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Aim The aim of this study was to provide a systematic approach to characterize DNA damage induction and repair in isolated peripheral blood mononuclear cells (PBMCs) after internal ex vivo irradiation with [I-131]NaI. In this appr...
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Aim The aim of this study was to provide a systematic approach to characterize DNA damage induction and repair in isolated peripheral blood mononuclear cells (PBMCs) after internal ex vivo irradiation with [I-131]NaI. In this approach, we tried to mimic ex vivo the irradiation of patient blood in the first hours after radioiodine therapy. Material and methods Blood of 33 patients of two centres was collected immediately before radioiodine therapy of differentiated thyroid cancer (DTC) and split into two samples. One sample served as non-irradiated control. The second sample was exposed to ionizing radiation by adding 1 ml of [I-131]NaI solution to 7 ml of blood, followed by incubation at 37 degrees C for 1 h. PBMCs of both samples were isolated, split in three parts each and (i) fixed in 70% ethanol and stored at - 20 degrees C directly (0 h) after irradiation, (ii) after 4 h and (iii) 24 h after irradiation and culture in RPMI medium. After immunofluorescence staining microscopically visible co-localizing gamma-H2AX + 53BP1 foci were scored in 100 cells per sample as biomarkers for radiation-induced double-strand breaks (DSBs). Results Thirty-two of 33 blood samples could be analysed. The mean absorbed dose to the blood in all irradiated samples was 50.1 +/- 2.3 mGy. For all time points (0 h, 4 h, 24 h), the average number of gamma-H2AX + 53BP1 foci per cell was significantly different when compared to baseline and the other time points. The average number of radiation-induced foci (RIF) per cell after irradiation was 0.72 +/- 0.16 at t = 0 h, 0.26 +/- 0.09 at t = 4 h and 0.04 +/- 0.09 at t = 24 h. A monoexponential fit of the mean values of the three time points provided a decay rate of 0.25 +/- 0.05 h(-1), which is in good agreement with data obtained from external irradiation with gamma- or X-rays. Conclusion This study provides novel data about the ex vivo DSB repair in internally irradiated PBMCs of patients before radionuclide therapy. Our findings show, in a large patient sample, that efficient repair occurs after internal irradiation with 50 mGy absorbed dose, and that the induction and repair rate after I-131 exposure is comparable to that of external irradiation with gamma- or X-rays.
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We studied the formation of double-strand DNA breaks (DNA DSB) induced by femtosecond laser radiation in A549 human lung adenocarcinoma cells using immunocytochemical staining of the resulting tracks of a specific DSB marker prote...
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We studied the formation of double-strand DNA breaks (DNA DSB) induced by femtosecond laser radiation in A549 human lung adenocarcinoma cells using immunocytochemical staining of the resulting tracks of a specific DSB marker protein phosphorylated ATM kinase (phospho-ATM). Additionally, colocalization of phospho-ATM tracks with gamma H2AX protein tracks was studied. The results of immunocytochemical analysis showed that 30 min after irradiation of cells with femtosecond pulses with energies of 1 and 2 nJ (radiation power density 2 x10(11) and 4 x10(11) W x cm(-2), respectively), the formation of tracks consisting of phospho-ATM and gamma H2AX proteins located in sites where the laser beam passes through the cell nuclei was observed. The presence of phospho-ATM tracks co-localized with gamma H2AX allows us to conclude that exposure to focused femtosecond infrared laser radiation with a pulse energy of 1-2 nJ leads to the formation of DNA DSB in irradiated cells.
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Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiologic...
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Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (gamma-H2AX, p53(ser15)) and cell-cycle specific cyclins (cyclin B1 and cyclin Dl). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53(ser15), gamma-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks.
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Immunofluorescence quantification of gamma H2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the gamma H2...
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Immunofluorescence quantification of gamma H2AX foci is a powerful approach to quantify DNA double-strand breaks induced by cancer therapy or accidental exposure to ionizing radiation. Here we report a modification to the gamma H2AX immunofluorescence labeling method, whereby cells are stained in-solution before being spotted and fixed onto microscope slides. Our modified method allows arraying of 16 patient samples/slide ready for foci counting in 2 h and demonstrated reliably detection of gamma H2AX foci in mononuclear cells prepared from patients who had undergone radiation therapy.
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Toxicity screening and risk assessment of an overwhelmingly large and ever-increasing number of chemicals are vitally essential for ecological safety and human health. Genotoxicity is particularly important because of its associat...
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Toxicity screening and risk assessment of an overwhelmingly large and ever-increasing number of chemicals are vitally essential for ecological safety and human health. Genotoxicity is particularly important because of its association with mutagenicity, carcinogenicity and cancer. Phosphorylated histone H2AX (gamma H2AX) is an early sensitive genotoxic biomarker. It is therefore highly desirable to develop analytical methods for the detection of trace gamma H2AX to enable screening and assessment of genotoxicity. Here, we developed a novel cathodic photo -electrochemical (PEC) immunoassay with dual signal amplification for the rapid and ultrasensitive detection of gamma H2AX in cell lysates. A sandwich immuno-reaction targeting gamma H2AX was first carried out on a 96-well plate, using a secondary antibody/gold nanoparticle/glucose oxidase conjugate as the labeled detection antibody. The conjugate increased the production of H2O2 and thus provided the first mechanism of signal amplification. The immuno-reaction product containing H2O2 was then detected on a photocathode prepared from Bi2+xWO6 rich in oxygen vacancies, with H2O2 acting as electron acceptor. The oxygen vacancies acted as both adsorption and activation sites of H2O2 and thus enhanced the photocurrent, which provided another mechanism of signal amplification. As a result, an ultrasensitive immunoassay for gamma H2AX determination was established with a limit of detection of 6.87 pg/mL (S/N = 3) and a wide linear range from 0.01 to 500 ng/mL. The practicability of this assay was verified by detecting gamma H2AX in cell lysates exposed to known genotoxic chemicals. Our work offers a promising tool for the screening of genotoxic chemicals and opening a new avenue toward environmental risk assessment.
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H2AX is a histone H2A variant that becomes phosphorylated upon genotoxic stress. The phosphorylated H2AX (gamma-H2AX) plays an antioncogenic role in the DNA damage response and its foci patterns are highly variable, in terms of in...
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H2AX is a histone H2A variant that becomes phosphorylated upon genotoxic stress. The phosphorylated H2AX (gamma-H2AX) plays an antioncogenic role in the DNA damage response and its foci patterns are highly variable, in terms of intensities and sizes. However, whether characteristic gamma-H2AX foci patterns are associated with oncogenesis (oncogenic-specific gamma-H2AX foci patterns) remains unknown. We previously reported that a defect in the acetyltransferase activity of TIP60 promotes cancer cell growth in human cell lines. In this study, we compared gamma-H2AX foci patterns between TIP60 wild-type cells and TIP60 HAT mutant cells by using machine learning. When focused solely on the intensity and size of gamma-H2AX foci, we extracted the TIP60 HAT mutant-like oncogenic-specific gamma-H2AX foci pattern among all datasets of gamma-H2AX foci patterns. Furthermore, by using the dimensionality reduction method UMAP, we also observed TIP60 HAT mutant-like oncogenic-specific gamma-H2AX foci patterns in TIP60 wild-type cells. In summary, we propose the existence of an oncogenic-specific gamma-H2AX foci pattern and the importance of a machine learning approach to extract oncogenic signaling among the gamma-H2AX foci variations.
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We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of ?-H2AX, a well-known marker of DNA damage. To investigate the potential application of ?-H2AX ...
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We have developed an early detection method for bladder carcinogens with high sensitivity and specificity using immunohistochemistry of ?-H2AX, a well-known marker of DNA damage. To investigate the potential application of ?-H2AX as a biomarker for early detection of hepatocarcino-gens, we examined ?-H2AX formation in the liver of rats treated with several different chemicals for 28 days. Six-week-old male F344 rats were orally treated for 28 days with five hepatocarcinogens: N-nitros-odiethylamine (DEN), di(2-ethylhexyl) phthalate, 1,4-dioxane (DO), 3,3'-dimethylbenzidine dihydro-chloride, or thioacetamide (TAA), or with two non-hepatocarcinogens: 4-chloro-o-phenylenediamine and N-ethyl-N-nitrosourea. At the end of the treatment period, immunohistochemistry for ?-H2AX and Ki67 and expression analysis of DNA repair-related genes were performed. Significant increases in ?-H2AX-positive hepatocytes with upregulation of Rad51 mRNA expression were induced by three of five hepatocarcinogens (DEN, DO, and TAA), whereas no changes were seen for the other two hepatocarcinogens and the two non-hepatocarcinogens. Significant increases in Ki67 expression with upregulation of Bripl, Xrcc5, and Lig4 were observed in rats treated with TAA, a nongenotoxic hepatocarcinogen, suggesting that both direct DNA damage and secondary DNA damage due to cell replication stress may be associated with ?-H2AX formation. These results suggest that ?-H2AX immunostaining has potential value for early detection of hepatocarcinogens, but examination of the effects of more chemicals is needed, as is whether ?-H2AX immunostaining should be combined with other markers to increase sensitivity. ?-H2AX immu-nostaining using formalin-fixed paraffin-embedded specimens can be easily incorporated into existing 28-day repeated-dose toxicity studies, and further improvements in this method are expected.
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Background: Cisplatin is a widely used cancer chemotherapeutic drug that causes DNA crosslinking and stimulates H2AX phosphorylation. Our goal was to assess the potential of gamma H2AX to help predict tumor response to cisplatin t...
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Background: Cisplatin is a widely used cancer chemotherapeutic drug that causes DNA crosslinking and stimulates H2AX phosphorylation. Our goal was to assess the potential of gamma H2AX to help predict tumor response to cisplatin treatment. Methods: The kinetics of cisplatin-induced DNA interstrand crosslinks was measured using the alkaline comet assay and compared with gamma H2AX formation and clonogenic cell survival in several DNA repair proficient or deficient human and rodent cell lines. Results: The comet assay was effective in ranking cell lines according to their relative sensitivity to cisplatin based on reduced crosslink formation measured 6 h after drug exposure or by the failure of irs3 and UV41 cell lines to subsequently remove crosslinks. In comparison, the initial rate of phosphorylation of H2AX measured over the first 6 It after cisplatin treatment was unrelated to drug sensitivity or crosslinking proficiency. However, for proliferating cell cultures, the fraction of cells that retained gamma H2AX foci 24 h after cisplatin treatment was correlated with the fraction of cells that lost clonogenic potential (slope = 1.1, r(2) = 0.85). Conclusions: H2AX phosphorylation occurs in response to replication fork damage caused by cisplatin induced DNA lesions, probably interstrand crosslinks. Although early kinetics of gamma H2AX formation was uninformative, retention of gamma H2AX foci 24 h after treatment was shown to be a useful indicator of cell response to killing by cisplatin. However, for gamma H2AX to serve as an indicator of cell viability after cisplatin treatment, cells must have the opportunity to transit S phase during the recovery period.
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The mitogen-activated protein kinase/ERK kinase (MEK)/ERK pathway was shown to be constitutively activated in a large number of acute myelogenous leukemia (AML) cells, suggesting the important roles of this pro-survival signaling ...
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The mitogen-activated protein kinase/ERK kinase (MEK)/ERK pathway was shown to be constitutively activated in a large number of acute myelogenous leukemia (AML) cells, suggesting the important roles of this pro-survival signaling in leukemogenesis and proliferation of AML cells. This study explored the impact of the MEK inhibitor AZD6244 on the effect of cytarabien (AraC), one of the most commonly used anti-leukemia agents, to induce growth arrest and apoptosis of AML cells. AZD6244 effectively blocked AraC-induced MEK/ERK activation and enhanced its ability to induce growth arrest and apoptosis of NB4 and HL60 cells in parallel with induction of DNA damage as measured by detection of gamma-H2AX by Western Blot analysis, resulting in enhanced expression of p21 (waf1) and downregulation of c-Myc and Bcl-xl in these cells. Enhanced induction of apoptosis mediated by combination of AZD6244 and AraC was also shown in freshly isolated AML cells (n = 3). Taken together, concomitant administration of AraC and the inhibitor of MEK/ERK signaling may be useful for treatment of individuals with AML.
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Dibromoacetonitrile (DBAN) is a carcinogenic disinfection byproduct (DBP) but how it precipitates cancer is unknown. Nucleotide excision repair (NER) is a versatile repair mechanism for removing bulky DNA lesions to maintain genom...
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Dibromoacetonitrile (DBAN) is a carcinogenic disinfection byproduct (DBP) but how it precipitates cancer is unknown. Nucleotide excision repair (NER) is a versatile repair mechanism for removing bulky DNA lesions to maintain genome stability, and impairment of this process is associated with cancer development. In this study, we found that DBAN inhibited NER and investigated its mechanism with other DNA damage responses. Human keratinocytes HaCaT were treated with DBAN followed by ultraviolet (UV) as a model inducer of DNA damage, pyrimidine dimers, which require NER for the removal. DBAN pretreatment exacerbated UV-cytotoxicity, and inhibited the repair of pyrimidine dimers. DBAN treatment delayed the recruitment of NER proteins, transcription factor IIH (TFIIH) and xeroderma pigmentosum complementation group G (XPG), to DNA damaged sites, and subsequent gap filling process. Moreover, DBAN suppressed the UV-induced double strand breaks (DSBs) formation, as well as phosphorylated histone H2AX (gamma-H2AX), a widely used DNA damage marker. Altogether, DBAN could negatively impact the NER process and phosphorylation pathway responding to DNA damage. This study was the first to identify the inhibition of NER and damage response signaling as a genotoxicity mechanism of a class of DBPs and it may serve as a foundation for DBP carcinogenesis.
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