Quantification Of DNA Double-strand Breaks Research Articles

Discovery of novel DNA-damaging agents through phenotypic screening for DNA double-strand break.

DNA double-strand breaks (DSBs) seriously damage DNA and promote genomic instability that can lead to cell death. They are the source of conditions such as carcinogenesis and aging, but also have important applications in cancer therapy. Therefore, rapid detection and quantification of DSBs in cells are necessary for identifying carcinogenic and anticancer factors. In this study, we detected DSBs using a flow cytometry-based high-throughput method to analyze γH2AX intensity. We screened a chemical library containing 9600 compounds and detected multiple DNA-damaging compounds, although we could not identify mechanisms of action through this procedure. Thus, we also profiled a representative compound with the highest DSB potential, DNA-damaging agent-1 (DDA-1), using a bioinformatics-based method we termed "molecular profiling." Prediction and verification analysis revealed DDA-1 as a potential inhibitor of topoisomerase IIα, different from known inhibitors such as etoposide and doxorubicin. Additional investigation of DDA-1 analogs and xenograft models suggested that DDA-1 is a potential anticancer drug. In conclusion, our findings established that combining high-throughput DSB detection and molecular profiling to undertake phenotypic analysis is a viable method for efficient identification of novel DNA-damaging compounds for clinical applications.

Experimental Setup for Irradiation of Cell Cultures at L2A2

Laser–plasma proton sources and their applications to preclinical research has become a very active field of research in recent years. In addition to their small dimensions as compared to classical ion accelerators, they offer the possibility to study the biological effects of ultra-short particle bunches and the correspondingly high dose rates. We report on the design of an experimental setup for the irradiation of cell cultures at the L2A2 laboratory at the University of Santiago de Compostela, making use of a 1.2 J Ti: Sapphire laser with a 10 Hz repetition rate. Our setup comprises a proton energy separator consisting of two antiparallel magnetic fields realized by a set of permanent magnets. It allows for selecting a narrow energy window around an adaptable design value of 5 MeV out of the initially broad spectrum typical for Target Normal Sheath Acceleration (TNSA). At the same time, unwanted electrons and X-rays are segregated from the protons. This part of the setup is located inside the target vessel of the L2A2 laser. A subsequent vacuum flange sealed with a thin kapton window allows for particle passage to external sample irradiation. A combination of passive detector materials and real-time monitors is applied for measurement of the deposited radiation dose. A critical point of this interdisciplinary project is the manipulation of biological samples under well-controlled, sterile conditions. Cell cultures are prepared in sealed flasks with an ultra-thin entrance window and analysed at the nearby Fundación Pública Galega Medicina Xenómica and IDIS. The first trials will be centred at the quantification of DNA double-strand breaks as a function of radiation dose.

Considering Cell Proliferation to Optimize Detection of Radiation-Induced 53BP1 Positive Foci in 15 Mouse Strains Ex Vivo.

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double-strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1 (53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of their proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 72 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ∼10-fold decrease from low to full confluence that is independent of mouse strain. On the other hand, the baseline level of spontaneous 53BP1+ foci in a fully confluent cell population was strain-dependent, suggesting genomic associations, and correlated with radiation sensitivity based on previous measurements in the same cell lines. Finally, we have developed an online open-access tool to correct for the effect of cell confluence on 53BP1+ foci-based quantification of DSB. This tool provides guidelines for the number of cells required to reach statistical significance for the detection of DSB induced by low doses of ionizing radiation as a function of confluence and time postirradiation.

Accumulation of DNA Damage and Alteration of the DNA Damage Response in Chronic Myeloid Leukemia

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

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.

DNA damage in blood leucocytes of prostate cancer patients during therapy with 177Lu-PSMA

PurposeThe aim of this study was to investigate the time- and dose-dependency of DNA double-strand break (DSB) induction and repair in peripheral blood leucocytes of prostate cancer patients during therapy with 177Lu-PSMA.MethodsBlood samples from 16 prostate cancer patients receiving their first 177Lu-PSMA therapy were taken before and at seven time-points (between 1 h and 96 h) after radionuclide administration. Absorbed doses to the blood were calculated using integrated time–activity curves of the blood and the whole-body. For DSB quantification, leucocytes were isolated, fixed in ethanol and immunostained with γ-H2AX and 53BP1 antibodies. Colocalizing foci of both DSB markers were manually counted in a fluorescence microscope.ResultsThe average number of radiation-induced foci (RIF) per cell increased within the first 4 h after administration, followed by a decrease indicating DNA repair. The number of RIF during the first 2.6 h correlated linearly with the absorbed dose to the blood (R2 = 0.58), in good agreement with previously published in-vitro data. At late time-points (48 h and 96 h after administration), the number of RIF correlated linearly with the absorbed dose rate (R2 = 0.56). In most patients, DNA DSBs were repaired effectively. However, in some patients RIF did not disappear completely even 96 h after administration.ConclusionThe general pattern of the time- and dose-dependent induction and disappearance of RIF during 177Lu-PSMA therapy is similar to that of other radionuclide therapies.

Detection of DNA Double-Strand Breaks Using Pulsed-Field Gel Electrophoresis.

DNA is one of the most biologically important targets of exogenous and endogenous toxicants as well as carcinogens. Damage to DNA can be of different types (e.g., DNA adducts, DNA protein cross-links, single-strand breaks, oxidized bases, abasic sites, and double-strand breaks (DSBs)). DSBs are considered the most lethal form of DNA damage for eukaryotic cells, and if left unrepaired or misrepaired, can cause cell death, chromosome instability, and cancer. DSBs can arise in the cells through different sources and can be distinguished as endogenous or exogenous DSBs. Exogenous sources can be chemotherapeutic drugs, irradiation, and environmental chemicals. The endogenous causes of DNA DSBs in the cells are mainly reactive oxygen species and faulty repair of oxidative clustered DNA lesions. Qualitative and quantitative analysis of DNA DSBs is of utmost importance to understand physiologically relevant cellular processes as well as to investigate the genotoxic or clastogenic effects of toxicants. Pulsed-field gel electrophoresis (PFGE) is a widely used method for direct quantification of DNA DSBs. In this method, the cells exposed to DSB-inducing agents are embedded in the agarose blocks and lysed. These agarose blocks containing DNA are then run under multiple electric fields which are at 120° angle, to aid in the movement of large DNA strands. It gives a direct and specific measure of DSBs unlike the foci-based assays. This chapter provides a brief overview of the various commonly used approaches to analyze DNA DSBs and describes the theory, advantages and method of PFGE, for use in cells exposed to DNA DSB inducing agents.

A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor

To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([CuII(phen)2]2+), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [CuII(phen)2]2+, a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [CuII(phen)2]2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [CuI(phen)2]+, with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [CuII(phen)2]2+.

Quantification of DNA double-strand breaks in peripheral blood mononuclear cells from healthy donors exposed to bendamustine by an automated γH2AX assay—an exploratory study

Background: DNA double strand breaks (DSBs) are the most severe form of DNA damage in eukaryotic cells treated with ionizing radiation or chemotherapeutic drugs. They can be quantitatively assessed by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX), where the number of γH2AX foci represents the number of DNA DSB. Real-time assessment of DSB could help tailoring cytotoxic therapies to individual patients regarding both response and adverse events. This would require reliable automated quantification technology not yet routinely available. Here we explore this concept in the context of malignant lymphoma. Methods: To investigate the DSB response to cytotoxic treatment in vitro , peripheral lymphocytes of healthy donors were incubated with bendamustine, an alkylating drug commonly used in lymphoma therapy. To mimic the clinical setting, the drug concentration per number of donor cells was either calculated as a standard dose or based on the body surface area of the individual donor. DNA DSB were quantified by an automatized immunofluorescence γH2AX assay using the AKLIDES NUK ® system. Results: Across all donors, the mean number of γH2AX foci per cell was 1.29, IQR (1.08) after bendamustine treatment as opposed to 0.04, IQR (0.125) in untreated freshly isolated peripheral blood mononuclear cells (PBMCs). The standardized incubation dosage resulted in a mean of 0.89, IQR (0.51) foci per cell, while individualized dose calculation yielded 1.57, IQR (0.5) foci per cell. The difference in γH2AX foci between the two dosage calculations was significant (P=0.036). In addition, we observed a trend towards a negative correlation between the donors’ body surface area and the number of foci per cell. Between donors, no significant correlation of the number of foci in response to a given dose was observed. Dose titrations on the cells of individual donors demonstrated a significant response (P Conclusions: The automatic AKLIDES analysis platform can assess γH2AX foci for routine use. The individual γH2AX foci response to in vitro bendamustine is dose-dependent and can be monitored timely. Calculating individual in vitro dosage from the donor’s body surface area resulted in a broad variation of foci counts between individuals, suggesting that this dosage method does not result in equivalent biological effects among different individuals. Adjusting the dose individually based on biological responses such as DNA DSB could offer a way of personalized medicine with conventional substances, reducing toxicity while increasing therapeutic efficacy.

Abstract ID: 75 Validating Geant4-DNA for Double Strand Brakes (DSB): A preliminary study

The investigation of biological effects in living tissues requires the modeling of physical, chemical, and physico-chemical interactions. Strong effort has been given in the 3D modeling of DNA structure, such as to describe the cell nucleus. In 2015, a new application, Geant4-DNA was presented, enabling the atomic level description of DNA molecules and the possibility of evaluating the direct damage induced on the DNA molecule by ionizing radiation [1] . Recently, a DNA Double-Strand Break (DSB) dosimeter was developed for the detection and quantification of DNA-DSBs after irradiation [2] . Our goal is to validate the simulated data, and standardize the experimental procedure for measuring the biological damage. In the present study, we investigated the PDB4DNA tool and developed a new G4-DNA class for the quantification of DSBs after irradiation. Experimental measures were used for testing the G4-DNA classes. A DNA molecule (1329 bp, the largest in PDBlib) was irradiated with 3 different absorbed doses equal to 10, 25 and 50 Gy. In our simulation study, we used a structure of a single DNA molecule (based on experimental data), and irradiated it multiple times to quantify the probability of DSBs. The simulations, on G4-DNA DSB quantification, resulted in DSB probabilities equal to 9.0%, 17.0%, and 31% for doses 10 Gy, 25 Gy, and 50 Gy respectively. Thus, the statistical difference between experimental and simulated DSBs was calculated to 8.09%, 9.23%, and 13.43% accordingly. Comparing our simulations with the standard used PDB4DNA class, differences varied from 40–70%. This is a preliminary study for the modeling, and the quantification of DNA-DSBs based on experimental data. The limitations of this study need to be addressed, such as the modeling of multiple DNA molecules for accurate statistical simulations, and chemical reactions need to be taken into account for the total quantification of the biological damage on DNA structures.

BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Visual quantification of DNA double-strand breaks in bacteria

In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.