DNA damage in circulating leukocytes measured with the comet assay may predict the risk of death

AbstractThe comet assay or single cell gel electrophoresis, is the most common method used to measure strand breaks and a variety of other DNA lesions in human populations. To estimate the risk of overall mortality, mortality by cause, and cancer incidence associated to DNA damage, a cohort of 2,403 healthy individuals (25,978 person-years) screened in 16 laboratories using the comet assay between 1996 and 2016 was followed-up. Kaplan–Meier analysis indicated a worse overall survival in the medium and high tertile of DNA damage (p < 0.001). The effect of DNA damage on survival was modelled according to Cox proportional hazard regression model. The adjusted hazard ratio (HR) was 1.42 (1.06–1.90) for overall mortality, and 1.94 (1.04–3.59) for diseases of the circulatory system in subjects with the highest tertile of DNA damage. The findings of this study provide epidemiological evidence encouraging the implementation of the comet assay in preventive strategies for non-communicable diseases.

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Imatinib (STI571) Induces DNA Damage in BCR/ABL-Expressing Leukemic Cells but Not in Normal Lymphocytes.

Blood ◽

10.1182/blood.v104.11.4353.4353 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4353-4353

Author(s):

Janusz Blasiak ◽

Jozef Drzewoski ◽

Tomasz Poplawski ◽

Agnieszka Czechowska

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Human Lymphocytes ◽

Direct Interaction ◽

K562 Cells ◽

Dna Lesions ◽

Dna Glycosylase ◽

Leukemic Cells ◽

Drug Induced ◽

Strand Breaks

Abstract Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets specifically the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.05 to 2 μM induced DNA damage in human leukemic K562 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the plasmid relaxation assay. K562 cells were unable to repair H2O2-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes.

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Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells.

Blood ◽

10.1182/blood.v106.11.2878.2878 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2878-2878 ◽

Cited By ~ 1

Author(s):

Mateusz Koptyra ◽

Scott Houghtaling ◽

Marcus Grompe ◽

Tomasz Skorski

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Chronic Phase ◽

Dna Lesions ◽

Double Strand Breaks ◽

Leukemia Cells ◽

Transformed Cells ◽

Strand Breaks ◽

Abl Kinase ◽

D2 Protein

Abstract Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.

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Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay

Alternatives to Laboratory Animals ◽

10.1177/026119299602400315 ◽

1996 ◽

Vol 24 (3) ◽

pp. 405-411 ◽

Cited By ~ 17

Author(s):

Mária Dušinská ◽

Andrew Collins

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Single Cells ◽

Dna Strand Breaks ◽

Human Populations ◽

Dna Damage And Repair ◽

Strand Breaks ◽

Dna Lesion ◽

Dna Strand ◽

Uv Photoproducts

The comet assay (single cell gel electrophoresis) is a rapid, very sensitive method for the detection of DNA strand breaks at the level of single cells, which is now being applied in genotojricity testing. We modified this method for the detection of a variety of kinds of DNA lesion, by treating nucleoid DNA in the gel with either formamidopyrimidine-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguanine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and UV photoproducts). By using this modified assay, we demonstrate the removal of DNA strand breaks and oxidised purines upon incubating cells after treatment with hydrogen peroxide. This modification clearly increases the usefulness of the assay for the analysis of DNA damage and repair, for screening human populations for DNA damage, and for testing novel chemicals for genotoxicity.

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DNA damage in lymphocytes after irradiation with 211At and 188Re

Nuklearmedizin ◽

10.3413/nukmed-0262 ◽

2009 ◽

Vol 48 (06) ◽

pp. 221-226 ◽

Cited By ~ 8

Author(s):

M. Wendisch ◽

G. Wunderlich ◽

R. Freudenberg ◽

J. Kotzerke ◽

R. Runge

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Mononuclear Cells ◽

Dna Lesions ◽

Alpha Particles ◽

Alkaline Comet Assay ◽

Peripheral Blood Mononuclear ◽

Strand Breaks ◽

Neutral Comet Assay ◽

Dna Strand

Summary Aim: Ionising radiation produces many types of DNA lesions of different complexity. High linear energy transfer (LET) types of radiation are biological more effective than low LET radiation. In the present work we applied the single cell gel electrophoreses (comet assay) to study the induction of initial DNA damage, efficiency of repair and residual DNA damage in lymphocytes after treatment with 211At and 188Re. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and irradiated with 211At and 188Re at different doses. The comet assay was performed under alkaline and neutral conditions in order to detect the initial DNA damage and its repair. The measure of damage was % tail DNA (percentage of DNA in the tail). Results: After treatment of cells with 188Re the initial DNA damage (% tail DNA) detected with the alkaline comet assay was higher than the damage measured for 211At. The neutral comet assay estimated higher tail intensities for 211At in contrast to 188Re. Compared with the complete repair (10%) after irradiation with 188Re, the radiotoxicity of alpha particles indicated reduced rejoining of DNA strand breaks (60–80% residual damage). Rejoining of DNA damage measured by the neutral comet method detected about 70% unrepaired strand breaks for 211At and 188Re. Conclusions: There are major differences between the repair of strand breaks caused by 188Re and 211At detected by the alkaline comet assay. The DNA-damage induced by the high LET Emitter 211At remains nearly unrepaired detected by both alkaline and neutral comet assay. Represented data following irradiation of lymphocytes with alpha and beta particles demonstrated higher biological effectiveness of 211At by factors of 2.0–2.5.

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Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

International Journal of Molecular Sciences ◽

10.3390/ijms22147638 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7638

Author(s):

Yvonne Lorat ◽

Judith Reindl ◽

Anna Isermann ◽

Christian Rübe ◽

Anna A. Friedl ◽

...

Keyword(s):

Electron Microscopy ◽

Dna Damage ◽

Spatial Clustering ◽

Dna Lesions ◽

Double Strand Breaks ◽

Stimulated Emission ◽

Nuclear Chromatin ◽

Carbon Ions ◽

Dna Double Strand Breaks ◽

Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

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Characterization of Escherichia coli DNA Lesions Generated within J774 Macrophages

Journal of Bacteriology ◽

10.1128/jb.182.18.5225-5230.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5225-5230 ◽

Cited By ~ 90

Author(s):

Eliana Schlosser-Silverman ◽

Maya Elgrably-Weiss ◽

Ilan Rosenshine ◽

Ron Kohen ◽

Shoshy Altuvia

Keyword(s):

Escherichia Coli ◽

Dna Damage ◽

Respiratory Burst ◽

Defense Mechanism ◽

Dna Lesions ◽

Intracellular Bacteria ◽

Bacterial Killing ◽

Strand Breaks ◽

E Coli ◽

Dna Strand

ABSTRACT Macrophages are armed with multiple oxygen-dependent and -independent bactericidal properties. However, the respiratory burst, generating reactive oxygen species, is believed to be a major cause of bacterial killing. We exploited the susceptibility of Escherichia coli in macrophages to characterize the effects of the respiratory burst on intracellular bacteria. We show that E. coli strains recovered from J774 macrophages exhibit high rates of mutations. We report that the DNA damage generated inside macrophages includes DNA strand breaks and the modification 8-oxo-2′-deoxyguanosine, which are typical oxidative lesions. Interestingly, we found that under these conditions, early in the infection the majority of E. coli cells are viable but gene expression is inhibited. Our findings demonstrate that macrophages can cause severe DNA damage to intracellular bacteria. Our results also suggest that protection against the macrophage-induced DNA damage is an important component of the bacterial defense mechanism within macrophages.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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Tetrahymena Meiotic Nuclear Reorganization Is Induced by a Checkpoint Kinase–dependent Response to DNA Damage

Molecular Biology of the Cell ◽

10.1091/mbc.e08-10-1058 ◽

2009 ◽

Vol 20 (9) ◽

pp. 2428-2437 ◽

Cited By ~ 32

Author(s):

Josef Loidl ◽

Kazufumi Mochizuki

Keyword(s):

Dna Damage ◽

Kinase Inhibitors ◽

Signal Transduction Pathway ◽

Dna Lesions ◽

Meiotic Pairing ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Nuclear Reorganization ◽

Bivalent Formation ◽

Atr Kinase

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 spo11Δ 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.

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The Dark Side of UV-Induced DNA Lesion Repair

Genes ◽

10.3390/genes11121450 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1450

Author(s):

Wojciech Strzałka ◽

Piotr Zgłobicki ◽

Ewa Kowalska ◽

Aneta Bażant ◽

Dariusz Dziga ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Uv Light ◽

Genetic Material ◽

Dna Lesions ◽

Dark Side ◽

Repair System ◽

Strand Breaks ◽

Dna Lesion ◽

Pyrimidine Dimers

In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers. In this review, we have focused on how plants cope with deleterious DNA damage that cannot be repaired by photoreactivation. The current understanding of light-independent mechanisms, classified as dark DNA repair, indispensable for the maintenance of plant genetic material integrity has been presented.

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The role of Sp1 in the detection and elimination of cells with persistent DNA strand breaks

NAR Cancer ◽

10.1093/narcan/zcaa004 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Polina S Loshchenova ◽

Svetlana V Sergeeva ◽

Sally C Fletcher ◽

Grigory L Dianov

Keyword(s):

Dna Damage ◽

Cancer Therapy ◽

Genome Stability ◽

Dna Strand Breaks ◽

Dna Lesions ◽

Exogenous Dna ◽

Strand Breaks ◽

Specificity Factor ◽

Dna Strand

Abstract Maintenance of genome stability suppresses cancer and other human diseases and is critical for organism survival. Inevitably, during a life span, multiple DNA lesions can arise due to the inherent instability of DNA molecules or due to endogenous or exogenous DNA damaging factors. To avoid malignant transformation of cells with damaged DNA, multiple mechanisms have evolved to repair DNA or to detect and eradicate cells accumulating unrepaired DNA damage. In this review, we discuss recent findings on the role of Sp1 (specificity factor 1) in the detection and elimination of cells accumulating persistent DNA strand breaks. We also discuss how this mechanism may contribute to the maintenance of physiological populations of healthy cells in an organism, thus preventing cancer formation, and the possible application of these findings in cancer therapy.

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