Bestimmung der DNA-Schädigung in vitro

2010 ◽  
Vol 49 (S 01) ◽  
pp. S64-S68
Author(s):  
E. Dikomey
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Chromosome Aberrations ◽  
Genetic Factors ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cell Inactivation ◽  
Repair Mechanisms ◽  
Break Repair

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

scholarly journals Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

2021 ◽  
Author(s):  
Xinrui Zhang ◽  
Mariana Bobeica ◽  
Michael Unger ◽  
Anastasia Bednarz ◽  
Bjoern Gerold ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Cancer Cells ◽  
Metabolic Activity ◽  
Focused Ultrasound ◽  
Double Strand Breaks ◽  
High Intensity Focused Ultrasound ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

scholarly journals Live-cell chromosome dynamics in Arabidopsis thaliana reveals increased chromatin mobility in response to DNA damage

2021 ◽  
Author(s):  
Anis Meschichi ◽  
Adrien Sicard ◽  
Frédéric Pontvianne ◽  
Svenja Reeck ◽  
Stefanie Rosa
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Genome Instability ◽  
High Mobility ◽  
Double Strand Breaks ◽  
Nuclear Space ◽  
Strand Breaks ◽  
Homologous Sequences ◽  
Damage Response ◽  
Repair Mechanisms

Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage potentially leading to translocations and genome instability. Homologous recombination (HR) is a conservative repair pathway in which intact homologous sequences are used as a template for repair. How damaged DNA molecules search for homologous sequences in the crowded space of the cell nucleus is, however, still poorly understood, especially in plants. Here, we measured global chromosome and DSB site mobility, in Arabidopsis thaliana, by tracking the motion of specific loci using the lacO/LacI tagging system and two GFP-tagged HR regulators, RAD51 and RAD54. We observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Interestingly, we observed that DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease of DSB mobility is observed, which seems to be associated to the relocation of DSBs to the nucleus periphery. Altogether, our data suggest that changes in chromatin mobility are triggered in response to DNA damage, and that this may act as a mechanism to enhance the physical search within the nuclear space to locate a homologous template during homology-directed DNA repair.

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

Homologous Recombination Repair of DNA Strand Breaks Induced by the Nucleoside Analogue CNDAG.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1189-1189
Author(s):  
Xiaojun Liu ◽  
Yingjun Jiang ◽  
Akira Matsuda ◽  
William Plunkett
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
T Cell ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Double Strand Breaks ◽  
Myelogenous Leukemia ◽  
Nucleoside Analogues ◽  
Strand Breaks ◽  
Damage Repair

Abstract Abstract 1189 We hypothesize that the novel deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl) guanine] may share the common action mechanism with its cytosine congener CNDAC [2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine], a prodrug of which, sapacitabine, is undergoing clinical trials in myeloid leukemias. CNDAC induces single strand breaks following incorporation into DNA. Subsequent processing or DNA replication across the unrepaired nicks would generate double strand breaks (DSBs) [1]. Because cytosine and guanine nucleoside congeners have remarkably different clinical activities, e.g., cytarabine (acute myelogenous leukemia) and nelarabine (T-cell malignancies), it will be useful to pursue investigations to fully characterize the metabolism and actions of CNDAG. This study was aimed at defining cellular response and damage repair mechanisms for two CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each prodrug is a substrate for adenosine deaminase (ADA), the action of which generates CNDAG. First, growth inhibition by both CNDAG prodrugs was dependent upon both concentration and time of exposure; the proliferation of T-cell malignant lines (CCRF-CEM and Jurkat) was suppressed more to B-cell lines (Raji and IM-9). This may be attributed to relatively low activity of deoxycytidine kinase in the latter cell lines. Second, p53 knocked-out and parental HCT116 cells were equally sensitive to CNDAG 6-NH2 in a clonogenic assay, indicating that cytotoxicity of CNDAG is independent of p53 status. Third, similar to CNDAC, CNDAG prodrugs activated repair proteins in multiple DNA damage response pathways, as revealed by immunoblotting. 24-hr incubation of CCRF-CEM cells with 50 microM either prodrug increased the phosphorylation of Ser-1981 on ATM, Ser-345 on Chk1, Thr-68 on Chk2, Ser-966 on SMC1, Ser-343 on Nbs1 and g-H2AX. In contrast, there was no increase in phosphorylation of two other sensor kinases, DNA-PKcs (Ser-2056) which participates in repair of double strand breaks by non-homologous end-joining, and ATR (Ser-428) which senses stalled DNA replication forks. Fourth, we investigated the role of components of homologous recombination (HR) in CNDAG-induced DNA damage repair. The clonogenic survival of human fibroblasts deficient in ATM or those transfected with an empty vector were approximately 20- to 30-fold more sensitive to CNDAG prodrugs than cells complemented with full-length ATM cDNA. Chinese hamster cells deficient in Rad51D or either of the two Rad51-interacting proteins, Xrcc3 and Brca2, conferred greater than 30-fold sensitivity to CNDAG prodrugs relative to respective wild type lines. Similar sensitization was also observed with CNDAC. In contrast, cells lacking HR function were not more sensitive to ara-C or ara-G compared to their parental and complemented cells, indicating HR is a unique repair mechanism for 2`-C-cyano-2`-deoxy-nucleoside analogues. Finally, a cytogenetic approach was used to analyze sister chromatid exchange (SCE, a hallmark for HR) formation in metaphase cells exposed to 2 microM CNDAG 6-NH2. The frequencies of SCEs in AA8 cells incubated with CNDAG for two cell cycles (mean 14.2 per metaphase) were 2-fold of those exposed for one cell cycle (mean 7.4 per metaphase, n>20, p<0.001), the latter greater than control (mean 6 per metaphase, p<0.05). Together these results demonstrate that DNA damage caused by CNDAG activates ATM-dependent signaling pathways and is repaired through homologous recombination. Thus, this is a class effect caused by 2`-C-cyano-2`-deoxy-nucleoside analogues. Our study suggests that despite relatively less potency, CNDAG might have distinct clinical activity from that of CNDAC. [1] Liu X, et al. Blood, Blood. 2010 May 17, Epub ahead of print, PMID: 20479284. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Attenuating the DNA damage response to double-strand breaks restores function in models of CNS neurodegeneration

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Richard I Tuxworth ◽  
Matthew J Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

Abstract DNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double-strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double-strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals Attenuating the DNA damage response to double strand breaks restores function in models of CNS neurodegeneration

2018 ◽  
Author(s):  
Richard I. Tuxworth ◽  
Matthew J. Taylor ◽  
Ane Martin Anduaga ◽  
Alaa Hussien-Ali ◽  
Sotiroula Chatzimatthaiou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nervous System ◽  
Dna Damage Response ◽  
Neurological Disorders ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Ganglion Neurons

AbstractDNA double-strand breaks are a feature of many acute and long-term neurological disorders, including neurodegeneration, following neurotrauma and after stroke. Persistent activation of the DNA damage response in response to double strand breaks contributes to neural dysfunction and pathology as it can force post-mitotic neurons to re-enter the cell cycle leading to senescence or apoptosis. Mature, non-dividing neurons may tolerate low levels of DNA damage, in which case muting the DNA damage response might be neuroprotective. Here, we show that attenuating the DNA damage response by targeting the meiotic recombination 11, Rad50, Nijmegen breakage syndrome 1 complex, which is involved in double strand break recognition, is neuroprotective in three neurodegeneration models in Drosophila and prevents Aβ1-42-induced loss of synapses in embryonic hippocampal neurons. Attenuating the DNA damage response after optic nerve injury is also neuroprotective to retinal ganglion cells and promotes dramatic regeneration of their neurites both in vitro and in vivo. Dorsal root ganglion neurons similarly regenerate when the DNA damage response is targeted in vitro and in vivo and this strategy also induces significant restoration of lost function after spinal cord injury. We conclude that muting the DNA damage response in the nervous system is neuroprotective in multiple neurological disorders. Our results point to new therapies to maintain or repair the nervous system.

scholarly journals YGR042W/MTE1 Functions in Double-Strand Break Repair with MPH1

2015 ◽  
Author(s):  
Askar Yimit ◽  
TaeHyung Kim ◽  
Ranjith Anand ◽  
Sarah Meister ◽  
Jiongwen Ou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Break Repair

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

scholarly journals Synthetic Lethal Approaches to Exploit Replicative Stress in Aggressive Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 173-173
Author(s):  
Francesca Cottini ◽  
Teru Hideshima ◽  
Rikio Suzuki ◽  
Paul G. Richardson ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Replicative Stress ◽  
Aggressive Disease ◽  
Stress Markers ◽  
Similar Phenotype

Abstract Background: Multiple myeloma (MM) cells show a variable combination of chromosomal translocations, copy-number variations, somatic mutations and clonal heterogeneity, which makes every patient unique. We have recently shown that MM cells have signs of ongoing DNA-damage, which activates an ATM/ABL1-dependent DNA damage response (DDR) without overt apoptosis (Cottini et al., Nat Med, 2014). Here we further characterize the mechanisms of DNA damage and replicative stress in MM, which provide the basis for a novel synthetic lethality treatment approach. Results: MM cell lines with active DNA damage have enrichment in pathways of DNA replication and cell cycle. These same MM cell lines also present 53BP1, RPA and RAD51 foci with activated ATR and CHK1. Of note, 53BP1, RPA and RAD51 foci are markers of replicative stress, associated with DNA hyper-replication and stalled replication forks. Importantly, replicative stress markers are also present in primary MM cells. We also demonstrated a gene expression signature specific for increased chromosomal instability and DNA damage in a cohort of MM patients versus normal plasma cells. Remarkably, a subset (20 percent) of patients with myeloma overexpress genes belonging to the instability signature; this group also shows an unfavorable prognosis due to a more aggressive disease. These findings suggest that some patients present a similar phenotype to the cell lines, characterized by extensive replicative stress and activation of hyper-replicative pathways. We therefore hypothesized that MM cells might be sensitive to replicative stress overload, which occurs when cells fail to endure the presence of an excess of unrepaired DNA. To evaluate this hypothesis, we used shRNAs to silence ATR, a protein involved in the control of stalled replication origins, in two myeloma cell lines, one with normal TP53 (H929) and another with mutant TP53 (OPM-2). Inhibition of ATR caused a reduction in cell growth and induction of apoptosis, both more evident in MM cell lines with mutant TP53. A similar phenotype was observed when MM cell lines were incubated with VE-821, a specific ATR inhibitor. The strongest response occurred in TP53 mutant cell lines, which are representative of a model of aggressive MM, consistent with the concept of replicative stress overload. Indeed, p53 is normally phosphorylated and active in MM, while TP53 loss in the context of hyper-replication may prevent activation of salvage checkpoint, thereby favoring cell death in the absence of ATR. ATR inhibition also induces an increase in DNA double strand breaks, as evidenced by the higher number of γ-H2A.X foci. Reactive oxygen species (ROS) can also mediate DNA damage, and treatment with an antioxidant reagent N-Acetylcysteine (NAC), which helps scavenging ROS by replenishing glutathione stores, was indeed capable of reducing DNA double strand breaks and replicative stress markers. Since cancer cells are particularly sensitive to oxidative stress, we then evaluated the anti-MM activity of piperlongumine (PL), a drug that induces ROS accumulation. MM cell lines were sensitive to PL treatment, while PBMCs were minimally affected. As expected, the apoptotic effects of PL were abrogated upon co-incubation with NAC, indicating the specific activity of PL on ROS. We next exploited the possibility of combining replicative and oxidative stress in myeloma cells, hoping to overcome the threshold of tolerance to unrepaired DNA. H929 and OPM-2 cells were transfected with ATR shRNAs or treated with VE-821 and incubated with DMSO or 1-2.5 μM PL; synergic effects by the combination treatment were evident in both myeloma cell lines and also in patient MM cells. Conclusion: Replicative stress is present in a group of MM patients, who have aggressive disease, myeloma cell hyperproliferation and poor prognosis. Strategies aimed to shift the balance towards high DNA damage by ROS production and reduced DNA repair can decrease MM growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

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