Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

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.

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.

Increasing genomic instability during cancer therapy in a patient with Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome (LFS) is a cancer predisposition disorder characterized by germline mutations of the p53 tumor-suppressor gene. In response to DNA damage, p53 stimulates protective cellular processes including cell-cycle arrest and apoptosis to prevent aberrant cell proliferation. Current cancer therapies involve agents that damage DNA, which also affect non-cancerous hematopoietic stem/progenitor cells. Here, we report on a child with LFS who developed genomic instability during craniospinal irradiation for metastatic choroid plexus carcinoma (CPC). CASE PRESENTATION: This previously healthy 4-year-old boy presented with parieto-temporal brain tumor, diagnosed as CPC grade-3. Screening for cancer-predisposing syndrome revealed heterozygous p53 germline mutation, leading to LFS diagnosis. After tumour resection and systemic chemotherapy, entire craniospinal axis was irradiated due to leptomeningeal seeding, resulting in disease stabilization for nearly 12 months. Blood lymphocytes of LFS patient (p53-deficient) and age-matched tumor-children (p53-proficient) were collected before, during and after craniospinal irradiation and compared with asymptomatic carriers for identical p53 mutation, not exposed to DNA-damaging treatment. In p53-deficient lymphocytes of LFS patient radiation-induced DNA damage failed to induce cell-cycle arrest or apoptosis. Although DNA repair capacity was not impaired, p53-deficient blood lymphocytes of LFS patient showed significant accumulation of 53BP1-foci during and even several months after irradiation, reflecting persistent DNA damage. Electron microscopy revealed DNA abnormalities ranging from simple unrepaired lesions to chromosomal abnormalities. Metaphase spreads of p53-deficient lymphocytes explored by mFISH revealed high amounts of complex chromosomal aberrations after craniospinal irradiation. CONCLUSIONS: Tumor suppressor p53 plays a central role in maintaining genomic stability by promoting cell-cycle checkpoints and apoptosis. Here, we demonstrate that a patient with LFS receiving craniospinal irradiation including large volumes of bone marrow developed progressive genomic instability of the hematopoietic system. During DNA-damaging radiotherapy, genome-stabilizing mechanisms in proliferating stem/progenitor cells are perturbed by p53 deficiency, increasing the risk of cancer initiation and progression.

Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation.

BACKGROUND AND PURPOSE: High linear energy transfer (LET) radiotherapy offers superior dose conformity and biological effectiveness compared with low-LET radiotherapy, representing a promising alternative for radioresistant tumours. A prevailing hypothesis is that energy deposition along the high-LET particle trajectories induces DNA lesions that are more complex and clustered and therefore more challenging to repair. The precise molecular mechanisms underlying the differences in radiobiological effects between high-LET and low-LET radiotherapies remain unclear. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.5h and 5h post exposure, the DNA-damage pattern in the chromatin ultrastructure was visualised using gold-labelled DNA-repair factors. The induction and repair of single-strand breaks, double-strand breaks (DSBs), and clustered lesions were analysed in combination with terminal dUTP nick-end labelling of DNA breaks. RESULTS: High-LET irradiation induced clustered lesions with multiple DSBs along ion trajectories predominantly in heterochromatic regions. The cluster size increased over time, suggesting inefficient DSB repair. Low-LET irradiation induced many isolated DSBs throughout the nucleus, most of which were efficiently rejoined. CONCLUSIONS: The clustering of DSBs in heterochromatin following high-LET irradiation perturbs efficient DNA repair, leading to greater biological effectiveness of high-LET irradiation versus that of low-LET irradiation.

Ultrastructural Insights into the Biological Significance of Persisting DNA Damage Foci after Low Doses of Ionizing Radiation.

PURPOSE: Intensity-modulated radiotherapy (IMRT) enables the delivery of high doses to target volume while sparing surrounding nontargeted tissues. IMRT treatment, however, substantially increases the normal tissue volume receiving low-dose irradiation, but the biologic consequences are unclear. EXPERIMENTAL DESIGN: Using mouse strains that varied in genetic DNA repair capacity, we investigated the DNA damage response of cortical neurons during daily low-dose irradiation (0.1 Gy). Using light and electron microscopic approaches, we enumerated and characterized DNA damage foci as marker for double-strand breaks (DSBs). RESULTS: During repeated low-dose irradiation, cortical neurons in brain tissues of all mouse strains had a significant increase of persisting foci with cumulative doses, with the most pronounced accumulation of large-sized foci in repair-deficient mice. Electron microscopic analysis revealed that persisting foci in repair-proficient neurons reflect chromatin alterations in heterochromatin, but not persistently unrepaired DSBs. Repair-deficient SCID neurons, by contrast, showed high numbers of unrepaired DSBs in eu- and heterochromatin, emphasizing the fundamental role of DNA-PKcs in DSB rejoining, independent of chromatin status. In repair-deficient ATM-/- neurons, large persisting damage foci reflect multiple unrepaired DSBs concentrated at the boundary of heterochromatin due to disturbed KAP1 phosphorylation. CONCLUSION: Repeated low-dose irradiation leads to the accumulation of persisting DNA damage foci in cortical neurons and thus may adversely affect brain tissue and increase the risk of carcinogenesis. Multiple unrepaired DSBs account for large-sized foci in repair-deficient neurons, thus quantifying foci alone may underestimate extent and complexity of persistent DNA damage. Clin Cancer Res; 22(21); 5300-11. ©2016 AACR.

Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy--the heavy burden to repair.

Low- and high-linear energy transfer (LET) ionising radiation are effective cancer therapies, but produce structurally different forms of DNA damage. Isolated DNA damage is repaired efficiently; however, clustered lesions may be more difficult to repair, and are considered as significant biological endpoints. We investigated the formation and repair of DNA double-strand breaks (DSBs) and clustered lesions in human fibroblasts after exposure to sparsely (low-LET; delivered by photons) and densely (high-LET; delivered by carbon ions) ionising radiation. DNA repair factors (pKu70, 53BP1, γH2AX, and pXRCC1) were detected using immunogold-labelling and electron microscopy, and spatiotemporal DNA damage patterns were analysed within the nuclear ultrastructure at the nanoscale level. By labelling activated Ku-heterodimers (pKu70) the number of DSBs was determined in electron-lucent euchromatin and electron-dense heterochromatin. Directly after low-LET exposure (5 min post-irradiation), single pKu70 dimers, which reflect isolated DSBs, were randomly distributed throughout the entire nucleus with a linear dose correlation up to 30 Gy. Most euchromatic DSBs were sensed and repaired within 40 min, whereas heterochromatic DSBs were processed with slower kinetics. Essentially all DNA lesions induced by low-LET irradiation were efficiently rejoined within 24h post-irradiation. High-LET irradiation caused localised energy deposition within the particle tracks, and generated highly clustered DNA lesions with multiple DSBs in close proximity. The dimensions of these clustered lesions along the particle trajectories depended on the chromatin packing density, with huge DSB clusters predominantly localised in condensed heterochromatin. High-LET irradiation-induced clearly higher DSB yields than low-LET irradiation, with up to ∼ 500 DSBs per µm(3) track volume, and large fractions of these heterochromatic DSBs remained unrepaired. Hence, the spacing and quantity of DSBs in clustered lesions influence DNA repair efficiency, and may determine the radiobiological outcome.

Accumulation of DNA damage in complex normal tissues after protracted low-dose radiation.

The biological consequences of low levels of radiation exposure and their effects on human health are unclear. Ionizing radiation induces a variety of lesions of which DNA double-strand breaks (DSBs) are the most biologically significant, because unrepaired or misrepaired DSBs can lead to genomic instability and cell death. Using repair-proficient mice as an in vivo system we monitored the accumulation of DNA damage in normal tissues exposed to daily low-dose radiation of 100mGy or 10mGy. Radiation-induced foci in differentiated and tissue-specific stem cells were quantified by immunofluorescence microscopy after 2, 4, 6, 8, and 10 weeks of daily low-dose radiation and DNA lesions were characterized using transmission electron microscopy (TEM) combined with immunogold-labeling. In brain, long-living cortical neurons had a significant accumulation of foci with increasing cumulative doses. In intestine and skin, characterized by constant cell renewal of their epithelial lining, differentiated enterocytes and keratinocytes had either unchanged or only slightly increased foci levels during protracted low-dose radiation. Significantly, analysis of epidermal stem cells in skin revealed a constant increase of 53BP1 foci during the first weeks of low-dose radiation even with 10mGy, suggesting substantial accumulations of DSBs. However, TEM analysis suggests that these remaining 53BP1 foci, which are predominantly located in compact heterochromatin, do not co-localize with phosphorylated Ku70 or DNA-PKcs, core components of non-homologous end-joining. The biological relevance of these persistent 53BP1 foci, particularly their contribution to genomic instability by genetic and epigenetic alterations, has to be defined in future studies.

Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

PURPOSE: DNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.

DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy.

The recognition and repair of DNA double-strand breaks (DSBs) occurs in the context of highly structured chromatin. Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic versus heterochromatic regions. While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of γH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Collectively, this TEM approach provides fascinating insights into the dynamic events of the DSB repair process that depend decisively upon the actual chromatin structure around the break.

iGentifier: indexing and large-scale profiling of unknown transcriptomes.

Development and refinement of methods to analyse differential gene expression has been essential in the progress of molecular biology. A novel approach called iGentifier is presented for profiling known and unknown transcriptomes, thus bypassing a major limitation in microarray analysis. The iGentifier technology combines elements of fragment display (e.g. Differential Display or RMDD) and tag sequencing (e.g. SAGE, MPSS) and allows for analysis of samples in high throughput using current capillary electrophoresis equipment. Application to epidermal tissue of wild-type and mlo5 barley (Hordeum vulgare) plants, infected with powdery mildew [Blumeria graminis (DC.) E.O. Speer f.sp.hordei], led to the identification of several 100 genes induced or repressed upon infection with many well known for their response to fungal pathogens or other stressors. Ten of these genes are suggested to be classified as marker genes for durable resistance mediated by the mlo5 resistance gene.