Cs-131 as an experimental tool for the investigation and quantification of the radiotoxicity of intracellular Auger decays in vitro.

PURPOSE: In this work, we set out to provide an experimental setup, using Cs-131, with associated dosimetry for studying relative biological effectiveness (RBE) of Auger emitters. MATERIAL AND METHODS: Cs-131 decays by 100% electron capture producing K- (9%) and L- (80%) Auger electrons with mean energies of 26 keV and 3.5 keV, respectively, plus ≈ 9.4 very low energy electrons (<0.5 keV) per decay. Cs-131 accumulates in the cells through the Na+/K+-ATPase. By this uptake mechanism and the alkali chemistry of Cs+, we argue for its intracellular homogeneous distribution. Cs-131 was added to the cell culture medium of HeLa and V79 Cells. The bio-kinetics of Cs-131 (uptake, release, intracellular distribution) was examined by measuring its intracellular activity concentration over time. Taking advantage of the 100% confluent cellular monolayer, we developed a new and robust dosimetry that is entrusted to a quantity called SC-value. RESULTS: The SC-values evaluated in the cell nucleus are almost independent of the nuclear size and geometry. We obtained dose-rate controlled RBE-values for intracellular Cs-131 decay. Using the γH2AX assay, the RBE was 1 for HeLa cells. Using the clonogenic cell survival, it was 3.9 for HeLa cells and 3.2 for V79 cells. CONCLUSION: This experimental setup and dosimetry provides reliable RBE-values for Auger emitters in various cell lines.

Cell colony counter called CoCoNut.

Clonogenic assays are powerful tools for testing cell reproductive death after biological damage caused by, for example, ionizing radiation. Traditionally, the methods require a cumbersome, slow and eye-straining manual counting of viable colonies under a microscope. To speed up the counting process and minimize those issues related to the subjective decisions of the scoring personnel, we developed a semi-automated, image-based cell colony counting setup, named CoCoNut (Colony Counter developed by the Nutech department at the Technical University of Denmark). It consists in an ImageJ macro and a photographic 3D-printed light-box, conceived and demonstrated to work together for Crystal Violet-stained colonies. Careful attention was given to the image acquisition process, which allows background removal (i.e. any unwanted element in the picture) in a minimally invasive manner. This is mainly achieved by optimal lighting conditions in the light-box and dividing the image of a flask that contains viable colonies by the picture of an empty flask. In this way, CoCoNut avoids using aggressive background removal filters that usually lead to suboptimal colony count recovery. The full method was tested with V79 and HeLa cell survival samples. Results were compared to other freely available tools. CoCoNut proved able to successfully distinguish between single and merged colonies and to identify colonies bordering on flask edges. CoCoNut software calibration is fast; it requires the adjustment of a single parameter that is the smallest colony area to be counted. The employment of a single parameter reduces the risk of subjectivity, providing a robust and user-friendly tool, whose results can be easily compared over time and among different bio-laboratories. The method is inexpensive and easy to obtain. Among its advantages, we highlight the possibility of combining the macro with a perfectly reproducible 3D-printed light-box. The CoCoNut software and the 3D-printer files are provided as supporting information (S1 CoCoNut Files).

Radiobiological effects of tritiated water short-term exposure on V79 clonogenic cell survival.

PURPOSE: We set out to improve the accuracy of absorbed dose calculations for in vitro measurements of the relative biological effectiveness (RBE) of tritiated water (HTO) for the clonogenic cell survival assay, also considering the influence of the end-of-track linear energy transfer (LET) of low-energy electrons. MATERIALS AND METHODS: The COmputation Of Local Electron Release (COOLER) program was adopted to investigate the cell geometry and the tritium full beta-decay spectrum impact on the S-values and subsequently on the RBE of HTO for clonogenic cell survival at similar high dose rates (HDR). RESULTS: S-values for cells growing in suspension are usually comparable to those for adherent cells. RBEs calculated at the 10% survival fraction through the use of the average energy are almost similar to those obtained with the beta-spectrum. For adherent cells, an RBE of 1.6 was found when HTO cell survival curves were compared to acute γ-ray exposures. Irrespective of the geometrical configuration, the RBE was 2.0 when the comparison was made with similar dose rates. CONCLUSIONS: These results underline the importance of irradiating at equal dose rates and cell culture conditions when measuring in vitro RBE-values.

The COOLER Code: A Novel Analytical Approach to Calculate Subcellular Energy Deposition by Internal Electron Emitters.

COmputation Of Local Electron Release (COOLER), a software program has been designed for dosimetry assessment at the cellular/subcellular scale, with a given distribution of administered low-energy electron-emitting radionuclides in cellular compartments, which remains a critical step in risk/benefit analysis for advancements in internal radiotherapy. The software is intended to overcome the main limitations of the medical internal radiation dose (MIRD) formalism for calculations of cellular S-values (i.e., dose to a target region in the cell per decay in a given source region), namely, the use of the continuous slowing down approximation (CSDA) and the assumption of a spherical cell geometry. To this aim, we developed an analytical approach, entrusted to a MATLAB-based program, using as input simulated data for electron spatial energy deposition directly derived from full Monte Carlo track structure calculations with PARTRAC. Results from PARTRAC calculations on electron range, stopping power and residual energy versus traveled distance curves are presented and, when useful for implementation in COOLER, analytical fit functions are given. Example configurations for cells in different culture conditions (V79 cells in suspension or adherent culture) with realistic geometrical parameters are implemented for use in the tool. Finally, cellular S-value predictions by the newly developed code are presented for different cellular geometries and activity distributions (uniform activity in the nucleus, in the entire cell or on the cell surface), validated against full Monte Carlo calculations with PARTRAC, and compared to MIRD standards, as well as results based on different track structure calculations (Geant4-DNA). The largest discrepancies between COOLER and MIRD predictions were generally found for electrons between 25 and 30 keV, where the magnitude of disagreement in S-values can vary from 50 to 100%, depending on the activity distribution. In calculations for activity distribution on the cell surface, MIRD predictions appeared to fail the most. The proposed method is suitable for Auger-cascade electrons, but can be extended to any energy of interest and to beta spectra; as an example, the 3H case is also discussed. COOLER is intended to be accessible to everyone (preclinical and clinical researchers included), and may provide important information for the selection of radionuclides, the interpretation of radiobiological or preclinical results, and the general establishment of doses in any scenario, e.g., with cultured cells in the laboratory or with therapeutic or diagnostic applications. The software will be made available for download from the DTU-Nutech website: http://www.nutech.dtu.dk/ .

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.

NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability.

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Inference of causal networks from time-varying transcriptome data via sparse coding.

Temporal analysis of genome-wide data can provide insights into the underlying mechanism of the biological processes in two ways. First, grouping the temporal data provides a richer, more robust representation of the underlying processes that are co-regulated. The net result is a significant dimensional reduction of the genome-wide array data into a smaller set of vocabularies for bioinformatics analysis. Second, the computed set of time-course vocabularies can be interrogated for a potential causal network that can shed light on the underlying interactions. The method is coupled with an experiment for investigating responses to high doses of ionizing radiation with and without a small priming dose. From a computational perspective, inference of a causal network can rapidly become computationally intractable with the increasing number of variables. Additionally, from a bioinformatics perspective, larger networks always hinder interpretation. Therefore, our method focuses on inferring the simplest network that is computationally tractable and interpretable. The method first reduces the number of temporal variables through consensus clustering to reveal a small set of temporal templates. It then enforces simplicity in the network configuration through the sparsity constraint, which is further regularized by requiring continuity between consecutive time points. We present intermediate results for each computational step, and apply our method to a time-course transcriptome dataset for a cell line receiving a challenge dose of ionizing radiation with and without a prior priming dose. Our analyses indicate that (i) the priming dose increases the diversity of the computed templates (e.g., diversity of transcriptome signatures); thus, increasing the network complexity; (ii) as a result of the priming dose, there are a number of unique templates with delayed and oscillatory profiles; and (iii) radiation-induced stress responses are enriched through pathway and subnetwork studies.

Persistence of γ-H2AX and 53BP1 foci in proliferating and non-proliferating human mammary epithelial cells after exposure to γ-rays or iron ions.

PURPOSE: To investigate γ-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionising radiation under different cell culture conditions. MATERIAL AND METHODS: HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. RESULTS: Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced γ-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both γ-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after γ-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. CONCLUSIONS: The disappearance of radiation-induced γ-H2AX and 53BP1 foci in HMEC has different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent γ-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double-strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodelling.

Lack of bystander effects from high-LET radiation for early cytogenetic end points.

The aim of this work was to study radiation-induced bystander effects for early cytogenetic end points in various cell lines using the medium transfer technique after exposure to high- and low-LET radiation. Cells were exposed to 20 MeV/ nucleon nitrogen ions, 968 MeV/nucleon iron ions, or 575 MeV/nucleon iron ions followed by transfer of the conditioned medium from the irradiated cells to unirradiated test cells. The effects studied included DNA double-strand break induction, gamma-H2AX focus formation, induction of chromatid breaks in prematurely condensed chromosomes, and micronucleus formation using DNA repair-proficient and -deficient hamster and human cell lines (xrs6, V79, SW48, MO59K and MO59J). Cell survival was also measured in SW48 bystander cells using X rays. Although it was occasionally possible to detect an increase in chromatid break levels using nitrogen ions and to see a higher number of gamma-H2AX foci using nitrogen and iron ions in xrs6 bystander cells in single experiments, the results were not reproducible. After we pooled all the data, we could not verify a significant bystander effect for any of these end points. Also, we did not detect a significant bystander effect for DSB induction or micronucleus formation in these cell lines or for clonogenic survival in SW48 cells. The data suggest that DNA damage and cytogenetic changes are not induced in bystander cells. In contrast, data in the literature show pronounced bystander effects in a variety of cell lines, including clonogenic survival in SW48 cells and induction of chromatid breaks and micronuclei in hamster cells. To reconcile these conflicting data, it is possible that the epigenetic status of the specific cell line or the precise culture conditions and medium supplements, such as serum, may be critical for inducing bystander effects.

Relative biological effectiveness of high-energy iron ions for micronucleus formation at low doses.

Dose-response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80(-)) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05-1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/microm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 +/- 0.8 (LET = 151 keV/microm), 4.3 +/- 0.5 (LET = 176 keV/microm), and 5.7 +/- 0.6 (LET = 235 keV/microm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 +/- 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.

Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement.

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.