A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

An Assessment of Radiation Doses From Radon Exposures Using a Mouse Model System.

BACKGROUND: Radon and its progenies contribute significantly to the natural background radiation and cause several thousands of lung cancer cases per year worldwide. Moreover, patients with chronic inflammatory joint diseases are treated in radon galleries. Due to the complex nature of radon exposure, the doses associated with radon exposures are difficult to assess. Hence, there is a clear need to directly measure dose depositions from radon exposures to provide reliable risk estimates for radiation protection guidelines. OBJECTIVES: We aimed to assess tissue-specific radiation doses associated with radon activity concentrations, that deposit similar dose levels as the annual natural radon exposure or radon gallery visits. METHODS: We exposed mice to defined radon concentrations, quantified the number of 53BP1 foci as a measure of induced DNA damage, and compared it with the number of foci induced by known doses of reference-type radiations. An image-based analysis of the 3-dimensional foci pattern provided information about the radiation type inflicting the DNA damage. RESULTS: A 1-hour exposure to 440 kBq/m3 radon-induced DNA damage corresponding to a dose of ∼10 mGy in the lung and ∼3.3 mGy in the kidney, heart, and liver. A 1-hour exposure to 44 kBq/m3 provided values consistent with a linear relationship between dose and radon concentration. Two-thirds of the dose in the lung was caused by α-particles. The dose in the kidney, heart, and liver and one-third of the dose in the lung likely resulted from ß- and γ-rays. DISCUSSION: We found that radon exposures mainly lead to α-particle-induced DNA damage in the lung, consistent with the lung cancer risk obtained in epidemiologic studies. Our presented biodosimetric approach can be used to benchmark risk model calculations for radiation protection guidelines and can help to understand the therapeutic success of radon gallery treatments.

Assessment of DNA damage by 53PB1 and pKu70 detection in peripheral blood lymphocytes by immunofluorescence and high-resolution transmission electron microscopy.

PURPOSE: 53BP1 foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA double-strand break (DSB) marker. In addition, high-resolution transmission electron microscopy (TEM) with immunogold labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA repair process. To establish this TEM method in the PBLs of patients with cancer, we analyzed and characterized whether different modes of irradiation influence the formation of DSBs, and whether accompanying chemotherapy influences DSB formation. METHODS: We obtained 86 blood samples before and 0.1, 0.5, and 24 h after irradiation from patients (n = 9) with head and neck or rectal cancers receiving radiotherapy (RT; n = 4) or radiochemotherapy (RCT; n = 5). 53BP1 foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70 dimers and 53BP1 clusters within euchromatin and heterochromatin of PBLs. RESULTS: IFM analyses showed that during radiation therapy, persistent 53BP1 foci in PBLs accumulated with increasing numbers of administered RT fractions. This 53BP1 foci accumulation was not influenced by the irradiation technique applied (3D conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1 foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT patients than in PBLs of RT only patients. Moreover, not every residual 53BP1 focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1 clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion or, possibly, defective repair. CONCLUSION: IFM 53BP1 foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1 focus but not every 53BP1 focus represents a DSB.

53BP1 expression and immunoscore are associated with the efficacy of neoadjuvant chemoradiotherapy for rectal cancer.

PURPOSE: Considering the effects of P53 binding protein 1 (53BP1) expression and T lymphocyte infiltration density on tumor radiosensitivity, we investigated the relation of 53BP1 expression and immunoscore based on T lymphocyte infiltration density with the efficacy of neoadjuvant chemoradiotherapy (CRT) for rectal cancer. METHODS: Fifty-five patients with rectal cancer receiving neoadjuvant CRT followed by surgery were enrolled. The 53BP1 expression level and the density of CD3+, CD8+, and CD45RO+ T lymphocytes in the tumor tissues were examined by immunohistochemistry, and the relation of these findings to the rates of tumor regression, disease-free survival (DFS), and overall survival (OS) was analyzed. RESULTS: The levels of 53BP1 and the CD3/CD8 immunoscore were closely correlated with the response to CRT (p < 0.05), with an area under the receiver operating characteristic curve for CRT efficacy prediction of 0.626 and 0.717, respectively. Further survival analysis revealed that high 53BP1 expression effectively prolonged 2­year DFS compared with low 53BP1 expression (87.5% [95%CI 77.3-97.7] vs. 53.3% [95%CI 28.1-78.6]; p < 0.05), while the effect of immunoscore on survival was restricted by the expression status of 53BP1. Cox multivariate analysis confirmed 53BP1 as an independent prognostic factor in DFS. CONCLUSION: The pretreatment levels of 53BP1 and the immunoscore based on CD3+/CD8+ T cell infiltration density in tumor tissues are effective predictors for the CRT response, and 53BP1 has a more pronounced impact on prognosis.

Immunofluorescence analysis of DNA damage response protein p53-binding protein 1 in a case of uterine dedifferentiated leiomyosarcoma arising from leiomyoma.

AIMS: Genomic instability has been indicated during the dedifferentiation process from leiomyoma (LM) to leiomyosarcoma (LMS). Previously, we have described that nuclear expression pattern of DNA damage response protein p53-binding protein 1 (53BP1), detected by immunofluorescence, reflects the magnitude of genomic instability during malignancy. Here, we present a case of LMS arising from LM with molecular analysis of 53BP1, which showed transitional magnitude of DNA damage response within a tumor. METHODS AND RESULTS: A fifty-year-old female with abdominal mass underwent hysterectomy. Histologically, the tumor consisted of LMS with highly atypical multinucleated giant cells as well as an LM component with transitional atypical spindle cells in the border area. LMS showed diffuse nuclear staining of 53BP1 expression, which has been previously described as high DNA damage response pattern. In contrast, the LM component lacked 53BP1 immunoreactivity and focal expression was observed in transitional lesion. Furthermore, double-labelled immunofluorescence revealed co-localization of 53BP1 with p53 and Ki-67 in the LMS component, which indicated abnormal DNA damage response in proliferative state. CONCLUSIONS: This study revealed that diffuse-type 53BP1 expression may be beneficial to estimate genomic instability during dedifferentiation from LM to DLMS.

DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells.

PURPOSE: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). METHODS AND MATERIALS: Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. RESULTS: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs. CONCLUSIONS: We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.

DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis.

BACKGROUND AND PURPOSE: High-precision radiotherapy is an effective treatment modality for tumors. Intensity-modulated radiotherapy techniques permit close shaping of high doses to tumors, however healthy organs outside the target volume are repeatedly exposed to low-dose radiation (LDR). The inherent vulnerability of hippocampal neurogenesis is likely the determining factor in radiation-induced neurocognitive dysfunctions. Using preclinical in-vivo models with daily LDR we attempted to precisely define the pathophysiology of radiation-induced neurotoxicity. MATERIAL AND METHODS: Genetically defined mouse strains with varying DNA repair capacities were exposed to fractionated LDR (5×/10×/15×/20×0.1 Gy) and dentate gyri from juvenile and adult mice were analyzed 72 h after last exposure and 1, 3, 6 months after 20 × 0.1 Gy. To examine the impact of LDR on neurogenesis, persistent DNA damage was assessed by quantifying 53BP1-foci within hippocampal neurons. Moreover, subpopulations of neuronal stem/progenitor cells were quantified and dendritic arborization of developing neurons were assessed. To unravel molecular mechanisms involved in radiation-induced neurotoxicity, hippocampi were analyzed using mass spectrometry-based proteomics and affected signaling networks were validated by immunoblotting. RESULTS: Radiation-induced DNA damage accumulation leads to progressive decline of hippocampal neurogenesis with decreased numbers of stem/progenitor cells and reduced complexities of dendritic architectures, clearly more pronounced in repair-deficient mice. Proteome analysis revealed substantial changes in neurotrophic signaling, with strong suppression directly after LDR and compensatory upregulation later on to promote functional recovery. CONCLUSION: Hippocampal neurogenesis is highly sensitive to repetitive LDR. Even low doses affect signaling networks within the neurogenic niche and interrupt the dynamic process of generation and maturation of neuronal stem/progenitor cells.

A Novel Diagnostic Method for Thyroid Follicular Tumors Based on Immunofluorescence Analysis of p53-Binding Protein 1 Expression: Detection of Genomic Instability.

Background: The preoperative diagnosis of thyroid follicular carcinomas (FCs) by fine-needle aspiration cytology is almost impossible. It was previously demonstrated that p53-binding protein 1 (53BP1) expression, based on immunofluorescence (IF), can serve as a valuable biomarker to estimate the malignant potential of various cancers. 53BP1 belongs to a class of DNA damage response molecules that rapidly localize to the site of DNA double-strand breaks, forming nuclear foci (NF). This study aimed to elucidate the utility of 53BP1 NF expression as a biomarker to differentiate follicular tumors (FTs). Methods: Associations between 53BP1 expression based on IF and histological types of FTs were analyzed using 27 follicular adenomas (FAs), 28 minimally invasive FCs, and 14 widely invasive FCs. Furthermore, the study clarified the relationship between 53BP1 NF and copy number aberrations (CNAs) based on array comparative genomic hybridization, a hallmark of genomic instability (GIN). Results: This study demonstrates differences in 53BP1 NF expression between FA and FC. The incidence of 53BP1 at NF significantly increased with FT progression in the following order: normal follicle < FA < minimally invasive FCs < widely invasive FCs. In contrast, no significant differences were observed in CNAs among the FT samples. Furthermore, there was no significant correlation between CNAs and 53BP1 at NF in FTs. Thus, based on a comparison of these two indicators of GIN, 53BP1 NF (by IF) was better able to estimate the malignancy of FTs compared to CNA (by array comparative genomic hybridization). Interestingly, IF revealed a heterogenous distribution of 53BP1 NF, which occurred more frequently in the invasive or subcapsular area than in the center of the tumor, suggesting intratumoral heterogeneity of GIN in FTs. Conclusions: It is proposed that IF analysis of 53BP1 expression could be a novel diagnostic method to estimate the malignant potential of FTs. Because 53BP1 NF reflect DNA double-strand breaks, it is hypothesized that the incidence of 53BP1 at NF can represent the level of GIN in tumor cells. IF analysis of 53BP1 expression will not only be an auxiliary histologic technique to diagnose FTs accurately, but also a novel technique for preoperative diagnosis using fine-needle aspiration cytology.

Clustered DNA damage concentrated in particle trajectories causes persistent large-scale rearrangements in chromatin architecture.

BACKGROUND AND PURPOSE: High linear-energy-transfer (LET) irradiation (IR) is characterized by unique depth-dose distribution and advantageous biologic effectiveness compared to low-LET-IR, offering promising alternatives for radio-resistant tumors in clinical oncology. While low-LET-IR induces single DNA lesions such as double-strand breaks (DSBs), localized energy deposition along high-LET particle trajectories induces clustered DNA lesions that are more challenging to repair. During DNA damage response (DDR) 53BP1 and ATM are required for Kap1-dependent chromatin relaxation, thereby sustaining heterochromatic DSB repair. Here, spatiotemporal dynamics of chromatin restructuring were visualized during DDR after high-LET and low-LET-IR. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon/calcium ions or low-LET photons. At 0.1 h, 0.5 h, 5 h and 24 h post-IR fluorophore- and gold-labeled repair factors (53BP1, pATM, pKAP-1, pKu70) were visualized by immunofluorescence and transmission electron microscopy, to monitor formation and repair of DNA damage in chromatin ultrastructure. To track chromatin remodeling at damage sites, decondensed regions (DCR) were delineated based on local chromatin concentration densities. RESULTS: Low-LET-IR induced single DNA lesions throughout the nucleus, but nearly all DSBs were efficiently rejoined without visible chromatin decompaction. High-LET-IR induced clustered DNA damage and triggered profound changes in chromatin structure along particle trajectories. In DCR multiple heterochromatic DSBs exhibited delayed repair despite cooperative activity of 53BP1, pATM, pKap-1. These closely localized DSBs may disturb efficient repair and subsequent chromatin restoration, thereby affecting large-scale genome organization. CONCLUSION: Clustered damage concentrated in particle trajectories causes persistent rearrangements in chromatin architecture, which may affect structural and functional organization of cell nuclei.