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.

BCR/ABL Oncogenic Kinase Promotes Unfaithful Repair of the Reactive Oxygen Species - Dependent DNA Double-Strand Breaks.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 712-712 ◽  
Author(s):  
Tomasz Skorski ◽  
Michal O. Nowicki ◽  
Rafal Falinski ◽  
Mateusz Koptyra ◽  
Artur Slupianek ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Double Strand Breaks ◽  
Transformed Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Abl Kinase

Abstract The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph1)-positive leukemia cells. We find that BCR/ABL kinase - induced reactive oxygen species (ROS) cause chronic oxidative DNA damage as indicated by an enzymatic assay detecting oxidized bases. These DNA lesions result in DNA double-strand breaks (DSBs) detected by comet assay, immunofluorescent gamma-H2AX nuclear foci and linker-ligation PCR (LL-PCR). Combined analysis of the length of LL-PCR products and the sequences of two reference genes DR-GFP and Na+/K+ ATPase revealed that ROS dependent DSBs occurred in the regions containing multiple, 5–9bp long stretches of G/C, in concordance with the notion that oxidative DNA damage is predominantly detected in G/C-rich sequences. Elevated numbers of DSBs were detected in BCR/ABL cell lines, murine bone marrow cells transformed with BCR/ABL and in CML patient samples, in comparison to normal counterparts. Inhibition of the BCR/ABL kinase by STI571 and diminishion of ROS activity by the ROS scavenger PDTC reduced DSBs formation. Cell cycle analysis revealed that most of these DSBs occur during S and G2/M phases, and are probably associated with the stalled replication forks. Homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in S and G2/M cell cycle phase. Using the in vivo recombination assay consisting of the DSB-dependent reconstitution of the green fluorescent protein (GFP) gene we found that HRR is stimulated in BCR/ABL-positive cells. In addition, in vitro assay measuring the activity of NHEJ revealed that this repair process is also activated by the BCR/ABL kinase. RAD51 and Ku70 play a key role in HRR and NHEJ, respectively. The reaction sites of HRR and NHEJ in the nuclei could be visualized by double-immunofluorescence detecting co-localization of gamma-H2AX foci (DSBs sites) with RAD51 (HRR sites) or Ku70 (NHEJ sites). Equal co-localization frequency of gamma-H2AX foci with RAD51 and Ku70 was detected, suggesting that both HRR and NHEJ play an important role in reparation of ROS-dependent DSBs in BCR/ABL-transformed cells. Analysis of the DSBs repair products in the reporter DR-GFP gene in BCR/ABL cells identified ~40% of HRR and ~60% of NHEJ events. Sequencing revealed point-mutations in HRR products and large deletions in NHEJ products in BCR/ABL-positive cells, but not in non-transformed cells. We propose that the following series of events may contribute to genomic instability of Ph1-positive leukemias: BCR/ABL → ROS → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair. Since BCR/ABL share many similarities with other members of the fusion tyrosine kinases (FTKs) family, these events may contribute to genomic instability of hematological malignancies caused by FTKs.

Fanconi Anemia D2 (FANCD2) Is Required To Overcome Reactive Oxygen Species (ROS) - Induced DNA Double-Strand Breaks in BCR/ABL-Positive Leukemias.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2119-2119
Author(s):  
Mateusz Koptyra ◽  
Tomasz Skorski
Keyword(s):  
Bone Marrow Cells ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Dna Lesions ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Growth Defect ◽  
Cell Cycle Distribution ◽  
Murine Bone Marrow ◽  
Abl Kinase

Abstract BCR/ABL-mediated transformation is associated with elevation of ROS which, in addition to enhancing the cytoplasmic signaling pathways, may increase the number of oxidative DNA lesions including DSBs. Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS. 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 implicating the role in repair of ROS-dependent DSBs. In addition, higher levels of monoubiquitinated FANCD2 protein 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, FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. This speculation is supported by impaired transformation potential of the BCR/ABL kinase 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. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied with delayed leukemogenesis in SCID mice. 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 and +/+ counterparts display similar levels of ROS and oxidized DNA bases, however, the former cells accumulate more DSBs evaluated by neutral comet assay and detected by γ-H2AX foci immunostaining. This effect could be reversed by the expression of FANCD2 S222A, but not K561R mutant, again implicating HR in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by vitamin E and N-acetylcysteine reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in 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.

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.

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

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.

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 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.

CML Progenitor Cells Have Chromsomal Instability and Display Increased DNA Damage at DNA Fragile Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1989-1989 ◽  
Author(s):  
Jamil K. Dierov ◽  
David W. Schoppy ◽  
Martin Carroll
Keyword(s):  
Dna Damage ◽  
Progenitor Cells ◽  
Chromosomal Instability ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Fragile Sites ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cd34 Cells

Abstract Chronic myelogeneous leukemia (CML) is a two stage disease which progresses to blast crisis over a period of 3–5 years in untreated patients. The BCR/ABL oncogene induces the hyperproliferation associated with chronic phase CML but whether BCR/ABL induces chromosomal instability leading to blast crisis has been controversial. We have previously demonstrated that BCR/ABL delays the repair of DNA double strand breaks and increases chromosomal instability in a murine cell line. Furthermore, we have demonstrated in cell lines that BCR/ABL disrupts the function of the DNA damage sensing protein, ataxia telangiectasia and rad 3 related (ATR). One of the functions of ATR is to maintain the stability of DNA fragile sites, late replicating sites in the chromosome that are frequently involved in translocations. To determine if BCR/ABL affects the stability of DNA fragile sites in Ba/F3 cells that do or do not express BCR/ABL, cells were incubated in low dose aphidicolin for 24 hours to induce fragile site breakage. BCR/ABL expressing cells, but not control cells, demonstrated fragile site damage consistent with a disruption of ATR function in BCR/ABL expressing cells. In order to determine if primary patient cells display a genomic instability phenotype, we have analyzed the response to DNA damage in CD34+ cells from normal volunteers and from CML patients seen at the University of Pennsylvania Cancer Center. We first examined the DNA repair response by treating cells for two hours with etoposide. Both normal cells and CML progenitor cells demonstrate DNA double strand breaks as measured by the comet assay, a quantitative assay for DNA double strand breaks. However, in Ph+ cells from the patient sample there was a delay in the repair of DNA double strand breaks as indicated by a significant increase in the olive tail moment at 2 hours and 24 hours after treatment with etoposide. In addition, we analyzed the effect of a two hour exposure to etoposide on chromosome stability as measured by spectral karyotyping (SKY). Normal CD34+ cells and CD34+ cells from patients were treated with etoposide and then allowed to recover for 48 hours before analysis of metaphase spreads. Normal cells demonstrated no spontaneous DNA damage and, after etoposide treatment and repair, demonstrated only modest levels of DNA damage (2 translocations and 5 numerical alterations per 14 metaphases analyzed). In contrast, Ph+ cells demonstrated spontaneous DNA damage in these cell conditions. Furthermore, after etoposide treatment Ph+ cells demonstrated high levels of DNA damage with 9 translocations and 12 numerical alterations in 13 metaphases. These results suggest that Ph+ progenitor cells from patients with CML demonstrate chromosomal instability and suggest a mechanism for progression from CML chronic phase to blast crisis. Full analysis of additional patient samples will be presented. Taken together, we propose that BCR/ABL disrupts ATR function in cell lines and primary cells leading to an increase in chromosomal instability that leads to CML blast crisis.

Monoubiquitination of the Fanconi Anemia D2 (FANCD2) Protein Regulates the Transforming Potential of BCR/ABL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3189-3189
Author(s):  
Mateusz Koptyra ◽  
Tomasz Stoklosa ◽  
Grazyna Hoser ◽  
Ilona Seferynska ◽  
Eliza Glodkowska ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Chronic Phase ◽  
Dna Lesions ◽  
Growth Potential ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Growth Defect ◽  
Cell Cycle Distribution ◽  
Abl Kinase

Abstract We showed before that BCR/ABL-mediated leukemogenesis is associated with the elevation of reactive oxygen species (ROS), which in addition to enhancing signaling pathways may increase the number of oxidative DNA lesions including DNA double-strand breaks (DSBs) [Blood, 2005, 2006]. CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) contain higher number of DSBs induced by ROS in comparison to CD34+ cells from healthy donors [Cancer Res., 2008]. Recent studies also revealed that CD34+CD38− CML-CP and CML-BC stem cell-enriched populations display more DSBs than normal counterparts as measured by gamma-H2AX foci formation on DNA. Thus, BCR/ABL-positive leukemia cells appear to accumulate an excess of ROS-induced DSBs, which may cause apoptosis if not repaired. We reported before that homologous recombination repair (HRR), involving RAD51 protein, plays a pivotal role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS [Blood, 2005]. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 by FANCL ubiquitinase and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 (FANCD2-Ub) interacts with RAD51 to facilitate HRR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint. We detected an increased amount of FANCD2-Ub in BCR/ABL-positive leukemia cell line and CD34+ CML-CP and CML-BC cells in comparison to normal counterparts. This effect was not associated with up-regulation of FANCD2 ubiquitinase FANCL or down-regulation of FANCD2 deubiquitinase USP1, but was reversed after inhibition of BCR/ABL kinase with imatinib and reduction of ROS with antioxidant vitamin E (VE) or N-acetylcysteine (NAC). Therefore we postulate that BCR/ABL kinase-dependent ROS-induced FANCD2- Ub may play a role in leukemic transformation. This hypothesis is supported by impaired transformation potential of BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. Restoration of the expression of FANCD2 protein in FANCD2−/− cells rescued the transforming potential of BCR/ABL kinase. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. Again BCR/ABL kinase did not exert a negative effect on the proliferation of FANCD2-reconstituted cells. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied by delayed leukemogenesis in SCID mice. Growth potential of BCR/ABL-positive FANCD2−/− could be rescued by co-expression of FANCD2 wild-type and S222A mutant, but not the K561R mutant. This observation supports our hypothesis that K561 monoubiquitination, but not S222 phosphorylation of FANCD2 might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL employs RAD51-dependent HRR to repair numerous DSBs induced by ROS, elevated expression of FANCD2-Ub may facilitate this process. This speculation is supported by the observation that although BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, the former cells accumulate more DSBs evaluated by neutral comet assay and immunostained gamma-H2AX nuclear foci. This effect could be reversed by the expression of FANCD2 S222A mutant, but not K561R mutant, again implicating FANCD2-Ub in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging ROS by VE and NAC reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that FANCD2-Ub, but not FANCD2-phosphoS222 may play an important role in BCR/ABL-dependent leukemogenesis probably due to its ability to interact with RAD51 and facilitate HRR of the numerous ROS-induced DSBs.

BCR/ABL Modifies the Kinetics and Fidelity of DNA Double-Strand Breaks Repair in Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1982-1982
Author(s):  
Artur Slupianek ◽  
Michal O. Nowicki ◽  
Tomasz Skorski
Keyword(s):  
Genomic Instability ◽  
Mammalian Cells ◽  
Double Strand Breaks ◽  
Time Dependent ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Dna Double Strand Breaks ◽  
Γ Irradiation ◽  
Normal Cells ◽  
Strand Breaks

Abstract Clinical observations and experimental findings indicated that BCR/ABL stimulates genomic instability leading to mutations and chromosomal abnormalities. The accumulation of genetic errors is believed to be responsible for the transition from a relatively benign CML chronic phase (CML-CP) to the aggressive blast crisis phase (CML-BC) and the resistance to imatinib mesylate. BCR/ABL- positive leukemia cells accumulate an excess of potentially lethal DNA double-strand breaks (DSBs) caused by reactive oxygen species (ROS) or genotoxic treatment. However, BCR/ABL tyrosine kinase facilitates the repair of DSBs and promotes survival. Therefore, the infidelity of DSBs repair processes may contribute to genomic instability in leukemia cells exposed to elevated numbers of spontaneous and/or induced DSBs. To test this hypothesis DSBs repair efficiency and fidelity was examined and compared in parental and BCR/ABL-transformed cells. Nuclear foci detected by γ-H2AX (the form of H2AX histone that is quickly phosphorylated on Serine 139 by ATM, ATR and/or DNA-PKcs kinases on megabase-length fragments near DSB sites) immunofluorescence served as indicators of DSBs. We found that BCR/ABL-positive leukemia cells acquire more DSBs after γ-irradiation in comparison to normal cells. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in mammalian cells. HR and NHEJ reaction sites in the nuclei can be visualized by double-immunofluorescence detecting co-localization of γ-H2AX foci with RAD51 or Ku70, respectively. NHEJ and HR appear to work in a time-dependent fashion (NHEJ followed by HR) and be more active in BCR/ABL-transformed cells in comparison to normal counterparts. Time-dependent engagements of NHEJ and HR mechanisms in repair of DSBs after γ-irradiation are accompanied by elevated accumulation of Ku70 and RAD51 proteins in cell lysates obtained from BCR/ABL cells. Specific DSBs repair assays confirmed that BCR/ABL leukemia cells in comparison to normal cells displayed enhanced capability of HR and NHEJ. However, analysis of DSBs repair products revealed that the repair mechanisms were less faithful in former cells generating large deletions and point mutations during NHEJ and HR, respectively. In summary, BCR/ABL leukemia cells display facilitated, but unfaithful HR and NHEJ, which may contribute to accumulation of genetic errors in surviving leukemia cells leading to malignant disease progression.

BCR/ABL Kinase Disrupts Formation of Mismatch Repair Complex To Induce Genomic Instability.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2864-2864
Author(s):  
Tomasz Stoklosa ◽  
Artur Slupianek ◽  
Grzegorz Basak ◽  
Tomasz Skorski
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Mismatch Repair ◽  
Genomic Instability ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Abl Kinase ◽  
Mnng Treatment ◽  
Mmr Proteins

Abstract Among different genotoxic agents, BCR/ABL cells are more resistant to N-methyl-N’-nitro-N-nitrosoguanine (MNNG), which methylates 06 position of guanine. 06 -MeG bases pair with either T or C during replication giving rise to mismatches. Mismatch repair (MMR) proteins are responsible for detecting and removing misincorporated nucleotides, which escaped proofreading activity of DNA polymerases. MMR proteins assembled on the mismatch can signal to repair or apoptosis. Defects in expression of MMR genes leads to drug resistance and mutator phenotype, observed in several different solid tumors. The role of MMR system in drug resistance and/or genomic instability of leukemic cells remains poorly understood. Parental cells and BCR/ABL expressing clones were incubated with MNNG for 4 weeks resulting in their MNNG-resistant derivatives, which may accumulate mutations in their genomic DNA resulting from methylating activity of the drug. To investigate the mutation rate and phenotype, ouabain-resistance test was employed. Cells become resistant to ouabain, a glycoside that inhibits ATP1A1 subunit of Na/K ATP-ase, when mutations arise in the gene fragment encoding this subunit. The clonogenic assay revealed over 5 times more ouabain resistant colonies in MNNG-resistant BCR/ABL-positive cells than in parental counterparts. To investigate the type of mutations, a fragment of Na/K ATP-ase gene encoding the ATP1A1 subunit was sequenced from MNNG-resistant BCR/ABL-positive and parental cells. The dominating mutation in BCR/ABL MNNG-resistant cells was C to T, while A to G mutations were prevalent in parental cells. In order to check the status of MMR proteins, Western blotting and immunofluorescence studies were performed. Expression of MMR proteins in BCR/ABL transformed cells was similar to parental cells, however immunofluorescence visualized dramatic changes after DNA damage in the nuclear co-localization of MMR proteins in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. Co-localization of MSH2 and MSH6 proteins, forming a heterodimer homologous to bacterial MutS, remained similar in parental and leukemia cells upon MNNG treatment. However, co-localization of MLH1 and PMS2 proteins, which form a heterodimer homologous to bacterial MutL, was detected in non-transformed cells, but not in BCR/ABL leukemia cells. In addition, MLH1 and MSH2 co-localized in normal, but not BCR/ABL-positive cells after MNNG treatment. The defects in interaction of MMR proteins in leukemia cells were reversed by inhibition of BCR/ABL kinase by STI571. Thus, the assembly of MMR proteins on mismatched bases and subsequent signaling to repair and/or apoptosis may be impaired in BCR/ABL leukemia cells. These results suggests a novel mechanism of regulation of DNA damage response proteins by oncogenic tyrosine kinase that can lead to genomic instability and drug resistance of leukemic cells.

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