Hematopoietic Stem Cells Repair Ionizing Radiation-Induced DNA Double Strand Breaks in a Cell Cycle-Dependent Manner.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3241-3241
Author(s):  
Senthil Kumar Pazhanisamy ◽  
Ningfei An ◽  
Yong Wang ◽  
Daohong Zhou
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Adult Mouse ◽  
Double Strand Breaks ◽  
Hematopoietic Progenitor Cells ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Strand Breaks ◽  
Quiescent Hscs

Abstract Abstract 3241 Poster Board III-178 Mice with mutations in various DNA repair genes exhibit accelerated aging due to hematopoietic stem cell (HSC) premature exhaustion, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. In addition, some of these mutated mice are highly susceptible to the development of leukemia and lymphoma due to an increase in genomic instability in HSCs. However, how HSCs respond to genotoxic stress and repair DNA damage have not been well established and thus, were investigated in the present study using a mouse model. Specifically, DNA damage and repair were analyzed by gH2AX immunofluorescent staining and neutral comet assay to quantify IR-induced DNA double strand breaks (DSBs) in HSCs (Lin- c-kit+ Sca1+ cells or LKS+ cells) and hematopoietic progenitor cells (HPCs; Lin- c-kit+ Sca1- cells or LKS- cells) isolated from adult mouse bone marrow (BM) after they were exposed to ionizing radiation (IR). The results showed that exposure to IR induced a similar number of DSBs in HSCs and hematopoietic progenitor cells (HPCs) isolated from adult mouse BM. However, HPCs repaired the damage within 6 h after IR, whereas more than 50% DSBs were unrepaired by HSCs even at 24h after IR, indicating that HSCs are highly deficient in repair of IR-induced DSBs. The deficient DSBs repair in HSCs is attributable to their quiescence, as sorted quiescent Pyronin Ylow HSCs were more deficient in repairing the damage than cycling Pyronin Yhigh HSCs. This suggestion is further supported by the observations that proliferating HSCs such as fetal liver HSCs and HSCs isolated from 5-FU-treated adult mouse BM repaired the damage as efficiently as HPCs. In addition, incubation of quiescent Pyronin Ylow HSCs from adult BM with stem cell factor and thrombopoietin for 48 h stimulated the cell cycle entry and DNA damage repair function. These findings indicate that stimulation of cell cycling can promote HSCs to repair DNA damage. The difference in repair of IR-induced DSBs between quiescent and cycling HSCs is not because they express different levels of key proteins (such as Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, Dclre1c, Nhej1, Brac-1, Brac-2, MRE11a, Nbs1, Rad50, Rad51, and ATM) involved in non-homologous end joining (NHEJ) and homologous recombination (HR). Instead, quiescent HSCs exhibited an insignificant activation of DNA-PK and minimal formation of XRCC4 and Rad51 foci after exposure to IR, suggesting that quiescent HSCs are deficient in DSBs repair through the NHEJ and HR pathways. However, quiescent HSCs exhibited similar levels of phosphorylation of ATM and p53 after IR compared to cycling HSCs and HPCs, indicating that quiescent HSCs are proficient in sensing DNA damage to initiate DNA damage responses. These findings provide crucial insights into how HSCs respond to and repair DNA damage, which could significantly advance our understanding on how HSCs maintain their genomic stability. Disclosures No relevant conflicts of interest to declare.

Loss Of NIPA Leads To Defects In The Repair Of DNA Double Strand Breaks In Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2488-2488
Author(s):  
Anna Lena Illert ◽  
Cristina Antinozzi ◽  
Hiroyuki Kawaguchi ◽  
Michal Kulinski ◽  
Christine Klitzing ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Meiotic Prophase ◽  
Conflicts Of Interest ◽  
Cyclin B1 ◽  
Double Strand Breaks ◽  
Conditional Knockout ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Chromosome Axis

Abstract Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. Using a conditional knockout strategy we inactivated the gene encoding Nipa. Nipa-deficient animals are viable, but show a lower birth rate and a reduced body weight. Furthermore, Nipa-deficient males were sterile due to a block of spermatogenesis during meiotic prophase. Virtually no spermatocytes progress beyond a late-zygotene to early-pachytene stage and reach an aberrant stage, with synaptonemal complex disassembly and incomplete synapsis. Nipa-/- females are sub-fertile with an early and severe meiotic defect during embryogenesis with extensive apoptosis in early prophase (E13.5-E14.5). Here we report, that Nipa-/- meiocytes exhibit persistent cytological markers for DNA double strand break repair proteins (like DMC1, RAD51) in meiotic prophase with more than twice as many DMC1 foci as control animals. Kinetic analysis of the first wave of spermatogenesis showed increased DMC1/RAD51 foci in Nipa-/- cells as soon as early-pachynema cells appear (13-14 days post partum). Moreover, we show that Nipa deficiency does not lead to a defect in meiotic sex chromosome inactivation despite epithelial stage IV apoptosis. Nipa-deficient spermatocytes exhibit numerous abnormalities in staining of chromosome axis associated proteins (like SYCP3 and STAG3) indicating that chromosome axis defects were associated with compromised chromosome axis integrity leading to overt chromosome fragmentation. Further in vitro analyses with bleomycin treated MEFs displayed high pH2AX levels in cells lacking NIPA. Repair of DNA DSB seemed to be abolished in these cells as the pH2AX-level were sustained and still visible after 90 min of timecourse, where wildtype cells already repaired sides of DNA Damage. Consistent with these findings NIPA-deficient spleen cells showed compromised DNA Damage repair measured in a comet assay with a significantly longer olive tail moment in NIPA knockout cells under repair conditions. Taken together, the phenotype of Nipa-knockout mice is a definitive proof of the meiotic significance of NIPA and our results show a new, unsuspected role of NIPA in chromosome stability and the repair of DNA double strand breaks. Disclosures: No relevant conflicts of interest to declare.

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.

ANKHD1 Silencing Delays S Phase Progression in Multiple Myeloma Cells Via Activation of ATM/ATR -CDC25a Pathway

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5624-5624
Author(s):  
Dhyani Anamika ◽  
Patricia Favaro ◽  
Sara Teresinha Olalla Saad
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Dna Damage ◽  
S Phase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Myeloma Cells ◽  
Checkpoint Kinase ◽  
Strand Breaks ◽  
Phase Progression

Abstract Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mechanisms of different response to ionizing irradiation in isogenic head and neck cancer cell lines

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Vesna Todorovic ◽  
Ajda Prevc ◽  
Martina Niksic Zakelj ◽  
Monika Savarin ◽  
Andreja Brozic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Head And Neck ◽  
Double Strand Breaks ◽  
The Other ◽  
Dna Double Strand Breaks ◽  
Altered Expression ◽  
Strand Breaks ◽  
Other Hand ◽  
Dna Damage Signalling

Abstract Background Treatment options for recurrent head and neck tumours in the previously irradiated area are limited, including re-irradiation due to radioresistance of the recurrent tumour and previous dose received by surrounding normal tissues. As an in vitro model to study radioresistance mechanisms, isogenic cells with different radiosensitivity can be used. However, they are not readily available. Therefore, our objective was to establish and characterize radioresistant isogenic human pharyngeal squamous carcinoma cells and to evaluate early radiation response in isogenic parental, radioresistant and radiosensitive cells. Methods Radioresistant cells were derived from parental FaDu cells by repeated exposure to ionizing radiation. Radiosensitivity of the established isogenic radioresistant FaDu-RR cells was evaluated by clonogenic assay and compared to isogenic parental FaDu and radiosensitive 2A3 cells. Additional phenotypic characterization of these isogenic cells with different radiosensitivity included evaluation of chemosensitivity, cell proliferation, cell cycle, radiation-induced apoptosis, resolution of DNA double-strand breaks, and DNA damage and repair signalling gene expression before and after irradiation. Results In the newly established radioresistant cells in response to 5 Gy irradiation, we observed no alteration in cell cycle regulation, but delayed induction and enhanced resolution of DNA double-strand breaks, lower induction of apoptosis, and pronounced over-expression of DNA damage signalling genes in comparison to parental cells. On the other hand, radiosensitive 2A3 cells were arrested in G2/M-phase in response to 5 Gy irradiation, had a prominent accumulation of and slower resolution of DNA double-strand breaks, and no change in DNA damage signalling genes expression. Conclusions We concluded that the emergence of the radioresistance in the established radioresistant isogenic cells can be at least partially attributed to the enhanced DNA double-strand break repair, altered expression of DNA damage signalling and repair genes. On the other hand, in radiosensitive isogenic cells the reduced ability to repair a high number of induced DNA double-strand breaks and no transcriptional response in DNA damage signalling genes indicate on a lack of adaptive response to irradiation. Altogether, our results confirmed that these isogenic cells with different radiosensitivity are an appropriate model to study the mechanisms of radioresistance.

High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

2003 ◽  
Vol 285 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
Natalia I. Dmitrieva ◽  
Dmitry V. Bulavin ◽  
Maurice B. Burg
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

scholarly journals Role of the Saccharomyces cerevisiae Rad53 Checkpoint Kinase in Signaling Double-Strand Breaks during the Meiotic Cell Cycle

2008 ◽  
Vol 28 (14) ◽  
pp. 4480-4493 ◽  
Author(s):  
Hugo Cartagena-Lirola ◽  
Ilaria Guerini ◽  
Nicola Manfrini ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Meiotic Cell ◽  
Dna Double Strand Breaks ◽  
Protein Fusion ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Meiotic Dsbs ◽  
Recombination Checkpoint

ABSTRACT DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.

scholarly journals PS2 - 153 Dianhydrogalactitol (VAL-083) Treatment Causes Irreparable DNA Double-Strand Breaks, S/G2 Phase Cell-Cycle Arrest and Cell Death in Cancer Cells

2016 ◽  
Vol 43 (S4) ◽  
pp. S12-S13
Author(s):  
B. Zhai ◽  
A. Steino ◽  
J. Bacha ◽  
D. Brown ◽  
M. Daugaard
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Death ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Double Strand Breaks ◽  
Phase Cell ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Cycle Arrest

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. VAL-083 readily crosses the blood-brain barrier, accumulates in brain tumor tissue and has shown activity in prior NCI-sponsored clinical trials against various cancers, including glioblastoma (GBM) and medulloblastoma. VAL-083 is also active against GBM cancer stem cells and acts as a radiosensitizer independent of O6-methylguanine-DNA methyltransferase activity (in contrast to e.g. temozolomide and BCNU). Here we report new insights into VAL-083 mechanism of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA damage. In lung (H2122, H1792, H23, A549) and prostate (PC3, LNCaP) cancer cell lines VAL-083 treatment caused irreversible S/G2 cell-cycle arrest and cell death (IC50 range 3.06-25.7 µM). VAL-083 pulse-treatment led to persistent phosphorylation of DNA double-strand breaks (DSB) sensors ATM, single-strand DNA-binding Replication Protein A (RPA32), and histone variant H2A.X, suggesting persistent DNA lesions. After 10 months in culture with increasing VAL-083 concentrations, H1792 and LNCaP cells survive at concentrations up to 9.4 µM and 7.4 µM, respectively, suggesting that efficient resistance mechanisms are not easily acquired by the cancer cells. Taken together with previous results showing that VAL-083 circumvents cisplatin-resistance and is less dependent on p53 activity than cisplatin, these results suggest a molecular mechanism for VAL-083 that differs from both TMZ, BCNU and cisplatin. They further suggest that irreparable DNA damage induced by VAL-083 is impervious to common strategies employed by cancer cells to escape effects of alkylating drugs used in GBM treatment.

scholarly journals Checkpoint Responses to DNA Double-Strand Breaks

2020 ◽  
Vol 89 (1) ◽  
pp. 103-133 ◽  
Author(s):  
David P. Waterman ◽  
James E. Haber ◽  
Marcus B. Smolka
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Model System ◽  
Double Strand Breaks ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Dsbs ◽  
Cell Lethality

Cells confront DNA damage in every cell cycle. Among the most deleterious types of DNA damage are DNA double-strand breaks (DSBs), which can cause cell lethality if unrepaired or cancers if improperly repaired. In response to DNA DSBs, cells activate a complex DNA damage checkpoint (DDC) response that arrests the cell cycle, reprograms gene expression, and mobilizes DNA repair factors to prevent the inheritance of unrepaired and broken chromosomes. Here we examine the DDC, induced by DNA DSBs, in the budding yeast model system and in mammals.

scholarly journals Effect of pivaloyl-substituted pyrrole containing heterocyclic compounds on DNA repair pathways in Ewing sarcoma cells

2018 ◽  
Vol 99 (2) ◽  
pp. 245-248
Author(s):  
A R Galembikova ◽  
S V Boychuk ◽  
P D Dunaev ◽  
R R Khusnutdinov ◽  
S S Zykova
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Tumor Cells ◽  
Ewing Sarcoma ◽  
Heterocyclic Compounds ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Brca 1 ◽  
Sarcoma Cells

Aim. To examine deoxyribonucleic acid (DNA) damage repair and cell cycle regulatory mechanisms of Ewing sarcoma cells exposed to pivaloyl-substituted pyrrole containing heterocyclic compounds. Methods. The study was performed on A673 Ewing sarcoma cell line. The tumor cells were incubated for 48 h in the presence of pivaloyl-substituted pyrrole containing heterocyclic compounds (compounds №20 and №24). Western blot analysis was utilized to examine expression of the markers of DNA single-strand (phosphorylated forms of ATR and Chk1) and double-strand breaks (phosphorylated forms of H2AX, АТМ, DNA-PK, BRCA-1, Chk-2). Analysis of the cell cycle phases was performed by flow cytometry (BD FacsCanto, USA). Results. Pivaloyl-substituted pyrrole containing heterocyclic compounds substantially increased the expression of histone 2A phosphorylated on serine 138 (γ-H2AX) that indicates DNA damage (double-strand breaks). Under exposure to pivaloyl-substituted pyrrole containing heterocyclic compounds the studied cells increased expression of phosphorylated forms of ATM-kinase and BRCA-1. Also cell cycle disorders leading to substantial G2/M arrest and enhanced apoptosis of tumor cells were observed. Conclusion. Pivaloyl-substituted pyrrole containing heterocyclic compounds induced DNA double-strand breaks in A673 Ewing sarcoma cell line; in response to DNA damage in tumor cells, the mechanisms of DNA double-strand breaks repair were activated; despite activation of DNA repair mechanisms, A673 cells underwent cell cycle arrest in the G2/M-phase and apoptosis.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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