BCR-ABL1 Kinase Inhibits DNA Glycosylases to Enhance Oxidative DNA Damage and Stimulate Genomic Instability

Abstract Abstract 520 Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of BCR-ABL1 kinase-positive chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 15–25% of patients initially responding favorably to imatinib will develop acquired drug resistance, which in 40–90% of cases is caused by genomic instability resulting in the appearance of clones expressing TKI resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species (ROS) resulting in excessive oxidative DNA damage such as oxidized DNA bases (8-oxoguanine and 5-hydroxycytosine→uracil) (Nieborowska-Skorska et al., Blood, 2012). Unfaithful and/or inefficient repair of these lesions generates TKI resistant point mutations in BCR-ABL1 kinase. Oxidative DNA lesions may be removed by base excision repair (BER) or, if not removed, will create mismatches, which are repaired by mismatch repair (MMR). Since we found that MMR is inhibited in CML-CP (Stoklosa et al., Cancer Res., 2008), the activity of BER is critical to prevent the accumulation of point mutations. Using an array of specific substrates and inhibitors/blocking antibodies we found that two major glycosylases, uracil-DNA glycosylase UNG2 and 8-oxoguanine glycosylase (OGG1) responsible for the excision of uracil (product of oxidation of cytosine) and 8-oxoguanine (8-oxoG) from DNA, respectively, were inhibited in BCR-ABL1 –transformed cell lines and CD34+ CML cells. The inhibitory effect was even more pronounced in CML blast phase (CML-BP) in comparison to CML-CP, it depended on BCR-ABL1 kinase activity and was not accompanied by deregulation of nuclear expression and/or chromatin association of these glycosylases. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases such as TEL-ABL1, TEL-PDGFbetaR and NPM-ALK did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI we found that UNG2 activity diminished the number of oxidized DNA bases detected by modified comet assay and prevented accumulation of point mutations in reporter gene Na+/K+ATPase, which encode resistance to ouabain. In conclusion, we hypothesize that inhibition of UNG2 and OGG1, accompanied by reduced MMR activity is responsible for accumulation of TKI-resistant BCR-ABL1 kinase point mutations and perhaps also other point mutations facilitating malignant progression of CML. Disclosures: No relevant conflicts of interest to declare.

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BCR/ABL Kinase Elevates ROS-Mediated Oxidative DNA Damage in CML Stem/Progenitor Cells and Affects the Efficiency and Fidelity of DNA Repair To Induce Genetic Instability.

Blood ◽

10.1182/blood.v110.11.34.34 ◽

2007 ◽

Vol 110 (11) ◽

pp. 34-34

Author(s):

Margaret Nieborowska-Skorska ◽

Artur Slupianek ◽

Tomasz Stoklosa ◽

Tomasz Poplawski ◽

Kimberly Cramer ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genomic Instability ◽

Chromosomal Aberrations ◽

Oxidative Dna Damage ◽

Point Mutations ◽

Dna Lesions ◽

Leukemia Cells ◽

Abl Kinase ◽

Imatinib Resistant

Abstract BCR/ABL kinase transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but a progenitor-driven disease. In CML-CP leukemia stem (LSCs) and progenitor (LPCs) cells reside in CD34+CD38− and CD34+CD38+ populations, respectively, whereas in CML-BC LSCs are found also in CD34+CD38+ population. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations and chromosomal aberrations associated with progression to CML-BC. Genomic instability may result from enhanced DNA damage and/or aberrant DNA repair mechanisms. We showed that CD34+ stem/progenitor CML cells contain higher levels of reactive oxygen species (ROS) than these from healthy donors (CML-BC>CML-CP>Normal). In addition, ROS were elevated in CD34+CD38− and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to cells from healthy donor. Higher ROS levels induced more oxidative DNA lesions such as oxidized bases (e.g., 8-oxoG) and DNA double-strand breaks (DSBs). ROS and oxidative DNA damage in CML stem/progenitor cells could be diminished by an antioxidant N-acetyl-cysteine. Moreover, inhibition of ROS by vitamin E reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells in SCID mice. Cellular DNA repair systems act to remove DNA damage and ultimately preserve the informational integrity of the genome. Base excision repair (BER) and mismatch repair (MMR) are responsible for removal of oxidized bases. BER was assessed using single- and double-stranded DNA substrates containing 5-OH-U (a derivative of ROS-damaged hydroxy-deoxycytidine). MMR activity was measured by restoration of the expression of GFP from the construct containing T-G mismatch in the start codon. BCR/ABL kinase severely inhibited BER and MMR in cell lines and CD34+ CML cells, and promoted accumulation of point mutations in genes encoding BCR/ABL kinase and Na+/K+ ATPase. Inhibition of BCR/ABL kinase by imatinib restored BER and MMR activities. Oxidized bases, if not repaired, may lead to accumulation of DSBs observed in LSCs and LPCs. DSBs may be processed by homologous recombination (HR), non-homologous and-joininig (NHEJ), and single-strand annealing (SSA). HR represents faithful repair, NHEJ usually produces small deletions, and SSA causes very large deletions. Genome-integrated repair-specific reporter cassettes containing two disrupted fragments of the gene encoding GFP were used where a single DSB induced by I-SceI endonuclease in one of the fragments stimulated HR, NHEJ, or SSA. In general, BCR/ABL kinase enhanced DSBs repair activities, however at the expense of their fidelity. Numerous point mutations were introduced in HR repair products. NHEJ generated larger than usual deletions. SSA, rather rare but very unfaithful, was also induced in BCR/ABL-positive leukemia cells. In summary, BCR/ABL kinase enhanced ROS-mediated oxidative DNA damage in LSCs and LPCs. In addition, BCR/ABL inhibited BER and MMR of usually non-lethal oxidized DNA lesions leading to accumulation of point mutations. Moreover, BCR/ABL kinase stimulated HR, NHEJ and SSA of lethal DSBs, but compromised the fidelity of repair.

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Genomic Instability in CML-CP originates From the Most Primitive Imatinib-Refractory Leukemia Stem Cells

Blood ◽

10.1182/blood.v120.21.909.909 ◽

2012 ◽

Vol 120 (21) ◽

pp. 909-909

Author(s):

Elisabeth Bolton ◽

Mirle Schemionek ◽

Hans-Urlich Klein ◽

Grazyna Hoser ◽

Sylwia Flis ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Board Of Directors ◽

Genomic Instability ◽

Research Funding ◽

Oxidative Dna Damage ◽

Dna Lesions ◽

Leukemia Stem Cells ◽

Human Leukemia ◽

Advisory Committees

Abstract Abstract 909 Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in the appearance of clones carrying BCR-ABL1 kinase mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or those harboring additional chromosomal aberrations, eventually leading to disease relapse and/or malignant progression to blast phase (CML-BP) [Skorski, T., Leukemia and Lymphoma, 2011]. We found that Lin−CD34+CD38− human leukemia stem cells (huLSCs), including the quiescent sub-population, and Lin−CD34+CD38+ human leukemia progenitor cells (huLPCs) accumulate high levels of reactive oxygen species (ROS) resulting in numerous oxidative DNA lesions such as 8-oxoguanine (8-oxoG) and DNA double-strand breaks (DSBs) [Nieborowska-Skorska, Blood, 2012]. huLSCs and huLPCs treated with TKIs continue to exhibit ROS-induced oxidative DNA damage suggesting the persistence of genomic instability in TKI-treated patients. Furthermore, genomic instability in TKI-refractory huLSCs and TKI-sensitive huLPCs may have a varying impact on disease progression and determining novel treatment modalities. To determine if TKI-refractory huLSCs are a source of genomic instability we employed a tetracycline-inducible murine model of CML-CP: SCLtTA/p210BCR-ABL1. Mice exhibiting CML-CP -like disease demonstrated splenomegaly, leukocytosis, and expansion of mature Gr1+/CD11b+ cells. ROS were elevated in Lin−c-Kit+Sca-1+ cells (muLSCs), but not Lin−c-Kit+Sca-1− cells (muLPCs), which was associated with higher mRNA expression of BCR-ABL1 in muLSCs. In addition, ROS levels were directly proportional to BCR-ABL1 kinase expression in transduced CD34+ human hematopoietic cells, thus confirming the “dosage-dependent” effect of BCR-ABL1 on ROS. Among the Lin−c-Kit+Sca-1+ cells, enhanced ROS were detected in TKI-refractory quiescent muLSCs, in CD34−Flt3− long-term and CD34+Flt3− short-term muLSCs, and also in CD34+Flt3+ multipotent progenitors. High levels of ROS in muLSCs were accompanied by aberrant expression of genes regulating ROS metabolism (mitochondrial electron transport, oxidative phosphorylation, hydrogen peroxide synthesis, and detoxification). In addition, muLSCs, including the quiescent sub-population, displayed high levels of oxidative DNA lesions (8-oxoG, and DSBs). ROS-induced oxidative DNA damage in muLSCs was accompanied by genomic instability in CML-CP –like mice, which accumulated a broad range of genetic aberrations recapitulating the heterogeneity of sporadic mutations detected in TKI-naive CML-CP patients. These aberrations include TKI-resistant BCR-ABL1 kinase mutations, deletions in Ikzf1 and Trp53 and additions in Zfp423 and Idh1 genes, which have been associated with CML-CP relapse and progression to CML-BP. Imatinib caused only modest inhibition of ROS and oxidative DNA damage in TKI-refractory muLSCs. In concordance, CML-CP –like mice treated with imatinib continued to accumulate genomic aberrations. Since BCR-ABL1(K1172R) kinase-dead mutant expressed in CD34+ human hematopoietic cells did not enhance ROS, it suggests that BCR-ABL1 kinase-independent mechanisms contribute to genomic instability. In summary, we postulate that ROS-induced oxidative DNA damage resulting in genetic instability may originate in the most primitive TKI-refractory huLSCs in TKI-naive and TKI-treated patients. Disclosures: Lange: Novartis: Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Mechanisms Generating free Radicals in CML Stem/Progenitor Cell Populations Causing DNA Damage and Genomic Instability

Blood ◽

10.1182/blood.v112.11.192.192 ◽

2008 ◽

Vol 112 (11) ◽

pp. 192-192 ◽

Cited By ~ 1

Author(s):

Margaret Nieborowska-Skorska ◽

Mateusz Koptyra ◽

Grazyna Hoser ◽

Regina Ray ◽

Danielle Ngaba ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Free Radicals ◽

Genomic Instability ◽

Chromosomal Aberrations ◽

Point Mutations ◽

Leukemia Cells ◽

Abl Kinase ◽

Imatinib Resistant ◽

Transformed Cell Lines

Abstract BCR/ABL kinase is the founding member of a family of oncogenic tyrosine kinases (OTKs) also including TEL/JAK2, TEL/PDGFR, TEL/ABL, and JAK2V617F, which induce myeloproliferative disorders (MPDs). BCR/ABL transforms hematopoietic stem cells (HSCs) to induce chronic myelogenous leukemia in chronic phase (CML-CP), which eventually evolves into fatal blast crisis (CML-BC). CML is a stem cell-derived but progenitor-driven disease. In CML-CP, leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) reside in the CD34+CD38- and CD34+CD38+ populations, respectively, whereas in CML-BC, LSCs are also found in the CD34+CD38+ population. In addition, CD34+ CML cells belong to either proliferative or quiescent populations; the latter of which responds poorly to the ABL kinase inhibitors. BCR/ABL kinase stimulates genomic instability causing imatinib-resistant point mutations in the kinase domain and additional chromosomal aberrations associated with progression to CML-BC (Oncogene, 2007). Since genomic instability usually results from enhanced DNA damage, we investigated the mechanisms responsible for “spontaneous” DNA damage in cells transformed by BCR/ ABL and other OTKs. Much endogenous DNA damage arises from free radicals such as reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). We showed that CD34+ stem/progenitor CML cells contain higher levels of ROS (superoxide anion = ·O2−, hydrogen peroxide = H2O2 and hydroxyl radical = ·OH) and RNS (nitric oxide = NO·) than CD34+ cells from normal donors (CML-BC>CML-CP>Normal). Moreover, ROS levels were elevated in CD34+CD38- and CD34+CD38+ sub-populations isolated from CML-BC and CML-CP patients in comparison to the corresponding cells from healthy donor. In addition, both proliferative and quiescent CD34+ CML cell sub-populations contained more ROS than their normal counterparts. Interaction with the stromal cells further elevated ROS levels in BCR/ABL-positive cells. Higher ROS/RNS levels induced more oxidative/nitrative DNA lesions, such as 8-oxoG and DNA double-strand breaks (DSBs), in CML-CP cells resulting in induction of point mutations in BCR/ABL kinase causing imatinib resistance and accumulation of chromosomal aberrations characteristic of CML-BC. In addition, cells transformed by other OTKs also displayed elevated ROS/ RNS and oxidative/nitrative DNA damage, implicating their role in malignant progression of MPDs. Our previous studies showed that elevated levels of oxidative DNA damage in OTK-transformed cells could be diminished by scavenging of ROS with N-acetyl-cysteine and vitamin E, which reduced the frequency of imatinib-resistant BCR/ABL point mutants and chromosomal aberrations in leukemia cells cultured in vitro and growing in SCID mice (Blood, 2006; Leukemia, 2008). These studies highlighted the importance of identification of the sources of free radicals in CML and other MPDs. We found that elevated levels of ROS in BCR/ABL-transformed cell lines and CD34+ CML cells were generated by three major mechanisms: NADPH oxidase (NOX) complexes containing NOX1 and/or NOX2, complex III of the mitochondrial respiratory chain (MRC), and 5-lipoxygenase (LOX). In addition, inducible nitric oxygen synthase (iNOS) produced RNS in leukemia cells. Using selective inhibitors of NOX, MRC, LOX and iNOS we estimated the contribution of these pathways to accumulation of free radicals causing oxidative/nitrative DNA damage in CML cells. In summary, BCR/ABL kinase-dependent elevation of ROS/RNS depends on several mechanisms, which are now targeted to determine their actual role in genomic instability in CML.

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Obesity, oxidative DNA damage and vitamin D as predictors of genomic instability in children and adolescents

International Journal of Obesity ◽

10.1038/s41366-021-00879-2 ◽

2021 ◽

Author(s):

Moonisah Usman ◽

Maria Woloshynowych ◽

Jessica Carrilho Britto ◽

Ivona Bilkevic ◽

Bethany Glassar ◽

...

Keyword(s):

Vitamin D ◽

Dna Damage ◽

Genomic Instability ◽

Oxidative Dna Damage ◽

Nutritional Deficiencies ◽

Aetiological Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Childhood And Adolescence ◽

Paediatric Obesity ◽

Vitamin D Levels

Abstract Background/objectives Epidemiological evidence indicates obesity in childhood and adolescence to be an independent risk factor for cancer and premature mortality in adulthood. Pathological implications from excess adiposity may begin early in life. Obesity is concurrent with a state of chronic inflammation, a well-known aetiological factor for DNA damage. In addition, obesity has been associated with micro-nutritional deficiencies. Vitamin D has attracted attention for its anti-inflammatory properties and role in genomic integrity and stability. The aim of this study was to determine a novel approach for predicting genomic instability via the combined assessment of adiposity, DNA damage, systemic inflammation, and vitamin D status. Subjects/methods We carried out a cross-sectional study with 132 participants, aged 10–18, recruited from schools and paediatric obesity clinics in London. Anthropometric assessments included BMI Z-score, waist and hip circumference, and body fat percentage via bioelectrical impedance. Inflammation and vitamin D levels in saliva were assessed by enzyme-linked immunosorbent assay. Oxidative DNA damage was determined via quantification of 8-hydroxy-2′-deoxyguanosine in urine. Exfoliated cells from the oral cavity were scored for genomic instability via the buccal cytome assay. Results As expected, comparisons between participants with obesity and normal range BMI showed significant differences in anthropometric measures (p < 0.001). Significant differences were also observed in some measures of genomic instability (p < 0.001). When examining relationships between variables for all participants, markers of adiposity positively correlated with acquired oxidative DNA damage (p < 0.01) and genomic instability (p < 0.001), and negatively correlated with vitamin D (p < 0.01). Multiple regression analyses identified obesity (p < 0.001), vitamin D (p < 0.001), and oxidative DNA damage (p < 0.05) as the three significant predictors of genomic instability. Conclusions Obesity, oxidative DNA damage, and vitamin D deficiency are significant predictors of genomic instability. Non-invasive biomonitoring and predictive modelling of genomic instability in young patients with obesity may contribute to the prioritisation and severity of clinical intervention measures.

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Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Nanomaterials ◽

10.3390/nano11061502 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1502

Author(s):

Fátima Brandão ◽

Carla Costa ◽

Maria João Bessa ◽

Elise Dumortier ◽

Florence Debacq-Chainiaux ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Amorphous Silica ◽

Oxidative Dna Damage ◽

Intratracheal Instillation ◽

Dna Lesions ◽

Lung Cells ◽

Rat Lung ◽

Inhalation Study

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

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Apurinic endonuclease-1 preserves neural genome integrity to maintain homeostasis and thermoregulation and prevent brain tumors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809682115 ◽

2018 ◽

Vol 115 (52) ◽

pp. E12285-E12294 ◽

Cited By ~ 6

Author(s):

Lavinia C. Dumitrache ◽

Mikio Shimada ◽

Susanna M. Downing ◽

Young Don Kwak ◽

Yang Li ◽

...

Keyword(s):

Dna Damage ◽

Nervous System ◽

Brain Tumors ◽

Oxidative Dna Damage ◽

Early Death ◽

Etiologic Agent ◽

Dna Lesions ◽

Oxidative Modification ◽

Early Gene ◽

Content Type

Frequent oxidative modification of the neural genome is a by-product of the high oxygen consumption of the nervous system. Rapid correction of oxidative DNA lesions is essential, as genome stability is a paramount determinant of neural homeostasis. Apurinic/apyrimidinic endonuclease 1 (APE1; also known as “APEX1” or “REF1”) is a key enzyme for the repair of oxidative DNA damage, although the specific role(s) for this enzyme in the development and maintenance of the nervous system is largely unknown. Here, using conditional inactivation of murine Ape1, we identify critical roles for this protein in the brain selectively after birth, coinciding with tissue oxygenation shifting from a placental supply to respiration. While mice lacking APE1 throughout neurogenesis were viable with little discernible phenotype at birth, rapid and pronounced brain-wide degenerative changes associated with DNA damage were observed immediately after birth leading to early death. Unexpectedly, Ape1Nes-cre mice appeared hypothermic with persistent shivering associated with the loss of thermoregulatory serotonergic neurons. We found that APE1 is critical for the selective regulation of Fos1-induced hippocampal immediate early gene expression. Finally, loss of APE1 in combination with p53 inactivation resulted in a profound susceptibility to brain tumors, including medulloblastoma and glioblastoma, implicating oxidative DNA lesions as an etiologic agent in these diseases. Our study reveals APE1 as a major suppressor of deleterious oxidative DNA damage and uncovers specific and broad pathogenic consequences of respiratory oxygenation in the postnatal nervous system.

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Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3729 ◽

2003 ◽

Vol 50 (1) ◽

pp. 211-215 ◽

Cited By ~ 15

Author(s):

Marcin Kruszewski ◽

Teresa Iwaneńko

Keyword(s):

Hydrogen Peroxide ◽

Dna Damage ◽

Cell Lines ◽

Oxidative Dna Damage ◽

Transition Metal Ions ◽

Dna Lesions ◽

Labile Iron Pool ◽

Labile Iron ◽

Iron Pool ◽

Mouse Lymphoma

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).

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Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells

Cells ◽

10.3390/cells9071671 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1671 ◽

Cited By ~ 1

Author(s):

Marios G. Krokidis ◽

Mariarosaria D’Errico ◽

Barbara Pascucci ◽

Eleonora Parlanti ◽

Annalisa Masi ◽

...

Keyword(s):

Dna Damage ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Enzymatic Digestion ◽

Cockayne Syndrome ◽

Dna Lesions ◽

Premature Aging ◽

Neurological Features ◽

Oxygen Tensions ◽

Base Excision

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

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CML-CP Mouse Model of Genomic Instability.

Blood ◽

10.1182/blood.v116.21.1210.1210 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1210-1210

Author(s):

Elisabeth Bolton ◽

Linda Kamp ◽

Hardik Modi ◽

Ravi Bhatia ◽

Steffen Koschmieder ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Stem Cell ◽

Mouse Model ◽

Board Of Directors ◽

Genomic Instability ◽

Oxidative Dna Damage ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Dependent Effect

Abstract Abstract 1210 Background: BCR-ABL1 transforms hematopoietic stem cells to induce chronic myeloid leukemia in chronic phase (CML-CP). Although CML is stem cell-derived, it is a progenitor cell-driven disease. In CML-CP, leukemia stem cells (LSCs) are characterized by elevated BCR-ABL1 expression in comparison to leukemia progenitor cells (LPCs). Increased expression of BCR-ABL1 kinase is also associated with progression from CML-CP to CML-blast phase. Previously we showed that BCR-ABL1 kinase stimulates reactive oxygen species (ROS)-dependent DNA damage resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). Result: To examine the effects of BCR-ABL1 expression on genomic instability during in vivo leukemogenesis we employed an inducible transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly, leukocytosis, and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood, as detected by FACS analysis. BCR-ABL1 mRNA expression was higher in Lin-c-Kit+Sca1+ stem-enriched cells than in Lin-c-Kit+Sca1- progenitor-enriched cells, thus reminiscent of CML-CP (LSCs>LPCs). BCR-ABL1 increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA lesions (8-oxoG) in LSC-enriched Lin-c-Kit+Sca1+ cells. Preliminary data also suggested that quiescent (CFSEmax) Lin-c-Kit+Sca1+ cells from BCR-ABL1-induced mice exhibited greater ROS (superoxide) production than non-induced counter parts. Moreover, higher levels of ROS were detected in BCR-ABL1-positive Lin-c-Kit+Sca1+ stem-enriched population in comparison to BCR-ABL1-positive Lin-c-Kit+Sca1- progenitor population, suggesting a dosage-dependent effect of BCR-ABL1. To confirm that BCR-ABL1 exerts a dosage-dependent effect on ROS-induced oxidative DNA damage, we showed that the levels of ROS, 8-oxoG and DNA double-strand breaks were proportional to BCR-ABL1 kinase expression in murine 32Dc13 and human CD34+ cells. Conclusion: In summary, this mouse model recapitulates the BCR-ABL1 expression profile attributed to stem and progenitor populations in human CML-CP. It also shows that the BCR-ABL1-positive, stem cell-enriched Lin-c-Kit+Sca1+ population displays elevated levels of ROS and oxidative DNA damage in comparison to normal counterparts, which makes it suitable to study the mechanisms of genomic instability in LSCs. Single nucleotide polymorphism (SNP) arrays will shed more light on the genomic instability of this BCR-ABL1-induced transgenic model of CML-CP. Disclosures: Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.

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Viral Infection Sentinel Rig-I Maintains Hematopoietic Stem Cell Function Through Regulating DNA-Damage Response

Blood ◽

10.1182/blood.v122.21.1166.1166 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1166-1166

Author(s):

Wu Zhang ◽

Meng-Lei Ding ◽

Xian-Yang Li ◽

He-Zhou Guo ◽

Hong-Xin Zhang ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Cell Function ◽

Conflicts Of Interest ◽

Dna Lesions ◽

Type I ◽

Hematopoietic Stem ◽

Damage Response ◽

Underlying Mechanisms ◽

And Function

Abstract Throughout life hematopoietic stem cells (HSCs) have to cope with various kinds of insults from inflammation to DNA damage constantly to maintain the integrity of stemness. It is possible that certain core factors are commonly implicated in the maintenance of HSC pool and function under discrete physiological and pathological conditions. However, the underlying mechanisms remain largely unexplored. Previous works have demonstrated that retinoic acid inducible gene I (Rig-I) plays an essential role in recognizing viral RNA and activating type I IFN transcription, but whether Rig-I is involved in the core program governing HSCs’ behaviors is unclear. Here, we report that in the steady status Rig-I deficiency significantly increased HSC number by dysregulating the cell-cycling status of HSCs in mice. However, HSCs in Rig-I-/- mice were actually more sensitive to genotoxic treatments such as irradiation as compared to wild type HSCs, causing more Rig-I-/- mice to die of hematopoietic exhaustion. In accordance, HSC transplantation assays showed a significant impact of Rig-I loss on the hematopoietic regeneration capacity. Mechanistically, we found that Rig-I represented a pivotal component of the molecular pathways that mediate DNA-damage response and the repair of DNA lesions. Taken together, these data indicate a crucial role of innate immunity-regulatory factor Rig-I in the maintenance of HSCs. Disclosures: No relevant conflicts of interest to declare.

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