dos fármacos , Pareamento Cromossômico/efeitos da radiação , Cromossomos/metabolismo , Endodesoxirribonucleases , Esterases/metabolismo , Genótipo , Meiose/efeitos dos fármacos , Meiose/efeitos da radiação , Metanossulfonato de Metila/farmacologia , Micronúcleo Germinativo/efeitos dos fármacos , Micronúcleo Germinativo/efeitos da radiação , Mutação/genética , Fenótipo , Proteínas de Protozoários/metabolismo , Tetrahymena/efeitos dos fármacos , Tetrahymena/efeitos da radiação , Raios Ultravioleta , WortmaninaRESUMO
The availability of selenium and the levels of specific selenoproteins might affect cancer risk by influencing the ability of DNA damaging agents to cause genomic instability and mutations. Transgenic mice that express reduced levels of selenoproteins and previously shown to be more susceptible to pathology associated with cancer development were used to study this possibility. These mice were exposed to X-rays and DNA damage assessed in the erythrocytes, where micronuclei formation was higher compared to the same cells obtained from irradiated wild-type controls. To determine whether the selenoprotein glutathione peroxidase-1 (GPx-1) might be involved in this protection, its levels were reduced by siRNA targeting in LNCaP human prostate cells. UV-induced micronuclei frequency was higher in these cells compared to control-transfected cells. These results indicate a role for selenoproteins in protecting DNA from damage and support human data implicating GPx-1 as a possible target of the chemoprotective effect of selenium.
Assuntos
Glutationa Peroxidase/metabolismo , Micronúcleo Germinativo/efeitos da radiação , Selenoproteínas/deficiência , Animais , Sequência de Bases , Dano ao DNA , Glutationa Peroxidase/efeitos da radiação , Camundongos , Camundongos Transgênicos , Micronúcleo Germinativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Selenoproteínas/metabolismo , Selenoproteínas/efeitos da radiação , Raios Ultravioleta , Raios X , Glutationa Peroxidase GPX1RESUMO
BACKGROUND: Restenosis is the complete occlusion of the blood vessel leading to such complications as ischemia/angina, myocardial infarction, and death. It can be managed by endovascular brachytherapy with both gamma and beta sources. Endovascular brachytherapy is performed worldwide on several thousands of cases per year. The gamma-emitter 192Ir as well as the beta-emitters 32P and 90Sr are mainly used. The dose to the occluded endothelial wall is 20 Gy. Interestingly, no information with respect to the dose absorbed by the blood during the course of the treatment exists. The aim of the present investigation was to verify if the micronucleus test is suitable to detect the dose absorbed by lymphocytes in the course of endovascular brachytherapy with 32P. MATERIALS AND METHODS: Blood was drawn from 16 patients immediately before and 1 day after the treatment. Frequencies of micronuclei were assessed. In order to ensure that the micronuclei did not arise due to fluoroscopy or reperfusion, we analyzed lymphocytes of 16 control patients who underwent interventional cardiology with balloon angioplasty only. RESULTS AND CONCLUSIONS: Enhanced frequencies of micronuclei were observed in lymphocytes of some donors following brachytherapy. No correlation could be detected between the level of induced micronuclei and the absorbed dose. Also, no effect of fluoroscopy or reperfusion was seen. Thus, although brachytherapy of restenosis with 32P leads to weak enhancement of the micronucleus frequency in lymphocytes, the effect was not seen in all patients; the reason for this heterogeneous response remains to be elucidated.
Assuntos
Angioplastia Coronária com Balão/métodos , Braquiterapia/métodos , Reestenose Coronária/radioterapia , Linfócitos/ultraestrutura , Micronúcleo Germinativo/efeitos da radiação , Células Cultivadas , Reestenose Coronária/sangue , Seguimentos , Humanos , Linfócitos/efeitos da radiação , Micronúcleo Germinativo/ultraestrutura , Resultado do TratamentoRESUMO
The spectra and dose response for mutations at expanded simple tandem repeat (ESTR) loci in the germline of male mice acutely exposed to low-LET X or gamma rays at pre-meiotic stages of spermatogenesis were compared in five strains of laboratory mice. Most mutation events involved the gain or loss of a relatively small number of repeat units, and the distributions of length changes were indistinguishable between the exposed and control males. Overall, a significant bias toward gains of repeats was detected, with approximately 60% of mutants showing gains. The values for ESTR mutation induction did not differ substantially between strains. The highest values of doubling dose were obtained for two genetically related strains, BALB/c and C.B17 (mean value 0.98 Gy). The estimates of doubling dose for three other strains (CBA/H, C57BL/6 x CBA/H F1 and 129SVJ x C57BL/6) were lower, with a mean value of 0.44 Gy. The dose response for ESTR mutation across all five strains was very close to that for the specific loci (Russell 7-locus test). The mechanisms of ESTR mutation induction and applications of this system for monitoring radiation-induced mutation in the mouse germline are discussed.