Radiation-induced Double-strand Breaks Research Articles

Modeling the oxygen effect in DNA strand break induced by gamma-rays with TOPAS-nBio

Objective.To present and validate a method to simulate from first principles the effect of oxygen on radiation-induced double-strand breaks (DSBs) using the Monte Carlo Track-structure code TOPAS-nBio.Approach.Two chemical models based on the oxygen fixation hypothesis (OFH) were developed in TOPAS-nBio by considering an oxygen adduct state of DNA and creating a competition kinetic mechanism between oxygen and the radioprotective molecule WR-1065. We named these models 'simple' and 'detailed' due to the way they handle the hydrogen abstraction pathways. We used the simple model to obtain additional information for the •OH-DNA hydrogen abstraction pathway probability for the detailed model. These models were calibrated and compared with published experimental data of linear and supercoiling fractions obtained with R6K plasmids, suspended in dioxane as a hydroxyl scavenger, and irradiated with137Cs gamma-rays. The reaction rates for WR-1065 and O2with DNA were taken from experimental works. Single-Strand Breaks (SSBs) and DSBs as a function of the dose for a range of oxygen concentrations [O2] (0.021%-21%) were obtained. Finally, the hypoxia reduction factor (HRF) was obtained from DSBs.Main Results.Validation results followed the trend of the experimental within 12% for the supercoiled and linear plasmid fractions for both models. The HRF agreed with measurements obtained with137Cs and 200-280 kVp x-ray within experimental uncertainties. However, the HRF at an oxygen concentration of 2.1% overestimated experimental results by a factor of 1.7 ± 0.1. Increasing the concentration of WR-1065 from 1 mM to 10-100 mM resulted in a HRF difference of 0.01, within the 8% statistical uncertainty between TOPAS-nBio and experimental data. This highlights the possibility of using these chemical models to recreate experimental HRF results.Significance.Results support the OFH as a leading cause of oxygen radio-sensitization effects given a competition between oxygen and chemical DNA repair molecules like WR-1065.

Identification of 6-Anilino Imidazo[4,5-c]pyridin-2-ones as Selective DNA-Dependent Protein Kinase Inhibitors and Their Application as Radiosensitizers.

The dominant role of non-homologous end-joining in the repair of radiation-induced double-strand breaks identifies DNA-dependent protein kinase (DNA-PK) as an excellent target for the development of radiosensitizers. We report the discovery of a new class of imidazo[4,5-c]pyridine-2-one DNA-PK inhibitors. Structure-activity studies culminated in the identification of 78 as a nM DNA-PK inhibitor with excellent selectivity for DNA-PK compared to related phosphoinositide 3-kinase (PI3K) and PI3K-like kinase (PIKK) families and the broader kinome, and displayed DNA-PK-dependent radiosensitization of HAP1 cells. Compound 78 demonstrated robust radiosensitization of a broad range of cancer cells in vitro, displayed high oral bioavailability, and sensitized colorectal carcinoma (HCT116/54C) and head and neck squamous cell carcinoma (UT-SCC-74B) tumor xenografts to radiation. Compound 78 also provided substantial tumor growth inhibition of HCT116/54C tumor xenografts in combination with radiation. Compound 78 represents a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for cancer treatment.

Oleandrin enhances radiotherapy sensitivity in lung cancer by inhibiting the ATM/ATR-mediated DNA damage response.

Despite active clinical trials on the use of Oleandrin alone or in combination with other drugs for the treatment of solid tumors, the potential synergistic effect of Oleandrin with radiotherapy remains unknown. This study reveals a new mechanism by which Oleandrin targets ATM and ATR kinase-mediated radiosensitization in lung cancer. Various assays, including clonogenic, Comet, immunofluorescence staining, apoptosis and Cell cycle assays, were conducted to evaluate the impact of oleandrin on radiation-induced double-strand break repair and cell cycle distribution. Western blot analysis was utilized to investigate alterations in signal transduction pathways related to double-strand break repair. The efficacy and toxicity of the combined therapy were assessed in a preclinical xenotransplantation model. Functionally, Oleandrin weakens the DNA damage repair ability and enhances the radiation sensitivity of lung cells. Mechanistically, Oleandrin inhibits ATM and ATR kinase activities, blocking the transmission of ATM-CHK2 and ATR-CHK1 cell cycle checkpoint signaling axes. This accelerates the passage of tumor cells through the G2 phase after radiotherapy, substantially facilitating the rapid entry of large numbers of inadequately repaired cells into mitosis and ultimately triggering mitotic catastrophe. The combined treatment of Oleandrin and radiotherapy demonstrated superior inhibition of tumor proliferation compared to either treatment alone. Our findings highlight Oleandrin as a novel and effective inhibitor of ATM and ATR kinase, offering new possibilities for the development of clinical radiosensitizing adjuvants.

Modelling Heterogeneous Anomalous Dynamics of Radiation-Induced Double-Strand Breaks in DNA during Non-Homologous End-Joining Pathway.

The process of end-joining during nonhomologous repair of DNA double-strand breaks (DSBs) after radiation damage is considered. Experimental evidence has revealed that the dynamics of DSB ends exhibit subdiffusive motion rather than simple diffusion with rare directional movement. Traditional models often overlook the rare long-range directed motion. To address this limitation, we present a heterogeneous anomalous diffusion model consisting of subdiffusive fractional Brownian motion interchanged with short periods of long-range movement. Our model sheds light on the underlying mechanisms of heterogeneous diffusion in DSB repair and could be used to quantify the DSB dynamics on a time scale inaccessible to single particle tracking analysis. The model predicts that the long-range movement of DSB ends is responsible for the misrepair of DSBs in the form of dicentric chromosome lesions.

The Influence of Computed Tomography Contrast Agent on Radiation-Induced Gene Expression and Double-Strand Breaks.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07-0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03-0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations. To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022). This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN. To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage. To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death. In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.

Radiation-induced double-strand breaks by internal ex vivo irradiation of lymphocytes: Validation of a Monte Carlo simulation model using GATE and Geant4-DNA

This study describes a method to validate a radiation transport model that quantifies the number of DNA double-strand breaks (DSB) produced in the lymphocyte nucleus by internal ex vivo irradiation of whole blood with the radionuclides 90Y, 99mTc, 123I, 131I, 177Lu, 223Ra, and 225Ac in a test vial using the GATE/Geant4 code at the macroscopic level and the Geant4-DNA code at the microscopic level. MethodsThe simulation at the macroscopic level reproduces an 8 mL cylindrical water-equivalent medium contained in a vial that mimics the geometry for internal ex vivo blood irradiation. The lymphocytes were simulated as spheres of 3.75 µm radius randomly distributed, with a concentration of 125 spheres/mL. A phase-space actor was attached to each sphere to register all the entering particles. The simulation at the microscopic level for each radionuclide was performed using the Geant4-DNA tool kit, which includes the clustering example centered on a density-based spatial clustering of applications with noise (DBSCAN) algorithm. The irradiation source was constructed by generating a single phase space from the sum of all phase spaces. The lymphocyte nucleus was defined as a water sphere of a 3.1 µm radius. The absorbed dose coefficients for lymphocyte nuclei (dLymph) were calculated and compared with macroscopic whole blood absorbed dose coefficients (dBlood). The DBSCAN algorithm was used to calculate the number of DSBs. Lastly, the number of DSB∙cell−1∙mGy−1 (simulation) was compared with the number of radiation-induced foci per cell and absorbed dose (RIF∙cell−1∙mGy−1) provided by experimental data for gamma and beta emitting radionuclides. For alpha emitters, dLymph and the number of α-tracks∙100 cell−1∙mGy−1 and DBSs∙µm−1 were calculated using experiment-based thresholds for the α-track lengths and DBSs/track values. The results were compared with the results of an ex vivo study with 223Ra. ResultsThe dLymph values differed from the dBlood values by −1.0% (90Y), −5.2% (99mTc), −22.3% (123I), 0.35% (131I), 2.4% (177Lu), −5.6% (223Ra) and −6.1% (225Ac). The number of DSB∙cell−1∙mGy−1 for each radionuclide was 0.015 DSB∙cell−1∙mGy−1 (90Y), 0.012 DSB∙cell−1∙mGy−1 (99mTc), 0.014DSB∙cell−1∙mGy−1 (123I), 0.012 DSB∙cell−1∙mGy−1 (131I), and 0.016 DSB∙cell−1∙mGy−1 (177Lu). These values agree very well with experimental data. The number of α-tracks∙100 cells−1∙mGy−1 for 223Ra and 225Ac where 0.144 α-tracks∙100 cells−1∙mGy−1 and 0.151 α-tracks∙100 cells−1∙mGy−1, respectively. These values agree very well with experimental data. Moreover, the linear density of DSBs per micrometer α-track length were 11.13 ± 0.04 DSB/µm and 10.86 ± 0.06 DSB/µm for 223Ra and 225Ac, respectively. ConclusionThis study describes a model to simulate the DNA DSB damage in lymphocyte nuclei validated by experimental data obtained from internal ex vivo blood irradiation with radionuclides frequently used in diagnostic and therapeutic procedures in nuclear medicine.

Monte Carlo-Based Radiobiological Investigation of the Most Optimal Ion Beam Forming SOBP for Particle Therapy.

Proton (p) and carbon (C) ion beams are in clinical use for cancer treatment, although other particles such as He, Be, and B ions have more recently gained attention. Identification of the most optimal ion beam for radiotherapy is a challenging task involving, among others, radiobiological characterization of a beam, which is depth-, energy-, and cell type- dependent. This study uses the FLUKA and MCDS Monte Carlo codes in order to estimate the relative biological effectiveness (RBE) for several ions of potential clinical interest such as p, 4He, 7Li, 10Be, 10B, and 12C forming a spread-out Bragg peak (SOBP). More specifically, an energy spectrum of the projectiles corresponding to a 5-cm SOBP at a depth of 8 cm was used. All secondary particles produced by the projectiles were considered and RBE was determined based on radiation-induced Double Strand Breaks (DSBs), as calculated by MCDS. In an attempt to identify the most optimal ion beam, using the latter data, biological optimization was performed and the obtained depth-dose distributions were inter-compared. The results showed that 12C ions are more effective inside the SOBP region, which comes at the expense of higher dose values at the tail (i.e., after the SOBP). In contrast, p beams exhibit a higher DSOPB/DEntrance ratio, if physical doses are considered. By performing a biological optimization in order to obtain a homogeneous biological dose (i.e., dose × RBE) in the SOBP, the corresponding advantages of p and 12C ions are moderated. 7Li ions conveniently combine a considerably lower dose tail and a DSOPB/DEntrance ratio similar to 12C. This work contributes towards identification of the most optimal ion beam for cancer therapy. The overall results of this work suggest that 7Li ions are of potential interest, although more studies are needed to demonstrate the relevant advantages. Future work will focus on studying more complex beam configurations.

Glioblastoma cells have increased capacity to repair radiation-induced DNA damage after migration to the olfactory bulb.

The invasive nature of GBM combined with the diversity of brain microenvironments creates the potential for a topographic heterogeneity in GBM radioresponse. Investigating the mechanisms responsible for a microenvironment-induced differential GBM response to radiation may provide insights into the molecules and processes mediating GBM radioresistance. Using a model system in which human GBM stem-like cells implanted into the right striatum of nude mice migrate throughout the right hemisphere (RH) to the olfactory bulb (OB), the radiation-induced DNA damage response was evaluated in each location according to γH2AX and 53BP1 foci and cell cycle phase distribution as determined by flow cytometry and immunohistochemistry. RNAseq was used to compare transcriptomes of tumor cells growing in the OB and the RH. Protein expression and neuron-tumor interaction were defined by immunohistochemistry and confocal microscopy. After irradiation, there was a more rapid dispersal of γH2AX and 53BP1 foci in the OB versus in the RH, indicative of increased double strand break repair capacity in the OB and consistent with the OB providing a radioprotective niche. With respect to the cell cycle, by 6h after irradiation there was a significant loss of mitotic tumor cells in both locations suggesting a similar activation of the G2/M checkpoint. However, by 24h post-irradiation there was an accumulation of G2 phase cells in the OB, which continued out to at least 96h. Transcriptome analysis showed that tumor cells in the OB had higher expression levels of DNA repair genes involved in non-homologous end joining and genes related to the spindle assembly checkpoint. Tumor cells in the OB were also found to have an increased frequency of soma-soma contact with neurons. GBM cells that have migrated to the OB have an increased capacity to repair radiation-induced double strand breaks and altered cell cycle regulation. These results correspond to an upregulation of genes involved in DNA damage repair and cell cycle control. Because the murine OB provides a source of radioresistant tumor cells not evident in other experimental systems, it may serve as a model for investigating the mechanisms mediating GBM radioresistance.

A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci

Immunofluorescent tagging of DNA double-strand break (DSB) markers, such as γ-H2AX and other DSB repair proteins, are powerful tools in understanding biological consequences following irradiation. However, whilst the technique is widespread, there are many uncertainties related to its ability to resolve and reliably deduce the number of foci when counting using microscopy. We present a new tool for simulating radiation-induced foci in order to evaluate microscope performance within in silico immunofluorescent images. Simulations of the DSB distributions were generated using Monte Carlo track-structure simulation. For each DSB distribution, a corresponding DNA repair process was modelled and the un-repaired DSBs were recorded at several time points. Corresponding microscopy images for both a DSB and (γ-H2AX) fluorescent marker were generated and compared for different microscopes, radiation types and doses. Statistically significant differences in miscounting were found across most of the tested scenarios. These inconsistencies were propagated through to repair kinetics where there was a perceived change between radiation-types. These changes did not reflect the underlying repair rate and were caused by inconsistencies in foci counting. We conclude that these underlying uncertainties must be considered when analysing images of DNA damage markers to ensure differences observed are real and are not caused by non-systematic miscounting.

RAD51 Inhibitor and Radiation Toxicity in Vestibular Schwannoma.

To describe the RAD51 response (DNA repair) to radiation-induced DNA damage in patient-derived vestibular schwannoma (VS) cells and investigate the utility of RAD51 inhibitor (RI-1) in enhancing radiation toxicity. Basic and translational science. Tertiary academic facility. VS tumors (n = 10) were cultured on 96-well plates and 16-well slides, exposed to radiation (0, 6, 12, or 18 Gy), and treated with RI-1 (0, 5, or 10 µM). Immunofluorescence was performed at 6 hours for γ-H2AX (DNA damage marker), RAD51 (DNA repair protein), and p21 (cell cycle arrest protein). Viability assays were performed at 96 hours, and capillary Western blotting was utilized to determine RAD51 expression in naïve VS tumors (n = 5). VS tumors expressed RAD51. In cultured VS cells, radiation initiated dose-dependent increases in γ-H2AX and p21 expression. VS cells upregulated RAD51 to repair DNA damage following radiation. Addition of RI-1 reduced RAD51 expression in a dose-dependent manner and was associated with increased γ-H2AX levels and decreased viability in a majority of cultured VS tumors. VS may evade radiation injury by entering cell cycle arrest and upregulating RAD51-dependent repair of radiation-induced double-stranded breaks in DNA. Although there was variability in responses among individual primary VS cells, RAD51 inhibition with RI-1 reduced RAD51-dependent DNA repair to enhance radiation toxicity in VS cells. Further investigations are warranted to understand the mechanisms of radiation resistance in VS and determine whether RI-1 is an effective radiosensitizer in patients with VS.

A Combination of Iohexol Treatment and Ionizing Radiation Exposure Enhances Kidney Injury in Contrast-Induced Nephropathy by Increasing DNA Damage.

Contrast media has been shown to induce nephropathy (i.e., contrast-induced nephropathy) after various types of radiological examinations. The molecular mechanism of contrast-induced nephropathy has been unclear. In this study, we investigated the mechanism of contrast-induced nephropathy by examining the effects of combined treatment of contrast medium and ionizing radiation on kidney cells in vitro and kidney tissue in vivo. In human renal tubular epithelium cells, immunofluorescence analysis revealed that iohexol increased the numbers of radiation-induced γH2AX nuclear foci. The numbers of γH2AX nuclear foci remained high at 24 h, suggesting that some radiation-induced double-strand breaks remain unrepaired in the presence of iohexol. We established a mouse model of contrast-induced nephropathy, then showed that iohexol and ionizing radiation synergistically reduced renal function and induced double-strand breaks. Importantly, iohexol induced significant macrophage accumulation and oxidative DNA damage in the kidneys of contrast-induced nephropathy model mice in the absence of ionizing radiation; these effects were amplified by ionizing radiation. The results suggest that underlying inflammation and oxidative DNA damage caused by iohexol contribute to the enhancement of radiation-induced double-strand breaks, leading to contrast-induced nephropathy.

The Arabidopsis HOP2 gene has a role in preventing illegitimate connections between nonhomologous chromosome regions.

Meiotic homologous chromosomes synapse and undergo crossing over (CO). In many eukaryotes, both synapsis and crossing over require the induction of double stranded breaks (DSBs) and subsequent repair via homologous recombination. In these organisms, two key proteins are recombinases RAD51 and DMC1. Recombinase-modulators HOP2 and MND1 assist RAD51 and DMC1 and also are required for synapsis and CO. We have investigated the hop2-1 phenotype in Arabidopsis during the segregation stages of both meiosis and mitosis. Despite a general lack of synapsis during prophase I, we observed extensive, stable interconnections between nonhomologous chromosomes in diploid hop2-1 nuclei in first and second meiotic divisions. Using γH2Ax as a marker of unrepaired DSBs, we detected γH2AX foci from leptotene through early pachytene but saw no foci from mid-pachytene onward. We conclude that the bridges seen from metaphase I onward are due to mis-repaired DSBs, not unrepaired ones. Examining haploids, we found that wild type haploids produce only univalents, but hop2-1 haploids like hop2-1 diploids have illegitimate connections stable enough to produce bridged chromosomes during segregation. Our results suggest that HOP2 has a significant active role in preventing repairs that use nonhomologous chromosomes during meiosis. We also found evidence that HOP2 plays a role in preventing illegitimate repair of radiation-induced DSBs in rapidly dividing petal cells. We conclude that HOP2 in Arabidopsis plays both a positive role in promoting synapsis and a separable role in preventing DSB repair using nonhomologous chromosomes. SIGNIFICANCE STATEMENT : The fidelity of homologous recombination (HR) during meiosis is essential to the production of viable gametes and for maintaining genome integrity in vegetative cells. HOP2 is an important protein for accurate meiotic HR in plants. We have found evidence of high levels of illegitimate repairs between nonhomologous chromosomes during meiosis and in irradiated petal cells in hop2-1 mutants, suggesting a role for HOP2 beyond its established role in synapsis and crossing over.

Lanzhou Lily polysaccharide fragment protects human umbilical vein endothelial cells from radiation-induced DNA double-strand breaks.

Radiotherapy is widely used in the treatment of tumors. However, while killing tumor cells, radiation may also cause damage to the surrounding normal tissues. Therefore, it is very important to find safe and effective radiation protection agents. To investgate the radiation protection effect of Lanzhou Lily polysaccharide fragments (LLP). Methods: The crude polysaccharides of Lanzhou Lily were extracted from the dried bulb powder of Lilium lilium by ultrasonic-assisted hot water method, and then five different fragments were separated from the polysaccharides by DEAE-52-cellulose column. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, neutral comet and immunofluorescent staining were used to investigate the effect of LPe fragment on Human Umbilical Vein Endothelial Cells (HUVEC) survival and the possible radioprotective mechanism. The LPe fragment (composing of mannose and glucose, with a ratio of 5.5:2.9, and the average molecular weight is 8629.8 Da), significantly promoted the proliferation of HUVECs and protected cells from X-ray-induced double-strand breaks (DSBs) in DNA, in which pretreatment with the LPe fragment at 100 μg/mL showed the most pronounced protection. In addition, the occurrence of X-ray-induced γH2AX foci was significantly reduced by treatment with the LPe fragment at 50, 100, and 200 μg/mL. Furthermore, caffeine or wortmannin in combination with the LPe fragment at 25 μg/mL significantly reduced the number of X-ray-induced γH2AX foci, indicating phosphoinositide-3 kinases (PI3K) is involved in H2AX phosphorylation in HUVECs. These results indicate the LPe fragment has a protective effect against radiation-induced DSBs and may be used as a natural antioxidant agent.

Golgi Phosphoprotein 3 Confers Radioresistance via Stabilizing EGFR in Lung Adenocarcinoma

Radioresistance is a major cause of treatment failure in tumor radiation therapy, and the underlying mechanisms of radioresistance are still elusive. Golgi phosphoprotein 3 (GOLPH3) has been reported to associate tightly with cancer progression and chemoresistance. Herein, we explored whether GOLPH3 mediated radioresistance of lung adenocarcinoma (LUAD) and whether targeted suppression of GOLPH3 sensitized LUAD to radiation therapy. The aberrant expression of GOLPH3 was evaluated by immunohistochemistry in LUAD clinical samples. To evaluate the association between GOLPH3 and radioresistance, colony formation and apoptosis were assessed in control and GOLPH3 knockdown cells. γ-H2AX foci and level determination and micronucleus test were used to analyze DNA damage production and repair. The rescue of GOLPH3 knockdown was then performed by exogenous expression of small interfering RNA-resistant mutant GOLPH3 to confirm the role of GOLPH3 in DNA damage repair. Mechanistically, the effect of GOLPH3 on regulating stability and nuclear accumulation of epidermal growth factor receptor (EGFR) and the activation of DNA-dependent protein kinase (DNA-PK) were investigated by quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and coimmunoprecipitation. The role of GOLPH3 in vivo in radioresistance was determined in a xenograft model. In tumor tissues of 33 patients with LUAD, the expression of GOLPH3 showed significant increases compared with those in matched normal tissues. Knocking down GOLPH3 reduced the clonogenic capacity, impaired double-strand break (DSB) repair, and enhanced apoptosis after irradiation. In contrast, reversal of GOLPH3 depletion rescued the impaired repair of radiation-induced DSBs. Mechanistically, loss of GOLPH3 accelerated the degradation of EGFR in lysosome, causing the reduction in EGFR levels, thereby weakening nuclear accumulation of EGFR and attenuating the activation of DNA-PK. Furthermore, adenovirus-mediated GOLPH3 knockdown could enhance the ionizing radiation response in the LUAD xenograft model. GOLPH3 conferred resistance of LUAD to ionizing radiation via stabilizing EGFR, and targeted suppression of GOLPH3 might be considered as a potential therapeutic strategy for sensitizing LUAD to radiation therapy.

DNA damage and repair in peripheral blood mononuclear cells after internal ex vivo irradiation of patient blood with 131I

AimThe aim of this study was to provide a systematic approach to characterize DNA damage induction and repair in isolated peripheral blood mononuclear cells (PBMCs) after internal ex vivo irradiation with [131I]NaI. In this approach, we tried to mimic ex vivo the irradiation of patient blood in the first hours after radioiodine therapy.Material and methodsBlood of 33 patients of two centres was collected immediately before radioiodine therapy of differentiated thyroid cancer (DTC) and split into two samples. One sample served as non-irradiated control. The second sample was exposed to ionizing radiation by adding 1 ml of [131I]NaI solution to 7 ml of blood, followed by incubation at 37 °C for 1 h. PBMCs of both samples were isolated, split in three parts each and (i) fixed in 70% ethanol and stored at − 20 °C directly (0 h) after irradiation, (ii) after 4 h and (iii) 24 h after irradiation and culture in RPMI medium. After immunofluorescence staining microscopically visible co-localizing γ-H2AX + 53BP1 foci were scored in 100 cells per sample as biomarkers for radiation-induced double-strand breaks (DSBs).ResultsThirty-two of 33 blood samples could be analysed. The mean absorbed dose to the blood in all irradiated samples was 50.1 ± 2.3 mGy. For all time points (0 h, 4 h, 24 h), the average number of γ-H2AX + 53BP1 foci per cell was significantly different when compared to baseline and the other time points. The average number of radiation-induced foci (RIF) per cell after irradiation was 0.72 ± 0.16 at t = 0 h, 0.26 ± 0.09 at t = 4 h and 0.04 ± 0.09 at t = 24 h. A monoexponential fit of the mean values of the three time points provided a decay rate of 0.25 ± 0.05 h−1, which is in good agreement with data obtained from external irradiation with γ- or X-rays.ConclusionThis study provides novel data about the ex vivo DSB repair in internally irradiated PBMCs of patients before radionuclide therapy. Our findings show, in a large patient sample, that efficient repair occurs after internal irradiation with 50 mGy absorbed dose, and that the induction and repair rate after 131I exposure is comparable to that of external irradiation with γ- or X-rays.

Primary Vestibular Schwannoma Cells Activate p21 and RAD51-Associated DNA Repair Following Radiation-Induced DNA Damage.

Vestibular Schwannoma (VS) can avoid cell death following radiation injury by entering cell cycle arrest and activating RAD51-related DNA repair. Although the radiobiology of various cancers is well-studied, the radiobiological effects in VS are poorly understood. In this study, we describe how VS cells enter cell cycle arrest (through p21 expression), activate DNA repair (through RAD51 upregulation), and avoid cell death after radiation-induced double-stranded breaks (DSB) in DNA (as measured by γ-H2AX). Primary human VS cells were cultured on 96-well plates and 16-well culture slides at 10,000 cells/well and exposed to either 0 or 18 Gray of radiation. Viability assays were performed at 96 h in vitro. Immunofluorescence for γ-H2AX, RAD51, and p21 was performed at 6 h. Radiation (18 Gy) induced the expression of γ-H2AX, p21, and RAD51 in six cultured VS, suggesting that irradiated VS acquire DSBs, enter cell cycle arrest, and initiate RAD51 DNA repair to evade cell death. However, viability studies demonstrate variable responses in individual VS cells with 3 of 6 VS showing radiation resistance to 18 Gy. On further analyses, radiation-resistant VS cells expressed significantly more p21 than radiation-responsive tumors. In response to radiation-induced DNA damage, primary VS cells can enter cell cycle arrest and express RAD51 DNA repair mechanisms to avoid cell death. Radioresistant VS cells may mount a more robust p21 response to ensure sufficient time for DNA repair. Further investigation into DNA repair proteins and cell cycle checkpoints may provide important insight on the radiobiology of VS and mechanisms for resistance.

Abstract 1394: Poly ADP-ribose polymerase inhibitor promotes the immunoradiotherapeutical efficacy in small cell lung cancer

Abstract This study aimed to investigate the safety and efficacy of radiotherapy combined with poly ADP-ribose polymerase (PARP) inhibitors and immune checkpoint inhibitors in small cell lung cancer (SCLC). Tumor-bearing xenograft mouse model was used to examine the safety and anti-tumor effects of radiotherapy plus niraparib and PD-1 antibodies. SCLC cell lines were used to explore the underlying mechanisms in this combination. PARP inhibitor suppressed SCLC cell proliferation, induced cell apoptosis, and increased cell cycle arrest at G2/M phase. Niraparib treatment augmented radiation-induced double-strand DNA breaks, which subsequently activated the cGAS/STING pathway to increase the expression levels of CXCL10, CCL5 and PD-L1 in SCLC cells. Moreover, the combination of radiation, niraparib and PD-1 antibodies significantly inhibited SCLC tumor growth and prolonged the survival of immunocompetent C57BL/6 mice. The induced CCL5 and CXCL10 activated CD8+ T cells in vivo. Our study indicated that niraparib combined with radiation promoted immune checkpoint inhibitor functions via inducing PD-L1 expression, and activated CD8+ T cells via increasing CXCL10 and CCL5 levels through cGAS/STING pathway. The combination treatment of PARP inhibitors, radiotherapy and immunotherapy were safe in vivo, effectively suppressed SCLC tumor progression, increased survival and decreased the adverse events via lowering the dose of individual treatment. Citation Format: Nannan Zhang, Yanping Gao, Zihang Zeng, Yuan Luo, Xueping Jiang, Jianguo Zhang, Jiali Li, Zhengrong Huang, Yan Gong, Conghua Xie. Poly ADP-ribose polymerase inhibitor promotes the immunoradiotherapeutical efficacy in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1394.

Mechanistic Modelling of Slow and Fast NHEJ DNA Repair Pathways Following Radiation for G0/G1 Normal Tissue Cells.

Simple SummaryWhen cells are irradiated, their DNA can become damaged, this causes different types of repair processes to try and fix the DNA breaks. One of the most lethal types of DNA damage is double-strand breaks (DSBs). This work models the most used DSB repair process called Non-Homologous End Joining (NHEJ) and includes both its resection-independent and resection-dependent pathways. The models produced are benchmarked against experimental normal and deficient cell-types across a wide range of radiation qualities. We compare two approaches of modelling, the first is where the DSBs can repair in parallel and the second is where the DSBs repair is entwined. We find that it is necessary to consider both the resection-independent and resection-dependent pathways as entwined to produce a model which robustly matches experimental work. Through better modelling of NHEJ repair, it can improve our understanding of radiation response which has potential in biological optimisation for radiotherapy.Mechanistic in silico models can provide insight into biological mechanisms and highlight uncertainties for experimental investigation. Radiation-induced double-strand breaks (DSBs) are known to be toxic lesions if not repaired correctly. Non-homologous end joining (NHEJ) is the major DSB-repair pathway available throughout the cell cycle and, recently, has been hypothesised to consist of a fast and slow component in G0/G1. The slow component has been shown to be resection-dependent, requiring the nuclease Artemis to function. However, the pathway is not yet fully understood. This study compares two hypothesised models, simulating the action of individual repair proteins on DSB ends in a step-by-step manner, enabling the modelling of both wild-type and protein-deficient cell systems. Performance is benchmarked against experimental data from 21 cell lines and 18 radiation qualities. A model where resection-dependent and independent pathways are entirely separated can only reproduce experimental repair kinetics with additional restraints on end motion and protein recruitment. However, a model where the pathways are entwined was found to effectively fit without needing additional mechanisms. It has been shown that DaMaRiS is a useful tool when analysing the connections between resection-dependent and independent NHEJ repair pathways and robustly matches with experimental results from several sources.

Histone Variant H2A.J Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence.

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.