@inproceedings{HausmannNeitzelHahnetal.2021, author = {Hausmann, Michael and Neitzel, Charlotte and Hahn, Hannes and Winter, Ruth and Falkova, Iva and Heermann, Dieter W. and Pilarczyk, G{\"o}tz and Hildenbrand, Georg and Scherthan, Harry and Falk, Martin}, title = {Space and Time in the Universe of the Cell Nucleus after Ionizing Radiation Attacks: A Comparison of Cancer and Non-Cancer Cell Response}, series = {The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response}, booktitle = {The 1st International Electronic Conference on Cancers: Exploiting Cancer Vulnerability by Targeting the DNA Damage Response}, publisher = {MDPI}, address = {Basel Switzerland}, doi = {https://doi.org/10.3390/IECC2021-09219}, year = {2021}, subject = {Krebszelle}, language = {en} } @article{PagačovaŠtefančikovaSchmidtKaleretal.2019, author = {Pag{\´a}čov{\´a}, Eva and Štefanč{\´i}kov{\´a}, Lenka and Schmidt-Kaler, Franz and Hildenbrand, Georg and Vičar, Tom{\´a}š and Depeš, Daniel and Lee, Jin-Ho and Bestvater, Felix and Lacombe, Sandrine and Porcel, Erika and Roux, St{\´e}phane and Wenz, Frederik and Kopecna, Olga and Falkov{\´a}, Iva and Hausmann, Michael and Falk, Martin}, title = {Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {3}, publisher = {MDPI AG}, issn = {1422-0067}, doi = {https://doi.org/10.3390/ijms20030588}, year = {2019}, abstract = {From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2-3 nm). Next, we introduced a novel super-resolution approach—single molecule localization microscopy (SMLM)—to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.}, subject = {Krebs, Medizin}, language = {en} } @article{FalkHausmannLukasovaetal.2014, author = {Falk, Martin and Hausmann, Michael and Lukasova, Emilie and Biswas, Abin and Hildenbrand, Georg and Davidkova, Marie and Krasavin, Evgeny and Kleibl, Zdenek and Falkova, Iva and Jezkova, Lucie and Stefancikova, Lenka and Sevcik, Jan and Hofer, Michal and Bacikova, Alena and Matula, Pavel and Boreyko, Alla and Vachelova, Jana and Michaelidesova, Anna and Kozubek, Stanislav}, title = {Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part - Structuromics}, series = {Critical Reviews in Eukaryotic Gene Expression}, volume = {24}, journal = {Critical Reviews in Eukaryotic Gene Expression}, number = {3}, publisher = {Begell House}, issn = {1045-4403}, doi = {https://doi.org/10.1615/CritRevEukaryotGeneExpr.v24.i3.40}, pages = {225 -- 247}, year = {2014}, subject = {Radiologie}, language = {en} } @article{HausmannNeitzelBobkovaetal.2020, author = {Hausmann, Michael and Neitzel, Charlotte and Bobkova, Elizaveta and Nagel, David and Hofmann, Andreas and Chramko, Tatyana and Smirnova, Elena and Kopecna, Olga and Pag{\´a}čov{\´a}, Eva and Boreyko, Alla and Krasavin, Evgeny and Falkova, Iva and Heermann, Dieter W. and Pilarczyk, G{\"o}tz and Hildenbrand, Georg and Bestvater, Felix and Falk, Martin}, title = {Single Molecule Localization Microscopy Analyses of DNA-Repair Foci and Clusters Detected Along Particle Damage Tracks}, series = {Frontiers in Physics}, volume = {8}, journal = {Frontiers in Physics}, publisher = {Frontiers Media SA}, issn = {2296-424X}, doi = {https://doi.org/10.3389/fphy.2020.578662}, year = {2020}, subject = {Krebszelle}, language = {en} } @article{DobešovaGierKopecnaetal.2022, author = {Dobešov{\´a}, Lucie and Gier, Theresa and Kopecna, Olga and Pag{\´a}čov{\´a}, Eva and Vičar, Tom{\´a}š and Bestvater, Felix and Toufar, Jiř{\´i} and Bač{\´i}kov{\´a}, Alena and Kopel, Pavel and Fedr, Radek and Hildenbrand, Georg and Falkov{\´a}, Iva and Falk, Martin and Hausmann, Michael}, title = {Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells}, series = {Pharmaceutics}, volume = {14}, journal = {Pharmaceutics}, number = {1}, publisher = {MDPI AG}, issn = {1999-4923}, doi = {https://doi.org/10.3390/pharmaceutics14010166}, year = {2022}, abstract = {(1) Background: In oncology research, a long-standing discussion exists about pros and cons of metal nanoparticle-enhanced radiotherapy and real mechanisms behind the tumor cell response to irradiation (IR) in presence of gold nanoparticles (GNPs). A better understanding of this response is, however, necessary to develop more efficient and safety nanoparticle (NP) types designed to disturb specific processes in tumor cells. (2) Aims and Methods: We combined 3D confocal microscopy and super-resolution single molecule localization microscopy (SMLM) to analyze, at the multiscale, the early and late effects of 10 nm-GNPs on DNA double strand break (DSB) induction and repair in tumor cells exposed to different doses of photonic low-LET (linear energy transfer) radiation. The results were correlated to different aspects of short and long-term cell viability. SkBr3 breast cancer cells (selected for the highest incidence of this cancer type among all cancers in women, and because most breast tumors are treated with IR) were incubated with low concentrations of GNPs and irradiated with 60Co γ-rays or 6 MV X-rays. In numerous post-irradiation (PI) times, ranging from 0.5 to 24 h PI, the cells were spatially (3D) fixed and labeled with specific antibodies against γH2AX, 53BP1 and H3K9me3. The extent of DSB induction, multi-parametric micro- and nano-morphology of γH2AX and 53BP1 repair foci, DSB repair kinetics, persistence of unrepaired DSBs, nanoscale clustering of γH2AX and nanoscale (hetero)chromatin re-organization were measured by means of the mentioned microscopy techniques in dependence of radiation dose and GNP concentration. (3) Results: The number of γH2AX/53BP1 signals increased after IR and an additional increase was observed in GNP-treated (GNP(+)) cells compared to untreated controls. However, this phenomenon reflected slight expansion of the G2-phase cell subpopulation in irradiated GNP(+) specimens instead of enhanced DNA damage induction by GNPs. This statement is further supported by some micro- and nano-morphological parameters of γH2AX/53BP1 foci, which slightly differed for cells irradiated in absence or presence of GNPs. At the nanoscale, Ripley's distance frequency analysis of SMLM signal coordinate matrices also revealed relaxation of heterochromatin (H3K9me3) clusters upon IR. These changes were more prominent in presence of GNPs. The slight expansion of radiosensitive G2 cells correlated with mostly insignificant but systematic decrease in post-irradiation survival of GNP(+) cells. Interestingly, low GNP concentrations accelerated DSB repair kinetics; however, the numbers of persistent γH2AX/53BP1 repair foci were slightly increased in GNP(+) cells. (4) Conclusions: Low concentrations of 10-nm GNPs enhanced the G2/M cell cycle arrest and the proportion of radiosensitive G2 cells, but not the extent of DNA damage induction. GNPs also accelerated DSB repair kinetics and slightly increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. GNP-mediated cell effects correlated with slight radiosensitization of GNP(+) specimens, significant only for the highest radiation dose tested (4 Gy).}, subject = {Strahlentherapie}, language = {en} } @article{HausmannFalkNeitzeletal.2021, author = {Hausmann, Michael and Falk, Martin and Neitzel, Charlotte and Hofmann, Andreas and Biswas, Abin and Gier, Theresa and Falkova, Iva and Heermann, Dieter W. and Hildenbrand, Georg}, title = {Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {7}, publisher = {MDPI AG}, issn = {1422-0067}, doi = {https://doi.org/10.3390/ijms22073636}, year = {2021}, abstract = {In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a "double-edged sword"—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.}, subject = {Krebszelle}, language = {en} } @article{BobkovaDepesLeeetal.2018, author = {Bobkova, Elizaveta and Depes, Daniel and Lee, Jin-Ho and Jezkova, Lucie and Falkova, Iva and Pagacova, Eva and Kopecna, Olga and Zadneprianetc, Mariia and Bacikova, Alena and Kulikova, Elena and Smirnova, Elena and Bulanova, Tatiana and Boreyko, Alla and Krasavin, Evgeny and Wenz, Frederik and Bestvater, Felix and Hildenbrand, Georg and Hausmann, Michael and Falk, Martin}, title = {Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy}, series = {International Journal of Molecular Sciences}, volume = {19}, journal = {International Journal of Molecular Sciences}, number = {12}, publisher = {MDPI AG}, issn = {1422-0067}, doi = {https://doi.org/10.3390/ijms19123713}, year = {2018}, abstract = {DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10-20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.}, subject = {Genom}, language = {en} } @misc{LeeBobkovaGieretal.2019, author = {Lee, Jin-Ho and Bobkova, Elizaveta and Gier, Theresa and Gote, Martin and Schmidt-kaler, Fanz and Brieger, Emily and Maus, Emanuel and Krufczik, Matthias and Chojowski, Robert and Korn, Friederike and Sarah, Schumann and Scherthan, Harry and Falkova, Iva and Falk, Martin and Hausmann, Michael and Hildenbrand, Georg}, title = {Mechanisms and Challenges for Understanding Radiation Induced Changes in Chromatin Nanoarchitecture}, series = {DRO 2018}, journal = {DRO 2018}, doi = {https://doi.org/10.13140/RG.2.2.31391.20647}, year = {2019}, abstract = {The three-dimensional architecture of genomes acts as an additional level of mode for fundamental biological processes such as DNA damage response. In this context, nanoprobing and super-resolution microscopy are powerful methods for structural analyses of genomic targets in native chromatin of single cells at resolutions of single antibodies, proteins, histones, short DNA stretches, etc. We used multi-color chromatin nanoprobing and single molecule localization microscopy of established DNA damage and chromatin markers in 3D-conserved nuclei of different cell types exposed to various types and doses of ionizing radiation. Similarly, effect of gold nanoparticles on extent and direction of cellular radiation response was assessed. Our studies revealed the nanoarchitecture of damage foci with respect to γH2AX, Mre11 or 53BP1 and their molecular rearrangements during repair processes. Nanoscopy of genomic Alu resulted in linear-quadratic dose-effects for low to higher dose ranges and in changes of H3K9me3 distribution around Alu clusters upon radiation exposure. Preliminary results show post-irradiation time dependent changes in Alu chromatin. Moreover, gold nanoparticles incorporated into cells seem to act by an interplay of radiation enhancement and chromatin remodeling leading to altered radiosensitivity. Our results contribute to the understanding of cellular radiation responses, thereby laying the basis for improved biological dosimetry and radiotherapies in future.}, subject = {Genom}, language = {en} } @article{FalkHausmannLukasovaetal.2014, author = {Falk, Martin and Hausmann, Michael and Lukasova, Emilie and Biswas, Abin and Hildenbrand, Georg and Davidkova, Marie and Krasavin, Evgeny and Kleibl, Zdenek and Falkova, Iva and Jezkova, Lucie and Stefancikova, Lenka and Sevcik, Jan and Hofer, Michal and Bacikova, Alena and Matula, Pavel and Boreyko, Alla and Vachelova, Jana and Michaelidesova, Anna and Kozubek, Stanislav}, title = {Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: PART A-Radiomics}, series = {Critical Reviews in Eukaryotic Gene Expression}, volume = {24}, journal = {Critical Reviews in Eukaryotic Gene Expression}, number = {3}, publisher = {Begell House}, issn = {1045-4403}, doi = {https://doi.org/10.1615/CritRevEukaryotGeneExpr.2014010313}, pages = {205 -- 223}, year = {2014}, subject = {Radiologie}, language = {en} }