Incorporation of Low Concentrations of Gold Nanoparticles: Complex Effects on Radiation Response and Fate of Cancer Cells.

: 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. : 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.

DeepFoci: Deep learning-based algorithm for fast automatic analysis of DNA double-strand break ionizing radiation-induced foci.

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis.

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.

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 repair in head and neck tumor cells and possibilities of its monitoring to estimate individual tumor radioresistance and selection of optimal primary treatment.

In order to maximize post-therapeutic quality of life, radio(chemo)therapy becomes preferred over surgery in head-and-neck tumor (HNT) treatment. However, the therapy selection is only based on the clinical experience and patient's preferences as the radiosensitivity markers remain unknown. New possibilities of deciding on the best primary therapy, moving us towards personalized medicine based on quantifiable biomarkers, have been opened by studies on DNA radiation damage and repair in individual patients tumors.

In Vitro Comparison of Passive and Active Clinical Proton Beams.

Nowadays, the irradiation methodology in proton therapy is switching from the use of passively scattered beams to active pencil beams due to the possibility of more conformal dose distributions. The dose rates of active pencil beams are much higher than those of passive beams. The purpose of this study was to investigate whether there is any difference in the biological effectiveness of these passive and active irradiation modes.

Challenges and Contradictions of Metal Nano-Particle Applications for Radio-Sensitivity Enhancement in Cancer Therapy.

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.

Recruitment of 53BP1 Proteins for DNA Repair and Persistence of Repair Clusters Differ for Cell Types as Detected by Single Molecule Localization Microscopy.

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.

Changes in Cryopreserved Cell Nuclei Serve as Indicators of Processes during Freezing and Thawing.

The mechanisms underlying cell protection from cryoinjury are not yet fully understood. Recent biological studies have addressed cryopreserved cell survival but have not correlated the cryoprotection effectiveness with the impact of cryoprotectants on the most important cell structure, the nucleus, and the freeze/thaw process. We identified changes of cell nuclei states caused by different types of cryoprotectants and associate them with alterations of the freeze/thaw process in cells.

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells.

Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci.

Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored.

Theoretical and experimental study of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability.

In this work the physico-chemical properties of selected cryoprotectants (antifreeze protein TrxA-AFP752, trehalose and dimethyl sulfoxide) were correlated with their impact on the constitution of ice and influence on frozen/thawed cell viability. The freezing processes and states of investigated materials solutions were described and explained from a fundamental point of view using ab-initio modelling (molecular dynamics, DFT), Raman spectroscopy, Differential Scanning Calorimetry and X-Ray Diffraction. For the first time, in this work we correlated the microscopic view (modelling) with the description of the frozen solution states and put these results in the context of human skin fibroblast viability after freezing and thawing.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells.

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts.

Determining Omics spatiotemporal dimensions using exciting new nanoscopy techniques to assess complex cell responses to DNA damage: part B--structuromics.

Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation.

Determining Omics spatiotemporal dimensions using exciting new nanoscopy techniques to assess complex cell responses to DNA damage: part A--radiomics.

Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure.

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient.

Function of chromatin structure and dynamics in DNA damage, repair and misrepair: γ-rays and protons in action.

According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure-the problematics addressed in this work.

Prognostic impact of p53 aberrations for R-CHOP-treated patients with diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma in adults. There are specific alterations that appear repeatedly in DLBCL cases and play a role in lymphomagenesis or progression of the disease. Some aberrations were used as prognostic markers in the pre-rituximab era.

Loss of the p53 tumor suppressor activity is associated with negative prognosis of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is typified by translocation t(11;14)(q13;q32) causing upregulation of cyclin D1 and deregulation of cell cycle. The cyclin D1 activation plays a critical role in MCL pathogenesis but additional oncogenic events, such as aberrations of the ARF/MDM2/p53 pathway are also necessary for progression of the disease. We analyzed the p53 tumor suppressor in tumor tissue of 33 patients with MCL.

An unusual p53 mutation detected in Burkitt's lymphoma: 30 bp duplication.

Burkitt's lymphomas (BL) are aggressive rapidly growing tumors typified by a high c-myc expression resulting from t(8;14)(q24;q32), t(2;8)(p12;q24) or t(8;22)(q24;q11) translocations. Alterations of the p53 tumor suppressor are also relatively frequent in BL. Several approaches have been adopted for detection of the p53 aberrations such as immunohistochemical analyses, immunoblotting, DNA sequencing, fluorescence in situ hybridization (FISH), and functional assays.