TY - JOUR A1 - Schäfer, Myriam A1 - Hildenbrand, Georg A1 - Hausmann, Michael T1 - Impact of Gold Nanoparticles and Ionizing Radiation on Whole Chromatin Organization as Detected by Single-Molecule Localization Microscopy JF - International Journal of Molecular Sciences N2 - In radiation tumor therapy, irradiation, on one hand, should cause cell death to the tumor. On the other hand, the surrounding non-tumor tissue should be maintained unaffected. Therefore, methods of local dose enhancements are highly interesting. Gold nanoparticles, which are preferentially uptaken by very-fast-proliferating tumor cells, may enhance damaging. However, the results in the literature obtained from cell culture and animal tissue experiments are very contradictory, i.e., only some experiments reveal increased cell killing but others do not. Thus, a better understanding of cellular mechanisms is required. Using the breast cancer cell model SkBr3, the effects of gold nanoparticles in combination with ionizing radiation on chromatin network organization were investigated by Single-Molecule Localization Microscopy (SMLM) and applications of mathematical topology calculations (e.g., Persistent Homology, Principal Component Analysis, etc.). The data reveal a dose and nanoparticle dependent re-organization of chromatin, although colony forming assays do not show a significant reduction of cell survival after the application of gold nanoparticles to the cells. In addition, the spatial organization of γH2AX clusters was elucidated, and characteristic changes were obtained depending on dose and gold nanoparticle application. The results indicate a complex response of ALU-related chromatin and heterochromatin organization correlating to ionizing radiation and gold nanoparticle incorporation. Such complex whole chromatin re-organization is usually associated with changes in genome function and supports the hypothesis that, with the application of gold nanoparticles, not only is DNA damage increasing but also the efficiency of DNA repair may be increased. The understanding of complex chromatin responses might help to improve the gold nanoparticle efficiency in radiation treatment. KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2024 U6 - https://doi.org/https://doi.org/10.3390/ijms252312843 SN - 1422-0067 VL - 25 IS - 23 PB - MDPI AG ER - TY - JOUR A1 - Ngwa, Wilfred A1 - Boateng, Francis A1 - Kumar, Rajiv A1 - Irvine, Darrell J. A1 - Formenti, Silvia A1 - Ngoma, Twalib A1 - Herskind, Carsten A1 - Veldwijk, Marlon R. A1 - Hildenbrand, Georg A1 - Hausmann, Michael A1 - Wenz, Frederik A1 - Hesser, Jürgen T1 - Smart Radiation Therapy Biomaterials JF - International Journal of Radiation Oncology*Biology*Physics KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2017 U6 - https://doi.org/https://doi.org/10.1016/j.ijrobp.2016.10.034 SN - 0360-3016 VL - 97 IS - 3 SP - 624 EP - 637 PB - Elsevier BV ER - TY - JOUR A1 - Moser, Felipe A1 - Hildenbrand, Georg A1 - Müller, Patrick A1 - Al Saroori, Alexander A1 - Biswas, Abin A1 - Bach, Margund A1 - Wenz, Frederik A1 - Cremer, Christoph A1 - Burger, Nina A1 - Veldwijk, Marlon R. A1 - Hausmann, Michael T1 - Cellular Uptake of Gold Nanoparticles and Their Behavior as Labels for Localization Microscopy JF - Biophysical Journal KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2016 U6 - https://doi.org/https://doi.org/10.1016/j.bpj.2016.01.004 SN - 0006-3495 VL - 110 IS - 4 SP - 947 EP - 953 PB - Elsevier BV ER - TY - JOUR A1 - Hausmann, Michael A1 - Ilić, Nataša A1 - Pilarczyk, Götz A1 - Lee, Jin-Ho A1 - Logeswaran, Abiramy A1 - Borroni, Aurora A1 - Krufczik, Matthias A1 - Theda, Franziska A1 - Waltrich, Nadine A1 - Bestvater, Felix A1 - Hildenbrand, Georg A1 - Cremer, Christoph A1 - Blank, Michael T1 - Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research JF - International Journal of Molecular Sciences N2 - Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2017 U6 - https://doi.org/https://doi.org/10.3390/ijms18102066 SN - 1422-0067 VL - 18 IS - 10 PB - MDPI AG ER - TY - JOUR A1 - Hildenbrand, Georg A1 - Metzler, Philipp A1 - Pilarczyk, Götz A1 - Bobu, Vladimir A1 - Kriz, Wilhelm A1 - Hosser, Hiltraud A1 - Fleckenstein, Jens A1 - Krufczik, Matthias A1 - Bestvater, Felix A1 - Wenz, Frederik A1 - Hausmann, Michael ED - Baptista, Pedro V. T1 - Dose enhancement effects of gold nanoparticles specifically targeting RNA in breast cancer cells JF - PLOS ONE KW - Krebs, Medizin KW - Brustkrebs KW - Strahlentherapie Y1 - 2018 U6 - https://doi.org/https://doi.org/10.1371/journal.pone.0190183 SN - 1932-6203 VL - 13 IS - 1 PB - Public Library of Science (PLoS) ER - TY - JOUR A1 - Eryilmaz, Marion A1 - Schmitt, Eberhard A1 - Krufczik, Matthias A1 - Theda, Franziska A1 - Lee, Jin-Ho A1 - Cremer, Christoph A1 - Bestvater, Felix A1 - Schaufler, Wladimir A1 - Hausmann, Michael A1 - Hildenbrand, Georg T1 - Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines JF - Cancers N2 - In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell’s decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair. KW - Krebs, Medizin KW - Strahlentherapie KW - Brustkrebs Y1 - 2018 U6 - https://doi.org/https://doi.org/10.3390/cancers10010025 SN - 2072-6694 VL - 10 IS - 1 PB - MDPI AG ER - TY - JOUR A1 - Bobkova, Elizaveta A1 - Depes, Daniel A1 - Lee, Jin-Ho A1 - Jezkova, Lucie A1 - Falkova, Iva A1 - Pagacova, Eva A1 - Kopecna, Olga A1 - Zadneprianetc, Mariia A1 - Bacikova, Alena A1 - Kulikova, Elena A1 - Smirnova, Elena A1 - Bulanova, Tatiana A1 - Boreyko, Alla A1 - Krasavin, Evgeny A1 - Wenz, Frederik A1 - Bestvater, Felix A1 - Hildenbrand, Georg A1 - Hausmann, Michael A1 - Falk, Martin T1 - Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy JF - International Journal of Molecular Sciences N2 - 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. KW - Genom KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2018 U6 - https://doi.org/https://doi.org/10.3390/ijms19123713 SN - 1422-0067 VL - 19 IS - 12 PB - MDPI AG ER - TY - JOUR A1 - Pagáčová, Eva A1 - Štefančíková, Lenka A1 - Schmidt-Kaler, Franz A1 - Hildenbrand, Georg A1 - Vičar, Tomáš A1 - Depeš, Daniel A1 - Lee, Jin-Ho A1 - Bestvater, Felix A1 - Lacombe, Sandrine A1 - Porcel, Erika A1 - Roux, Stéphane A1 - Wenz, Frederik A1 - Kopecna, Olga A1 - Falková, Iva A1 - Hausmann, Michael A1 - Falk, Martin T1 - Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy JF - International Journal of Molecular Sciences N2 - 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. KW - Krebs, Medizin KW - Strahlentherapie Y1 - 2019 U6 - https://doi.org/https://doi.org/10.3390/ijms20030588 SN - 1422-0067 VL - 20 IS - 3 PB - MDPI AG ER - TY - JOUR A1 - Hausmann, Michael A1 - Neitzel, Charlotte A1 - Bobkova, Elizaveta A1 - Nagel, David A1 - Hofmann, Andreas A1 - Chramko, Tatyana A1 - Smirnova, Elena A1 - Kopecna, Olga A1 - Pagáčová, Eva A1 - Boreyko, Alla A1 - Krasavin, Evgeny A1 - Falkova, Iva A1 - Heermann, Dieter W. A1 - Pilarczyk, Götz A1 - Hildenbrand, Georg A1 - Bestvater, Felix A1 - Falk, Martin T1 - Single Molecule Localization Microscopy Analyses of DNA-Repair Foci and Clusters Detected Along Particle Damage Tracks JF - Frontiers in Physics KW - Krebszelle KW - Strahlentherapie KW - Genom KW - Zellkern Y1 - 2020 U6 - https://doi.org/https://doi.org/10.3389/fphy.2020.578662 SN - 2296-424X VL - 8 PB - Frontiers Media SA ER - TY - JOUR A1 - Hausmann, Michael A1 - Falk, Martin A1 - Neitzel, Charlotte A1 - Hofmann, Andreas A1 - Biswas, Abin A1 - Gier, Theresa A1 - Falkova, Iva A1 - Heermann, Dieter W. A1 - Hildenbrand, Georg T1 - Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach JF - International Journal of Molecular Sciences N2 - 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. KW - Krebszelle KW - Strahlentherapie Y1 - 2021 U6 - https://doi.org/https://doi.org/10.3390/ijms22073636 SN - 1422-0067 VL - 22 IS - 7 PB - MDPI AG ER -