scholarly journals DNA break clustering inside repair domains predicts cell death and mutation frequency in human fibroblasts and in Chinese hamster cells for a 103x range of linear energy transfers

2020 ◽  
Author(s):  
Eloise Pariset ◽  
Ianik Plante ◽  
Artem L. Ponomarev ◽  
Louise Viger ◽  
Trevor Evain ◽  
...  
Keyword(s):  
Experimental Data ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Cosmic Radiation ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Cell Orientation ◽  
Strand Breaks

ABSTRACTCosmic radiation, composed of high charged and energy (HZE) particles, causes cell death and mutations that can subsequently lead to cancers. Radiation-mediated mutations are induced by inter- and intra-chromosomal rearrangements (translocations, deletions, inversions) that are triggered by misrepaired DNA breaks, especially double-strand breaks (DSBs). In this work, we introduce a new model to predict radiation-mediated induction of cell death and mutation in two different cell lines across a large range of linear energy transfer (LET) values, based on the assumption that DSBs cluster into repair domains, as previously suggested by our group. Specifically, we propose that the probabilities of cell survival and cell mutation can be determined from the number of DSBs and the number of pairwise DSB interactions forming radiation-induced foci. We computed the distribution and locations of DSBs with the new simulation code RITCARD (relativistic ion tracks, chromosome aberrations, repair, and damage) and combined them with experimental data from HF19 human fibroblasts and V79 Chinese hamster cells to derive the parameters of our model and expand its predictions to the relative biological effectiveness (RBE) for cell survival and mutation in both cell lines in response to 9 different irradiation particles and energies ranging from 10 to 1,600 MeV/n. Our model generates the correct bell shape of LET dependence for RBE, as well as similar RBE values as experimental data, notably including data that were not used to set the model parameters. Interestingly, our results also suggest that cell orientation (parallel or perpendicular) with respect to the HZE beam can modulate the RBE for both cell death and mutation frequency. Cell orientation effects, if confirmed experimentally, would be another strong piece of evidence for the existence of DNA repair domains and their critical role in interpreting cellular sensitivity to cosmic radiation and hadron therapy.AUTHOR SUMMARYOne of the main hazards of human spaceflight beyond low Earth orbit is space radiation exposure. Galactic cosmic rays (GCRs), in particular their high-charge and high-energy particle component, induce a unique spatial distribution of DNA double strand breaks in the nucleus along their traversal in the cell [1], which result in significantly higher cancer risk than X-rays [2]. To mitigate this hazard, there is a significant need to better understand and predict the effects of cosmic radiation exposure at the cellular level. We have computationally predicted two biological endpoints – cell survival and probability of mutations, critical for cancer induction mechanisms – for the full spectrum of cosmic radiation types and energies, by modeling the distribution of DNA damage locations within the cell nucleus. From experimental results of cell survival and mutation probability in two standard cell lines, we were able to derive the parameters of the model for multiple radiation qualities, both biological endpoints, and two irradiation orientations. The model was validated against biological data and showed high predictive capability on data not used for tuning the model. Overall, this work opens new perspectives to predict multiple responses to cosmic radiation, even with limited experimental data available.

A Role for DNA Double Strand Breaks in the Sensitivity of AML Cells to Clofarabine: Enhanced In Vitro Sensitivity in Primary AML Cells with FLT3 Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2582-2582
Author(s):  
Monica Pallis ◽  
Martin Grundy ◽  
Claire Seedhouse ◽  
Heather Pimblett ◽  
Nigel Russell
Keyword(s):  
Cell Death ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Mitochondrial Membrane ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Cytochrome C Release ◽  
Strand Breaks ◽  
Flt3 Mutations ◽  
Kg1 Cells

Abstract Clofarabine is a purine nucleoside analogue which has been incorporated into several therapeutic trial protocols for the treatment of leukaemias including acute myeloid leukaemia (AML). The aims of the study are to ascertain mechanisms of clofarabine action in AML using cell lines and presentation samples from patients. We measured apoptosis by TdT assay, cytochrome C release and flow cytometric assays of the mitochondrial membrane potential probe DiOC6. To study the effects of clofarabine on DNA synthesis and DNA double strand breaks, we used bromodeoxyuridine (BrdU) and H2AX assays respectively. Equitoxic doses were established that caused approximately 20% cell death in the AML cell lines HL-60 (0.3 μM), KG1 (1 μM) and MV4.11 (1 μM) after 6 hours of continuous exposure. At these doses, clofarabine induced apoptotic DNA nicks, as measured by the TdT assay, and Cytochrome C release in all three cell lines. However, clofarabine-induced mitochondrial hyperpolarisation and depolarisation were found to be cell-type specific, occurring in HL-60 but not MV4.11 or KG1 cells, i.e. mitochondrial membrane depolarisation is not an essential part of the mechanism of clofarabine-induced apoptosis. Following clofarabine treatment, DNA synthesis was reduced by 91% within 1 hour in KG1 cells and by 80% in HL-60, but by only 9% in MV4.11. Out of the three cell lines, MV4.11 cells, which have a FLT3 internal tandem duplication (ITD), were the only ones to completely repair DNA double strand breaks following clofarabine removal. In 12 samples from patients with AML, the proportion of cells which incorporated BrdU in a 45 minute assay - a measure of the rate of cell cycling - differed considerably between samples, from 0.8% to 23%, median 13%. Cell death induced by clofarabine was correlated with the cycling rate of untreated cells (P=0.016). All 12 samples showed inhibition of DNA synthesis within 60 minutes of clofarabine treatment (range 20%–92% inhibition, median 54%). Mitochondrial membrane hyperpolarisation and depolarisation were not observed in patient cells. However, DNA double strand breaks were induced by clofarabine in patient cells. Paradoxically, i.e. in contrast to the results seen with the MV4.11 cell line, toxicity was greatest in samples with a FLT3 mutation (P=0.007). In conclusion, the FLT3 mutant MV4.11 cell line effectively repairs clofarabine-induced double strand breaks. Cell death in patient cells cultured with clofarabine is correlated with the presence of a FLT3 mutation. As we have previously established that AML samples with FLT3 mutations have upregulated DNA repair activity, this paradox might be explained by cycles of attempted DNA repair frustrated by renewed clofarabine incoporation into DNA, thus increasing the toxicity of the drug.

Targeting Aberrant Non-Homologous End Joining in Multiple Myeloma: Role of the Classical and Alternative Pathways in Genomic Instability

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3417-3417 ◽  
Author(s):  
Teresa Calimeri ◽  
Daniele Caracciolo ◽  
Nicola Amodio ◽  
Mehmet Kemal Samur ◽  
Marzia Leotta ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Genomic Instability ◽  
Cell Lines ◽  
Lethal Dose ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Nhej Pathway ◽  
Non Homologous End Joining

Abstract Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM. To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes. In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. Disclosures No relevant conflicts of interest to declare.

scholarly journals DNA Double-Strand Breaks Affect Chromosomal Rearrangements during Methotrexate-Mediated Gene Amplification in Chinese Hamster Ovary Cells

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 376
Author(s):  
Jong Youn Baik ◽  
Hye-Jin Han ◽  
Kelvin H. Lee
Keyword(s):  
Cell Lines ◽  
Gene Amplification ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chromosomal Rearrangements ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
The Impact

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes.

New Therapeutic Strategy for Sensitisation of CLL Cells with Inactivation of the DNA Damage Response by Targeting the Deubiquitylating Enzyme USP7-Dependent Pathways,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3865-3865
Author(s):  
Angelo Agathanggelou ◽  
Anastasia Zlatanou ◽  
Ahmed Gulshanara ◽  
Grant Stewart ◽  
Pamela R Kearns ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Cell Lines ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Pharmacological Inhibition ◽  
Caspase 7 ◽  
P53 Activation ◽  
Regulatory Loop

Abstract Abstract 3865 Chronic Lymphocytic Leukaemia (CLL) is characterised by marked clinical heterogeneity and tumours with deletion or mutation of the TP53 and ATM genes on chromosomes 17p and 11q are associated with poor outcome. ATM is a protein kinase that, following induction of DNA double strand breaks (DSBs), phosphorylates a number of downstream targets and synchronises a network of cellular responses including p53 activation that leads to induction of pro-apoptotic genes and activation of apoptosis. Consequently, loss of integrity of ATM/p53 pathway results in apoptotic resistance, retention of cells with genomic damage, and tumour progression. Cellular p53 levels are regulated through a p53-Mdm2 regulatory loop whereby Mdm2, a ubiquitin ligase, facilitates p53 polyubiquitination and targeting of p53 for proteasome degradation. It was recently shown that stabilisation of p53 can be achieved by manipulation of this regulatory loop through use of small molecule inhibitors of this p53 degradation pathway, termed nutlins, which prevent p53 ubiquitination. More recently, a class of deubiquitinating enzymes (DUBs) highlighted an additional level of p53-Mdm2 regulation. In particular, a specific DUB, (USP7/HAUSP), has a high affinity for Mdm2 and functions by antagonizing Mdm2 ubiquitination. Unlike nutlins, USP7 also has been implicated in the regulation of cell cycle, mitosis and DNA damage response and we reasoned that USP7 inhibition may sensitise CLL tumours with ATM and TP53 defects. Our analysis of 25 primary CLL tumours with different ATM and TP53 status indicated that USP7 was robustly expressed in all CLL tumour cells tested. Through collaboration with Hybrigenics we obtained a specific USP7 inhibitor, HBX19818. To determine if cell killing could be induced in ATM or TP53 deficient tumours by inhibiting the USP7-Mdm2 pathway, we analysed the induction of cell death over a range of HBX19818 concentrations using both isogenic CLL cell lines with and without ATM and/or p53, as well as 18 primary CLL tumours. We observed a significant cytotoxic effect of HBX19818 in isogenic CLL lines, at concentrations between 1–10μM, irrespective of their ATM and TP53 status. Strikingly, the majority of primary CLL tumours were sensitive to HBX19818 concentrations between 8μM and 16μM to which non-tumour PBMCs were resistant. Western blotting was used to monitor the induction of p53, apoptosis (associated with caspase 7 and PARP1 cleavage), and also whether DNA damage was induced (as measured by H2AX phosphorylation) in response to HBX19818. Our analysis revealed that pharmacological inhibition of USP7 led to p53 upregulation in the p53 proficient CLL cells associated with a robust induction of p21 indicating that the stabilised p53 was active. This response was absent in Mec-1 cell line with non-functional p53. Interestingly, caspase 7 and PARP1 were only cleaved in the ATM wild type CLL cell lines suggesting that activated p53 was capable of inducing cell death. In contrast, the ATM and p53 deficient CLL cell lines did not exhibit any markers suggesting an apoptotic mode of cell death. Rather, these cell lines displayed elevated levels of phospho-H2AX, suggesting the induction of DNA damage, possibly caused by an underlying DNA repair defect. Consistent with our hypothesis, both p53 proficient and p53 non-functional CLL cell lines failed to induce the recruitment of the HR protein Rad51 to sites of IR-induced DNA double strand breaks. Taken together, our data implies that in addition to p53 activation, pharmacological inhibition of USP7 can exert a cytotoxic effect by further mechanisms, possibly by modulating DNA double strand break repair. This is consistent with previous reports suggesting that USP7 regulates monoubiquitination of transcription factor FOXO4 and is involved in the regulation of DNA repair and mitotic progression via its interactions with Claspin and Chfr respectively. We suggest that pharmacological inhibition of USP7 represents a promising target for the treatment of tumours with defective ATM and p53 signalling. Disclosures: No relevant conflicts of interest to declare.

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.

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