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

Blood ◽  
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.

scholarly journals A comparison of the in vitro genotoxicity of anticancer drugs idarubicin and mitoxantrone.

2002 ◽  
Vol 49 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Janusz Błasiak ◽  
Ewa Gloc ◽  
Mariusz Warszawski
Keyword(s):  
Dna Damage ◽  
Human Lymphocytes ◽  
Dna Glycosylase ◽  
Oxygen Species ◽  
Anthracycline Antibiotic ◽  
Strand Breaks ◽  
Endonuclease Iii ◽  
Normal Human ◽  
In Vitro Genotoxicity

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Stefano Bonassi ◽  
Marcello Ceppi ◽  
Peter Møller ◽  
Amaya Azqueta ◽  
Mirta Milić ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Circulatory System ◽  
Dna Lesions ◽  
Human Populations ◽  
Strand Breaks ◽  
Risk Of Death ◽  
Kaplan Meier ◽  
Cox Proportional Hazard Regression ◽  
Overall Mortality

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.

scholarly journals Free radical scavengers can modulate the DNA-damaging action of alloxan.

2003 ◽  
Vol 50 (1) ◽  
pp. 205-210 ◽  
Author(s):  
Janusz Blasiak ◽  
Agnieszka Sikora ◽  
Agnieszka Czechowska ◽  
Józef Drzewoski
Keyword(s):  
Dna Damage ◽  
Free Radicals ◽  
Free Radical ◽  
Antioxidant Activities ◽  
Human Lymphocytes ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Free Radical Scavengers ◽  
Dependent Manner ◽  
Radical Scavengers

Alloxan can generate diabetes in experimental animals and its action can be associated with the production of free radicals. It is therefore important to check how different substances often referred to as free radical scavengers may interact with alloxan, especially that some of these substance may show both pro- and antioxidant activities. Using the alkaline comet assay we showed that alloxan at concentrations 0.01-50 microM induced DNA damage in normal human lymphocytes in a dose-dependent manner. Treated cells were able to recover within a 120-min incubation. Vitamins C and E at 10 and 50 microM diminished the extent of DNA damage induced by 50 microM alloxan. Pre-treatment of the lymphocytes with a nitrone spin trap, alpha-(4-pyridil-1-oxide)- N-t-butylnitrone (POBN) or ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which mimics glutathione peroxides, reduced the alloxan-evoked DNA damage. The cells exposed to alloxan and treated with formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), enzymes recognizing oxidized and alkylated bases, respectively, displayed greater extent of DNA damage than those not treated with these enzymes. The results confirmed that free radicals are involved in the formation of DNA lesions induced by alloxan. The results also suggest that alloxan can generate oxidized DNA bases with a preference for purines and contribute to their alkylation.

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

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2878-2878 ◽  
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.

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

2009 ◽  
Vol 48 (06) ◽  
pp. 221-226 ◽  
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.

scholarly journals Validation of the in vitro comet assay for DNA cross-links and altered bases detection

2021 ◽  
Author(s):  
Damián Muruzabal ◽  
Julen Sanz-Serrano ◽  
Sylvie Sauvaigo ◽  
Bertrand Treillard ◽  
Ann-Karin Olsen ◽  
...  
Keyword(s):  
Comet Assay ◽  
Human Health Risk ◽  
High Sensitivity ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Vitro Assay ◽  
Cytotoxic Compound ◽  
Endonuclease Iii ◽  
Cross Links

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

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

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.

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels

2002 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Shawna M. Jackman ◽  
Geraldine M. Grant ◽  
Christopher J. Kolanko ◽  
David A. Stenger ◽  
Joginder Nath
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Damage Assessment ◽  
Human Lymphocytes ◽  
Jet Propulsion

scholarly journals Characterization of Escherichia coli DNA Lesions Generated within J774 Macrophages

2000 ◽  
Vol 182 (18) ◽  
pp. 5225-5230 ◽  
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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