Number Of DNA Double-strand Breaks Research Articles

DNA double-strand breaks of human peripheral blood lymphocyte induced by CT examination of oral and maxillofacial region.

To explore whether a computed tomography (CT) examination of the head and neck region induces biological damage and whether the damage was correlated with the radiation dose. Peripheral blood was taken from 33 individuals who received head and neck CT examinations. Blood samples were divided into three groups: the control group and the in vivo and in vitro irradiation groups. The number of DNA double-strand breaks was estimated by comparing the changes in the rates of γ-H2AX foci formation in the peripheral blood before and after CT examination. The absorbed dose and effective dose were calculated with the software VirtualDose based on the Monte Carlo method, and the absorbed doses in blood were estimated accordingly. The γ-H2AX foci rates were increased in the in vivo (p < 0.001) and in vitro irradiation groups (p < 0.001) after CT examination when compared with those in the control group. The rate of γ-H2AX foci formation showed linear dose-responses for the CT dose index volume (CTDIvol), dose-length product (DLP), and blood dose after CT examination. A CT examination of the head and neck region provides a high enough radiation dose to induce DNA double-strand breaks in cells in the peripheral blood. There was a linear correlation between the formation of DNA double-strand breaks and radiation doses after CT examination. In addition to ensuring image quality, in a real clinical situation, the scanning area should be strictly administered, and repeated operations should be avoided to minimise the patient's radiation dose.

Protective Effect of Asiaticoside on Radiation-induced Proliferation Inhibition and DNA Damage of Fibroblasts and Mice Death.

BackgroundRadiation-induced injuries (RII) mainly result from reactive oxygen species (ROS), which are harmful compounds that can damage DNA. Asiaticoside (AC), one of the main functional components extracted from Centella asiatica, has potent pharmacological effects such as anti-inflammatory and anti-oxidant activity. However, its role in RII remains unclear.PurposeThe purpose of the current study is to investigate whether AC can mitigate RII in vitro and in vivo.Material and MethodsCell model of RII was successfully established by 5J/m2 radiation in vitro. For the in vivo RII model, mice were irradiated with 5 Gy to the thorax. The degree of damage to cells or mouse tissue was determined by measuring the numbers of DNA double-strand breaks (DSBs), oxidative stress, and mouse survival rates.ResultsIn the in vitro assay, AC administration significantly reduced radiation-induced growth inhibition of Escherichia coli and fibroblasts, DSBs and apoptosis of fibroblasts; in the in vivo study, AC could decrease antioxidant capacity (T-AOC) of plasma and protect mice from RII, thereby improving the survival rates of mice after radiation.ConclusionsThese novel data indicate that AC is able to prevent radiation-initiated genotoxicity by mitigating DNA damage, and might serve as a safe and effective radio-protective agent.

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

Anticancer drug and ionizing radiation-induced DNA damage differently influences transcription activity and DDR-related stress responses of an endothelial monolayer

The endothelium contributes to the pathophysiology of adverse effects caused by conventional (genotoxic) anticancer therapeutics (cAT). The relevance of structurally different types of cAT-induced DNA lesions for eliciting selected endothelial stress responses is largely unknown. Here, we analyzed the cAT-induced formation of DNA double-strand breaks (DSB), transcription blockage and DNA damage response (DDR) in time kinetic analyses employing a monolayer of primary human endothelial cells (HUVEC). We observed that the degree of cAT-induced transcription blockage, the number of DSB and activation of DDR-related factors diverge. For instance, ionizing radiation caused the formation of numerous DSB and triggerd a substantial activation of ATM/Chk2 signaling, which however were not accompanied by a significant transcription inhibition. By contrast, the DNA cross-linking cAT cisplatin triggered a rapid and substantial blockage of transcription, which yet was not reflected by an appreciable number of DSB or increased levels of pATM/pChk2. In general, cAT-stimulated ATM-dependent phosphorylation of Kap1 (Ser824) and p53 (Ser15) reflected best cAT-induced transcription blockage. In conclusion, cAT-induced formation of DSB and profound activation of prototypical DDR factors is independent of the inhibition of RNA polymerase II-regulated transcription in an endothelial monolayer. We suggest that DSB formed directly or indirectly following cAT-treatment do not act as comprehensive triggers of superior signaling pathways shutting-down transcription while, at the same time, causing an appreciable stimulation of the DDR. Rather, it appears that distinct cAT-induced DNA lesions elicit diverging signaling pathways, which separately control transcription vs. DDR activity in the endothelium.

Enhancement of Cytotoxicity of Enediyne Compounds by Hyperthermia: Effects of Various Metal Complexes on Tumor Cells.

Enediyne natural products are a class of compounds that were recognized for their potential as chemotherapeutic agents many years ago, but found to be highly cytotoxic due to their propensity for low thermal activation. Bergman cyclization of the enediyne moiety produces a diradical intermediate, and may subsequently induce DNA damage and account for the extreme cytotoxicity. While difficulties in controlling the thermal cyclization reaction have limited the clinical use of cyclic enediynes, we have previously shown that enediyne activity, and thus toxicity at physiological temperatures can be modulated by metallation of acyclic enediynes. Furthermore, the cytotoxicity of "metalloenediynes" can be potentiated by hyperthermia. In this study, we characterized a suite of novel metallated enediyne motifs that usually induced little or no cytotoxicity when two different human cancer cell lines were treated with the compounds at 37°C, but showed a significant enhancement of cytotoxicity after cells were exposed to moderate hyperthermia during drug treatment. Cultured U-1 melanoma or MDA-231 breast cancer cells were treated with various concentrations of Cu, Fe and Zn complexes of the enediyne (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) and clonogenic survival was assessed to determine the effects of the drugs at 37°C and 42.5°C. Toxicity at 37°C varied for each compound, but hyperthermia potentiated the cytotoxicity of each compound in both cell lines. Cytotoxicity was concentration-, time- and temperature-dependent. Heating cells during drug treatment resulted in enhanced apoptosis, but the role of cell cycle perturbation in the response of the cells to the drugs was less clear. Lastly, we showed that hyperthermia enhanced the number of DNA double-strand breaks (DSBs) induced by the compounds, and inhibited their repair after drug treatment. Thus, thermal enhancement of cytotoxicity may be due, at least in part, to the propensity of the enediyne moiety to induce DSBs, and/or a reduction in DSB repair efficiency. We propose that "tuning" of metalloenediyne toxicity through better-controlled reactivity could have potential clinical utility, since we envision that such compounds could be administered systemically as relatively non-toxic agents, but cytotoxicity could be enhanced in, and confined to a tumor volume when subjected to localized heating.

Inhibition of DNA Double-Strand Break Repair by Niclosamide in Human Colorectal Cancer Cells

The radiosensitizing effect of niclosamide against human rectal adenocarcinoma cells SW837 and human sigmoid colon carcinoma cells COLO 320 HSR and the underlying molecular mechanisms were investigated in this study. The survival rate of tumor cells under treatment with niclosamide, γ-radiation, and their combination was evaluated. The level of MDR1, Bcl-2, β-catenin, and γH2AX histone, a marker of DNA double-strand breaks (DSBs), was determined by flow cytometry using immunocytochemical methods. It was shown that the combined treatment with niclosamide and γ-radiation led to more intense tumor cell death than when niclosamide and γ-radiation were used separately. A decrease in the level of β-catenin was detected in the cells of the COLO 320 HSR line under treatment with niclosamide, which was more intense under combined treatment with niclosamide and irradiation. At the same time, the content of β-catenin in the cells of the SW837 line remained unchanged under treatment with niclosamide and γ-radiation and under their combined use. Niclosamide did not lead to the formation of DNA DSBs in unirradiated cells and did not increase their level 1 h after radiation exposure. The treatment of the cells with niclosamide before irradiation led to the fact that 24 h after irradiation the level of γH2AX histone, reflecting the number of DNA DSBs, was 1.6 times higher than that in intact cells and the cells exposed only to radiation. This evidences the inhibition of DNA DSB repair induced by γ-radiation. Thus, niclosamide possesses a radiosensitizing effect on human colorectal cancer cells, which is based on inhibition of DNA DSB repair. The inhibition of the Wnt/β-catenin signaling pathway under treatment with niclosamide is not detected in all types of tumor cells.

PTEN expression in U251 glioma cells enhances their sensitivity to ionizing radiation by suppressing DNA repair capacity.

Mutations in phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor gene, lead to altered sensitivity to drugs and radiation in various types of cancer. Restoring PTEN expression in tumor cells can increase radiosensitivity by inhibiting the repair of DNA double-strand breaks (DSBs). Thus, determining the mechanism of action of this protein may lead to novel therapeutic strategies. In this study, we transduced U251 cells with a lentiviral vector expressing PTEN to examine the mechanism of radiosensitization. Specifically, we examined the formation of radiation-induced DNA DSBs and apoptosis, as well as the expression of several proteins involved in repairing DSBs (p53, ataxia-telangiectasia mutated, DNA-dependent protein kinase C, Ku70-80). Our results showed that PTEN transduction sensitized U251 cells to X-rays, increasing the number of DSBs per cell and fraction of cells undergoing apoptosis. Additionally, the average size of γH2AX nuclear foci was increased following irradiation. These findings were accompanied by a PTEN-dependent irradiation-independent increase in p53 levels and decrease in phosphorylated Ku70/80 levels. Our results suggest that PTEN affects radiosensitivity by reducing DSB repair and by enhancing the p53 pathway, leading to increased apoptosis.

Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223.

Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

Highly Efficient Multiplexed Base Editing with Minimized Off-Targets for the Development of Universal CAR-T Cells to Treat Pediatric T-ALL

Autologous CAR-T therapies have demonstrated remarkable efficacy in treating some hematologic cancers. While these therapies can have substantial clinical benefit for patients, generating bespoke cell therapies creates manufacturing challenges, resulting in inconsistent products and delays in treatment that are often incompatible with effective clinical management of patients. Strategies to create universally-compatible CAR-T therapies, generated from single donors for the treatment of many patients, have been developed as a solution to these challenges, thereby reducing cost of goods, lot-to-lot variability and enabling timely treatment. Mitigating the risks of graft-versus-host-disease (GvHD) and host rejection of CAR-Ts are important components of any strategy to generate these universal therapies. Most first generation approaches utilize DNA double strand break (DSB)-inducing nucleases to ablate the expression of relevant genes in donor T cells to overcome these barriers. However, simultaneous induction of multiple DSBs results in a cell population containing genomic rearrangements, and can lead to significantly reduced cell proliferation. Approaches to develop CAR-T therapies for T cell malignancies, such as T-ALL, encounter additional challenges, including extensive fratricide caused by targeting T cell surface markers such as CD3 and CD7, which are present on both the malignant and the CAR-T cells. Resolving this issue requires additional gene editing, leading to, in the case of nuclease-based strategies, an increased number of DSBs, further genomic rearrangements, and decreased cell expansion. Base editors (BEs) are a novel class of gene editing reagents that enable programmable, single-base changes in genomic DNA without creating DSBs. Work in the Qasim lab has demonstrated proof of concept for an alternative means of producing universal, fratricide-resistant CAR-T cells for treating T cell malignancies by using cytosine base editor (CBE) technology. Here, we demonstrate a previously-unpublished CBE that produces greatly diminished off-target effects while preserving on-target activity. Using multiplex base editing, we demonstrate simultaneous knockout of four genes (TRAC, CD7, CD52, and PDCD1) with between 80-95% efficiency, producing engineered CAR-T cells with greatly diminished risk of GvHD, graft cell rejection, fratricide, and exhaustion. We show that, in contrast to nuclease editing, concurrent modification of four genetic loci using our reduced off-target CBE produces highly efficient gene knockouts with no detectable genomic rearrangements and no observable change in cell expansion compared to control conditions. T-ALL is a heterogeneous disease with variable expression of CD3 and CD7 across tumor cells in the same patient. To reduce the risk of antigen escape by tumor cells during the course of treatment with base edited CAR-Ts, we envisioned creating two independent CAR-T populations targeting CD3 (3CAR-Ts) or CD7 (7CAR-Ts). Alone or in combination, base edited 3CAR-Ts and 7CAR-Ts demonstrate robust cytokine release, potent in vitro cytotoxicity, and in vivo tumor control with antigen-positive tumor cells, and display minimal antigen-independent activity. Taken together, our approach addresses existing limitations in CAR-T cell manufacturing and demonstrates that simultaneous base editing using an improved specificity CBE at four target genes is a feasible strategy for generating universal, fratricide-resistant CAR-T cells for the potential treatment of T cell malignancies such as T-ALL. More generally, this program demonstrates the potential for base editing to create highly-engineered cell therapies featuring at least four simultaneous edits, which can confer a wide range of desirable therapeutic attributes. Disclosures Qasim: CellMedica: Research Funding; Bellicum: Research Funding; Servier: Research Funding; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Autolus: Equity Ownership.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Overall, the thyrocyte responses following exposure to β or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

1079P - Dose adjustment of chemotherapy in aggressive lymphoma using automated and standardized analysis and evaluation of DNA double strand breaks

BackgroundIn eukaryotic cells treated with chemotherapeutic drugs DNA double strand breaks (DSB) are the most severe form of DNA damage which usually leads to apoptotic cell death. Cytotoxic chemotherapy is still a mainstay of cancer therapy, but there are few established methods to individually predict efficacy and tolerability. In most regimens dosage is based on the body surface area, a dimension of little biological precision, and individual dose adaptions are usually performed by clinical guessing or in response to adverse events. This project sets out to examine biological parameters of cell damage. MethodsIn particular, we measure DNA double strand breaks by an automated yH2AX assay applying the Aklides NUK® system. In preliminary work, PBMCs were stimulated with bendamustine or with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone, the components of the R-CHOP protocol, to induce DSB. Recruitment of lymphoma patients under R-CHOP therapy and its cytotoxic effects on DSB is currently ongoing. ResultsThere was no inter-individual correlation between drug dose and number of foci, whereas does titrations on the cells of each individual donor demonstrated a clear correlation between dose and yH2AX assay result. Measurements of DSB in vivo using PBMCs of lymphoma patients is currently ongoing. First results show a rising number of DSBs in leucocytes under chemotherapy. ConclusionsThe lack of correlation across individuals between yH2AX foci and drug dosage based on standard calculation by body surface area suggests that standardized dosing of chemotherapeutic drugs based on gross physical determinants such as weight or body surface area does not correspond to the individual’s biological response and clinical effect. To confirm our hypothesis, different biomarkers should also to be involved in this project, like microRNAs and telomere length. The future plan of this project is the replacement of standard dosage regimens by biologically based, real-time-adapted individual dosage in lymphoma to reduce toxicity while increasing therapeutic efficacy. Legal entity responsible for the studyThe authors. FundingBrandenburgian Ministry of Science, Research and Culture (MWFK) Federal Ministry of Education and Research. DisclosureAll authors have declared no conflicts of interest.

Modeling gold nanoparticle radiosensitization using a clustering algorithm to quantitate DNA double-strand breaks with mixed-physics Monte Carlo simulation.

The radiosensitization properties of gold nanoparticles (GNPs) are investigated using a simple Geant4 cell model considering a realistic cell geometry and a clustering algorithm to characterize the number of DNA double-strand breaks (DSBs). A mixed-physics approach is taken for accurate modeling of low-energy photon interactions in the different regions of the model using Geant4-DNA physics within the cell, and Livermore physics within gold. Density-based spatial clustering of applications with noise (DBSCAN), a clustering algorithm, is used to directly quantitate DNA DSBs after irradiation. The simulation was run using different sizes of GNPs, different distances of GNPs from the cell nucleus, and several combinations of these two conditions. Four types of radiation were simulated in the work: 80-keV monoenergetic photons, 100-keV monoenergetic photons, a 250-kVp photon spectrum, and a 6-MV flattening filter free (FFF) photon spectrum. A variable enhancement in DSB yield, nucleus dose, and cell dose was observed when there are GNPs in the cell cytoplasm, and increases with larger GNPs and proximity to the nucleus. The distance of the GNPs from the nucleus has a large impact on the DSB yield and nucleus dose, but little to no effect on the cell dose. The cell dose enhancement factor of 80keV photons varies from 1.037-1.125 at 0.2µm for 30-100nm GNPs to 1.040-1.127 at 4μm. The DSB enhancement factor varies from 1.050 to 1.174 at 0.2µm to a marginal effect of <1.01 at 4μm. For 100keV, the dose enhancement factor is from 1.142-1.470 at 0.2µm to 1.106-1.371 at 4μm. The DSB enhancement factor varies from 1.249-1.813 at 0.2µm to almost no effect at 4μm. For 250kVp, the dose enhancement factor is from 1.117-1.393 at 0.2μm to 1.110-1.342 at 4μm. The DSB enhancement factor varies from 1.183-1.600 at 0.2μm to a marginal effect of ~1.03 at 4μm. A 6-MV FFF shows a dose enhancement factor of 1.061-1.103 at 0.2μm and 1.053-1.107 at 4μm. The DSB yield varies from 1.070-1.143 at 0.2μm to a marginal effect at 4μm. The stark difference in behavior for DSB yield when compared to cell dose highlights the importance of evaluating more complex radiobiological quantities rather than dose alone when evaluating the radiosensitization properties from metallic nanomaterials. The nucleus dose showed similar characteristics to the DSB yield demonstrating the ability of the method to predict DNA damage and its relationship with nuclear dose. The proposed method provides a way to explore the radiobiological mechanisms of radiation-induced DNA damages, and it aids to evaluate the physical radiosensitization properties of GNP-aided radiotherapy, which can be easily combined with radiochemical DSB quantitation in order to better understand the intricate DNA damage induction mechanisms that are involved in GNP-aided radiotherapy.

Quinacrine-Mediated Inhibition of Nrf2 Reverses Hypoxia-Induced 5-Fluorouracil Resistance in Colorectal Cancer.

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for the systemic treatment of colorectal cancer (CRC), but its effectiveness against CRC is limited by increased 5-FU resistance caused by the hypoxic tumor microenvironment. The purpose of our study was to assess the feasibility of using quinacrine (QC) to increase the efficacy of 5-FU against CRC cells under hypoxic conditions. QC reversed the resistance to 5-FU induced by hypoxia in CRC cell lines, as determined using ATP-Glo cell viability assays and clonogenic survival assays. Treatment of cells with 5-FU under hypoxic conditions had no effect on the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a regulator of cellular resistance to oxidative stress, whereas treatment with QC alone or in combination with 5-FU reduced Nrf2 expression in all CRC cell lines tested. Overexpression of Nrf2 effectively prevented the increase in the number of DNA double-strand breaks induced by QC alone or in combination with 5-FU. siRNA-mediated c-Jun N-terminal kinase-1 (JNK1) knockdown inhibited the QC-mediated Nrf2 degradation in CRC cells under hypoxic conditions. The treatment of CRC xenografts in mice with the combination of QC and 5-FU was more effective in suppressing tumor growth than QC or 5-FU alone. QC increases the susceptibility of CRC cells to 5-FU under hypoxic conditions by enhancing JNK1-dependent Nrf2 degradation.

Radiosensitizing effects of trabectedin on human A549 lung cancer cells and HT-29 colon cancer cells

Background and Purpose Trabectedin is a unique alkylating agent with promising effects against a range of solid tumors. In this study, we aimed to examine the cytotoxic and radiosensitizing effects of trabectedin on two human epithelial tumor cell lines in vitro, and its effects on DNA repair capacity. Methods Cancer cells (A549: human lung cancer cells, HT-29: colon cancer cells) were treated with either trabectedin alone for the determination of their growth, or in combination with radiation for the determination of their metabolic activity, proliferation, and clonogenic survival. Besides, the γH2AX foci assay was performed for the assessment of ionizing radiation-induced DNA damage and to evaluate the influence of trabectedin on DNA damage repair. Results Treatment with trabectedin resulted in a growth-inhibiting effect on both cell lines, with the IC50 values remaining within a low nanomolar range. Analyses of metabolic activity confirmed a cytotoxic influence of trabectedin and a BrdU assay demonstrated an antiproliferative effect. When combined with radiation, incubation with trabectedin was found to enhance the radiosensitivity of the tumor cells. The γH2AX foci assay resulted in an increased number of DNA double-strand breaks (DSBs) in cells treated with trabectedin. Conclusion The results of this study underline the antitumor activity of trabectedin at low nanomolar concentrations. We demonstrated that trabectedin enhanced radiation response in human lung (A549) cancer cells and colon (HT-29) cancer cells. Further studies are necessary to examine trabectedin as a potential candidate for future applications in radiotherapy.

Combined effect of alpha particles and cigarette smoke on human lung epithelial cells in vitro

Purpose: The combined toxicity of alpha particles and cigarette smoke to the critical cells in the lungs was investigated to assess the risk of smoking workers who handle naturally occurring radioactive materials.Materials and methods: The toxicity of alpha particles and cigarette smoke extract (CSE) was evaluated in terms of DNA double-strand break (DSB) induction and clonogenic cell death of human lung epithelial cells in vitro. The cells were exposed to alpha particles at doses of up to 0.25 Gy for gamma-H2AX assay and from 1.25 Gy to 5 Gy for clonogenic assay. CSE exposure of the cells was facilitated in the culture medium at CSE concentrations ranging from 1% to 12%. Additional experiments were performed using mouse endothelial cells for comparison.Results: The increases in the levels of DNA DSBs were linearly dependent on radiation dose and CSE concentration. The CSE-treated cells also responded with a linearly increasing number of DNA DSBs to the radiation dose. Both human lung epithelial cells and mouse endothelial cells showed exponential decreases in clonogenic surviving fraction as the dose from alpha particle exposure increased. Both cells responded with the clonogenic surviving fractions decreasing in a linear proportion to the CSE concentration in the culture medium.Conclusion: In our experimental in vitro setup, CSE treatment and alpha particle exposure affected the cells in an additive manner either for DNA DSB production or for clonogenic cell death induction.

The response of human mesenchymal stem cells to internal exposure to tritium β-rays.

There is no doubt that estimating the exposure risk of external and internal low-dose radiation is an imperative issue in radiobiological study. Human mesenchymal stem cells (hMSCs) are multipotent and self-renewing, supporting the regeneration of damaged tissue, including tissue damaged by radiation. However, the responses of hMSCs to internal exposure to radionuclides are still insufficiently understood. In order to evaluate the adverse effects produced by internal exposure to tritiated water (HTO) at a low dose, hMSCs were exposed to 2 × 107 Bq/ml HTO, and the biological effects after the exposure were examined. Apoptosis and DNA double-strand breaks (DSBs) were assayed to analyze the cellular response to the damage induced by HTO. Slight enhancement of apoptosis was found after treatment, except at the dose of 9 mGy. The number of DSBs at 24 h post-irradiation showed that the DNA damage was able to be efficiently repaired by the hMSCs. Moreover, the increasing proportion of the cell population in S phase proved that the persistence of residual γH2AX foci at lower concentrations of HTO was attributable to the secondary production of DSBs in DNA replication. Our work adds to the available data, helping us understand the risk of stem cell transformation due to internal exposure and its correlation with low-dose radiation–induced carcinogenesis.

QDSB-Seq is a general method for genome-wide quantification of DNA double-strand breaks using sequencing

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.

Open source bioimage informatics tools for the analysis of DNA damage and associated biomarkers

DNA double-strand breaks (DSBs) are critical cellular lesions that represent a high risk for genetic instability. DSBs initiate repair mechanisms by recruiting sensor proteins and mediators. DSB biomarkers, like phosphorylated histone 2AX (γH2AX) and p53-binding protein 1 (53BP1), are detectable by immunofluorescence as either foci or distinct patterns in the vicinity of DSBs. The correlation between the number of foci and the number of DSBs makes them a useful tool for quantification of DNA damage in precision medicine, forensics and cancer research. The quantification and characterization of foci ideally uses large cell numbers to achieve statistical validity. This requires software tools that are suitable for the analysis of large complex data sets, even for users with limited experience in digital image processing. This includes, for example, pre-processing, transformation and presentation of the data in a less complex structure for further data analysis. There are numerous software solutions for the analysis of foci. This review gives an overview of open source image processing software packages, including graphical user interfaces (GUIs) such as CellProfiler, Icy and ImageJ/Fiji. Software packages like CellProfiler and Icy enable to gain high-content information about DSB biomarkers. Programming languages, like Python, are discussed briefly.

Cross-Correlative Single-Cell Analysis Reveals Biological Mechanisms of Nanoparticle Radiosensitization.

Nanoparticle radiosensitization has been demonstrated well to enhance the effects of radiotherapy, motivate the improvement of therapeutic ratios, and decrease morbidity in cancer treatment. A significant challenge exists in optimizing formulations and translation due to insufficient knowledge of the associated mechanisms, which have historically been limited to physical concepts. Here, we investigated a concept for the role of biological mechanisms. The mere presence of gold nanoparticles led to a down-regulation of thymidylate synthase, important for DNA damage repair in the radioresistant S-phase cells. By developing a cross-correlative methodology to reveal probabilistic gold nanoparticle uptake by cell sub-populations and the associated sensitization as a function of the uptake, a number of revealing observations have been achieved. Surprisingly, for low numbers of nanoparticles, a desensitization action was observed. Sensitization was discovered to preferentially impact S-phase cells, in which impairment of the DNA damage response by the homologous recombination pathway dominates. This small but radioresistant cell population correlates with much greater proliferative ability. Thus, a paradigm is presented whereby enhanced DNA damage is not necessarily due to an increase in the number of DNA double-strand breaks (DSBs) created but can be from a nanoparticle-induced impairment of the damage response by down-regulating repair proteins such as thymidylate synthase.

Distinct contribution of Rac1 expression in cardiomyocytes to anthracycline-induced cardiac injury

Cardiotoxicity is the dose limiting adverse effect of anthracycline-based anticancer therapy. Inhibitor studies point to Rac1 as therapeutic target to prevent anthracycline-induced cardiotoxicity. Yet, supporting genetic evidence is still missing and the pathophysiological relevance of different cardiac cell types is unclear. Here, we employed a tamoxifen-inducible cardiomyocyte-specific rac1 knock-out mouse model (Rac1flox/flox/MHC-MerCreMer) to investigate the impact of Rac1 expression in cardiomyocytes on cardiac injury following doxorubicin treatment. Distinctive stress responses resulting from doxorubicin treatment were observed, including upregulation of systemic markers of inflammation (IL-6, IL-1α, MCP-1), cardiac damage (ANP, BNP), DNA damage (i.e. DNA double-strand breaks (DSB)), DNA damage response (DDR) and cell death. Measuring the acute doxorubicin response, the serum level of MCP-1 was elevated, cardiac mRNA expression of Hsp70 was reduced and cardiac DDR was specifically enhanced in Rac1 deficient mice. The frequency of apoptotic heart cells remained unaffected by Rac1. Employing a subactue model, the number of doxorubicin-induced DSB was significantly reduced if Rac1 is absent. Yet, the doxorubicin-triggered increase in serum ANP and BNP levels remained unaffected by Rac1. Overall, knock-out of rac1 in cardiomyocytes confers partial protection against doxorubicin-induced cardiac injury. Hence, the data provide first genetic evidence supporting the view that pharmacological targeting of Rac1 is useful to widen the therapeutic window of anthracycline-based anticancer therapy by alleviating acute/subacute cardiomyocyte damage. Furthermore, considering published data obtained from the use of pharmacological Rac1 inhibitors, the results of our study indicate that Rac1-regulated functions of cardiac cell types others than cardiomyocytes additionally influence the adverse outcomes of anthracycline treatment on the heart.