TY - JOUR A1 - Milić, Mirta A1 - Ceppi, Marcello A1 - Bruzzone, Marco A1 - Azqueta, Amaya A1 - Brunborg, Gunnar A1 - Godschalk, Roger A1 - Koppen, Gudrun A1 - Langie, Sabine A1 - Møller, Peter A1 - Teixeira, João Paulo A1 - Alija, Avdulla A1 - Anderson, Diana A1 - Andrade, Vanessa A1 - Andreoli, Cristina A1 - Asllani, Fisnik A1 - Bangkoglu, Ezgi Eyluel A1 - Barančoková, Magdalena A1 - Basaran, Nursen A1 - Boutet-Robinet, Elisa A1 - Buschini, Annamaria A1 - Cavallo, Delia A1 - Costa Pereira, Cristiana A1 - Costa, Carla A1 - Costa, Solange A1 - Da Silva, Juliana A1 - Del Boˊ, Cristian A1 - Dimitrijević Srećković, Vesna A1 - Djelić, Ninoslav A1 - Dobrzyńska, Malgorzata A1 - Duračková, Zdenka A1 - Dvořáková, Monika A1 - Gajski, Goran A1 - Galati, Serena A1 - García Lima, Omar A1 - Giovannelli, Lisa A1 - Goroshinskaya, Irina A. A1 - Grindel, Annemarie A1 - Gutzkow, Kristine B. A1 - Hernández, Alba A1 - Hernández, Carlos A1 - Holven, Kirsten B. A1 - Ibero-Baraibar, Idoia A1 - Ottestad, Inger A1 - Kadioglu, Ela A1 - Kažimirová, Alena A1 - Kuznetsova, Elena A1 - Ladeira, Carina A1 - Laffon, Blanca A1 - Lamonaca, Palma A1 - Lebailly, Pierre A1 - Louro, Henriqueta A1 - Mandina Cardoso, Tania A1 - Marcon, Francesca A1 - Marcos, Ricard A1 - Moretti, Massimo A1 - Moretti, Silvia A1 - Najafzadeh, Mojgan A1 - Nemeth, Zsuzsanna A1 - Neri, Monica A1 - Novotna, Bozena A1 - Orlow, Irene A1 - Paduchova, Zuzana A1 - Pastor, Susana A1 - Perdry, Hervé A1 - Spremo-Potparević, Biljana A1 - Ramadhani, Dwi A1 - Riso, Patrizia A1 - Rohr, Paula A1 - Rojas, Emilio A1 - Rossner, Pavel A1 - Safar, Anna A1 - Sardas, Semra A1 - Silva, Maria João A1 - Sirota, Nikolay A1 - Smolkova, Bozena A1 - Staruchova, Marta A1 - Stetina, Rudolf A1 - Stopper, Helga A1 - Surikova, Ekaterina I. A1 - Ulven, Stine M. A1 - Ursini, Cinzia Lucia A1 - Valdiglesias, Vanessa A1 - Valverde, Mahara A1 - Vodicka, Pavel A1 - Volkovova, Katarina A1 - Wagner, Karl-Heinz A1 - Živković, Lada A1 - Dušinská, Maria A1 - Collins, Andrew R. A1 - Bonassi, Stefano T1 - The hCOMET project: International database comparison of results with the comet assay in human biomonitoring. Baseline frequency of DNA damage and effect of main confounders JF - Mutation Research/Reviews in Mutation Research N2 - The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations. KW - comet assay KW - DNA damage KW - pooled analysis KW - human biomonitoring KW - biomarkers Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-371614 VL - 787 ER - TY - JOUR A1 - Henriksson, Sofia A1 - Calderón-Montaño, José Manuel A1 - Solvie, Daniel A1 - Warpman Berglund, Ulrika A1 - Helleday, Thomas T1 - Overexpressed c-Myc sensitizes cells to TH1579, a mitotic arrest and oxidative DNA damage inducer JF - Biomolecules N2 - Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation. KW - MTH1 KW - TH588 KW - TH1579 KW - c-Myc KW - replication stress KW - DNA damage KW - cell death KW - cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297547 SN - 2218-273X VL - 12 IS - 12 ER - TY - JOUR A1 - Djelić, Ninoslav A1 - Borozan, Sunčica A1 - Dimitrijević-Srećković, Vesna A1 - Pajović, Nevena A1 - Mirilović, Milorad A1 - Stopper, Helga A1 - Stanimirović, Zoran T1 - Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro JF - International Journal of Molecular Sciences N2 - Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM. KW - diabetes KW - oxidative stress KW - DNA damage KW - lymphocytes KW - thyroid hormone Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285988 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Ben Khaled, Najib A1 - Hammer, Katharina A1 - Ye, Liangtao A1 - Alnatsha, Ahmed A1 - Widholz, Sebastian A. A1 - Piseddu, Ignazio A1 - Sirtl, Simon A1 - Schneider, Julia A1 - Munker, Stefan A1 - Mahajan, Ujjwal Mukund A1 - Montero, Juan José A1 - Griger, Joscha A1 - Mayerle, Julia A1 - Reiter, Florian P. A1 - De Toni, Enrico N. T1 - TRAIL receptor targeting agents potentiate PARP inhibitor efficacy in pancreatic cancer independently of BRCA2 mutation status JF - Cancers N2 - Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists. KW - apoptosis KW - DNA damage KW - pancreatic neoplasms KW - poly(ADP-ribose) polymerase inhibitors KW - TNF-related apoptosis-inducing ligand Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290884 SN - 2072-6694 VL - 14 IS - 21 ER - TY - JOUR A1 - Winkelbeiner, Nicola A1 - Wandt, Viktoria K. A1 - Ebert, Franziska A1 - Lossow, Kristina A1 - Bankoglu, Ezgi E. A1 - Martin, Maximilian A1 - Mangerich, Aswin A1 - Stopper, Helga A1 - Bornhorst, Julia A1 - Kipp, Anna P. A1 - Schwerdtle, Tanja T1 - A multi-endpoint approach to base excision repair incision activity augmented by PARylation and DNA damage levels in mice: impact of sex and age JF - International Journal of Molecular Sciences N2 - Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2'-deoxyguanosine (8-oxodG), 5-hydroxy-2'-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery. KW - maintenance of genomic integrity KW - ageing KW - sex KW - DNA damage KW - base excision repair (incision activity) KW - DNA damage response KW - poly(ADP-ribosyl)ation KW - liver Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285706 SN - 1422-0067 VL - 21 IS - 18 ER - TY - JOUR A1 - Trifault, Barbara A1 - Mamontova, Victoria A1 - Burger, Kaspar T1 - In vivo proximity labeling of nuclear and nucleolar proteins by a stably expressed, DNA damage-responsive NONO-APEX2 fusion protein JF - Frontiers in Molecular Biosciences N2 - Cellular stress can induce DNA lesions that threaten the stability of genes. The DNA damage response (DDR) recognises and repairs broken DNA to maintain genome stability. Intriguingly, components of nuclear paraspeckles like the non-POU domain containing octamer-binding protein (NONO) participate in the repair of DNA double-strand breaks (DSBs). NONO is a multifunctional RNA-binding protein (RBP) that facilitates the retention and editing of messenger (m)RNA as well as pre-mRNA processing. However, the role of NONO in the DDR is poorly understood. Here, we establish a novel human U2OS cell line that expresses NONO fused to the engineered ascorbate peroxidase 2 (U2OS:NONO-APEX2-HA). We show that NONO-APEX2-HA accumulates in the nucleolus in response to DNA damage. Combining viability assays, subcellular localisation studies, coimmunoprecipitation experiments and in vivo proximity labeling, we demonstrate that NONO-APEX2-HA is a stably expressed fusion protein that mimics endogenous NONO in terms of expression, localisation and bona fide interactors. We propose that in vivo proximity labeling in U2OS:NONO-APEX2-HA cells is capable for the assessment of NONO interactomes by downstream assays. U2OS:NONO-APEX2-HA cells will likely be a valuable resource for the investigation of NONO interactome dynamics in response to DNA damage and other stimuli. KW - APEX2 KW - proximity labeling KW - NONO KW - paraspeckles KW - nucleolus KW - DNA damage Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276707 SN - 2296-889X VL - 9 ER - TY - JOUR A1 - Schumann, S. A1 - Eberlein, U. A1 - Lapa, C. A1 - Müller, J. A1 - Serfling, S. A1 - Lassmann, M. A1 - Scherthan, H. T1 - α-Particle-induced DNA damage tracks in peripheral blood mononuclear cells of [\(^{223}\)Ra]RaCl\(_{2}\)-treated prostate cancer patients JF - European Journal of Nuclear Medicine and Molecular Imaging N2 - Purpose One therapy option for prostate cancer patients with bone metastases is the use of [\(^{223}\)Ra]RaCl\(_{2}\). The α-emitter \(^{223}\)Ra creates DNA damage tracks along α-particle trajectories (α-tracks) in exposed cells that can be revealed by immunofluorescent staining of γ-H2AX+53BP1 DNA double-strand break markers. We investigated the time- and absorbed dose-dependency of the number of α-tracks in peripheral blood mononuclear cells (PBMCs) of patients undergoing their first therapy with [\(^{223}\)Ra]RaCl\(_{2}\). Methods Multiple blood samples from nine prostate cancer patients were collected before and after administration of [\(^{223}\)Ra]RaCl\(_{2}\), up to 4 weeks after treatment. γ-H2AX- and 53BP1-positive α-tracks were microscopically quantified in isolated and immuno-stained PBMCs. Results The absorbed doses to the blood were less than 6 mGy up to 4 h after administration and maximally 16 mGy in total. Up to 4 h after administration, the α-track frequency was significantly increased relative to baseline and correlated with the absorbed dose to the blood in the dose range < 3 mGy. In most of the late samples (24 h - 4 weeks after administration), the α-track frequency remained elevated. Conclusion The γ-H2AX+53BP1 assay is a potent method for detection of α-particle-induced DNA damages during treatment with or after accidental incorporation of radionuclides even at low absorbed doses. It may serve as a biomarker discriminating α- from β-emitters based on damage geometry. KW - γ-H2AX KW - DNA damage KW - nuclear medicine KW - dosimetry KW - α-Emitter KW - biokinetics KW - prostate cancer KW - [\(^{223}\)Ra]RaCl\(_{2}\) KW - 53BP1 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265462 SN - 1619-7089 VL - 48 IS - 9 ER - TY - JOUR A1 - Djuzenova, Cholpon S. A1 - Fischer, Thomas A1 - Katzer, Astrid A1 - Sisario, Dmitri A1 - Korsa, Tessa A1 - Streussloff, Gudrun A1 - Sukhorukov, Vladimir L. A1 - Flentje, Michael T1 - Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs JF - BMC Cancer N2 - Background: Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. Methods: Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by gamma H2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. Results: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (similar to 80% vs. similar to 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. Conclusions: The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism. KW - DNA damage KW - DNA-PK KW - Histone gamma H2AX KW - p53 KW - Radiation sensitivity Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265826 VL - 21 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Schuele, Carolin A1 - Stopper, Helga T1 - Cell survival after DNA damage in the comet assay JF - Archives of Toxicology N2 - The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided. KW - Cell death and comet assay KW - DNA damage KW - DNA repair Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265339 VL - 95 IS - 12 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Stipp, Franzisca A1 - Gerber, Johanna A1 - Seyfried, Florian A1 - Heidland, August A1 - Bahner, Udo A1 - Stopper, Helga T1 - Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay JF - Archives of Toxicology N2 - The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction. KW - human biomonitoring KW - DNA damage KW - DNA repair KW - comet assay KW - blood samples Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265326 VL - 95 IS - 5 ER -