Dual Nucleosomal Double-Strand Breaks Are the Key Effectors of Curative Radiation Therapy

Most ionizing radiation produces δ-rays of ≈1 keV that can impart MGy doses to 100 nm3 volumes of DNA. These events can produce severe dual double-strand breaks (DDSBs) on nucleosomes, particularly in dense heterochromatic DNA. This is the most common multiply damaged site, and their probabilities determine the biological effectiveness of different types of radiation. We discuss their frequency, effect on cell survival, DNA repair, and imaging by gold nanoparticle tracers and electron microscopy. This new and valuable nanometer resolution information can be used for determining the optimal tumor cure by maximizing therapeutic effects on tumors and minimizing therapeutic effects on normal tissues. The production of DDSBs makes it important to deliver a rather high dose and LET to the tumor (>2.5 Gy/Fr) and at the same time reach approximately 1.8–2.3 Gy of the lowest possible LET per fraction in TP53 intact normal tissues at risk. Therefore, their intrinsic low-dose hyper-sensitivity (LDHS)-related optimal daily fractionation window is utilized. Before full p53 activation of NHEJ and HR repair at ≈½ Gy, the low-dose apoptosis (LDA) and LDHS minimize normal tissue mutation probabilities. Ion therapy should thus ideally produce the lowest possible LET in normal tissues to avoid elevated DDSBs. Helium to boron ions can achieve this with higher-LET Bragg peaks, producing increased tumor DDSB densities. Interestingly, the highest probability of complication-free cure with boron or heavier ions requires a low LET round-up for the last 10–15 GyE, thereby steepening the dose response and further minimizing normal tissue damage. In conclusion, the new high-resolution DSB and DDSB diagnostic methods, and the new more accurate DNA-repair-based radiation biology, have been combined to increase our understanding of what is clinically important in curative radiation therapy. In fact, we must understand that we already passed the region of optimal LET and need to go back one step rather than forward, with oxygen being contemplated. As seen by the high overkill and severely high LET in the distal tumor and the increased LET to normal tissues (reminding of neutrons or neon ions), it is therefore preferable to use lithium–boron ions or combine carbon with an optimal 10–15 GyE photon, electron, or perhaps even a proton round-up, thus allowing optimized, fractionated, curative, almost complication-free treatments with photons, electrons, and light ions, introducing a real paradigm shift in curative radiation therapy with a potential 5 GyE tumor boost, 25% increase in complication-free cure and apoptotic–senescent Bragg Peak molecular light ion radiation therapy

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

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

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

Beyond Repair Foci: DNA Double-Strand Break Repair in Euchromatic and Heterochromatic Compartments Analyzed by Transmission Electron Microscopy

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.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.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.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

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 germlinemutations of the p53 tumor-suppressor gene. In response to DNA damage, p53 stimulates protective cellularprocesses including cell-cycle arrest and apoptosis to prevent aberrant cell proliferation. Currentcancer therapies involve agents that damage DNA, which also affect non-cancerous hematopoieticstem/progenitor cells. Here, we report on a child with LFS who developed genomic instability duringcraniospinal 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 germlinemutation, leading to LFS diagnosis. After tumour resection and systemic chemotherapy, entire craniospinalaxis was irradiated due to leptomeningeal seeding, resulting in disease stabilization for nearly12 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 asymptomaticcarriers for identical p53 mutation, not exposed to DNA-damaging treatment. In p53-deficientlymphocytes 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 showedsignificant accumulation of 53BP1-foci during and even several months after irradiation, reflecting persistentDNA damage. Electron microscopy revealed DNA abnormalities ranging from simple unrepairedlesions to chromosomal abnormalities. Metaphase spreads of p53-deficient lymphocytes explored bymFISH revealed high amounts of complex chromosomal aberrations after craniospinal irradiation.Conclusions: Tumor suppressor p53 plays a central role in maintaining genomic stability by promotingcell-cycle checkpoints and apoptosis. Here, we demonstrate that a patient with LFS receiving craniospinalirradiation including large volumes of bone marrow developed progressive genomic instability of thehematopoietic system. During DNA-damaging radiotherapy, genome-stabilizing mechanisms in proliferatingstem/progenitor cells are perturbed by p53 deficiency, increasing the risk of cancer initiation andprogression