G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells.

While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.

Interphase Cytogenetic Analysis of G0 Lymphocytes Exposed to α-Particles, C-Ions, and Protons Reveals their Enhanced Effectiveness for Localized Chromosome Shattering-A Critical Risk for Chromothripsis.

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by χ- or γ-rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by γ-rays; and the RBE values obtained are up to 28.6 for α-particles (92 keV/µm), 10.5 for C-ions (295 keV/µm), and 4.9 for protons (28.5 keV/µm). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event-posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements.

Chromosome breaks generated by low doses of ionizing radiation in G2-phase are processed exclusively by gene conversion.

Four repair pathways process DNA double-strand breaks (DSBs). Among these pathways the homologous recombination repair (HRR) subpathway of gene conversion (GC) affords error-free processing, but functions only in S- and G2-phases of the cell cycle. Classical non-homologous end-joining (c-NHEJ) operates throughout the cell cycle, but causes small deletions and translocations. Similar deficiencies in exaggerated form, combined with reduced efficiency, are associated with alternative end-joining (alt-EJ). Finally, single-strand annealing (SSA) causes large deletions and possibly translocations. Thus, processing of a DSB by any pathway, except GC, poses significant risks to the genome, making the mechanisms navigating pathway-engagement critical to genome stability. Logically, the cell ought to attempt engagement of the pathway ensuring preservation of the genome, while accommodating necessities generated by the types of DSBs induced. Thereby, inception of DNA end-resection will be key determinant for GC, SSA and alt-EJ engagement. We reported that during G2-phase, where all pathways are active, GC engages in the processing of almost 50 % of DSBs, at low DSB-loads in the genome, and that this contribution rapidly drops to nearly zero with increasing DSB-loads. At the transition between these two extremes, SSA and alt-EJ compensate, but at extremely high DSB-loads resection-dependent pathways are suppressed and c-NHEJ remains mainly active. We inquired whether in this processing framework all DSBs have similar fates. Here, we analyze in G2-phase the processing of a subset of DSBs defined by their ability to break chromosomes. Our results reveal an absolute requirement for GC in the processing of chromatid breaks at doses in the range of 1 Gy. Defects in c-NHEJ delay significantly the inception of processing by GC, but leave processing kinetics unchanged. These results delineate the essential role of GC in chromatid break repair before mitosis and classify DSBs that underpin this breakage as the exclusive substrate of GC.

Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects.

A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3-4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6-8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.

The lysine-specific methyltransferase KMT2C/MLL3 regulates DNA repair components in cancer.

Genome-wide studies in tumor cells have indicated that chromatin-modifying proteins are commonly mutated in human cancers. The lysine-specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination-mediated double-strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

Capabilities of the RENEB network for research and large scale radiological and nuclear emergency situations.

PURPOSE: To identify and assess, among the participants in the RENEB (Realizing the European Network of Biodosimetry) project, the emergency preparedness, response capabilities and resources that can be deployed in the event of a radiological or nuclear accident/incident affecting a large number of individuals. These capabilities include available biodosimetry techniques, infrastructure, human resources (existing trained staff), financial and organizational resources (including the role of national contact points and their articulation with other stakeholders in emergency response) as well as robust quality control/assurance systems. MATERIALS AND METHODS: A survey was prepared and sent to the RENEB partners in order to acquire information about the existing, operational techniques and infrastructure in the laboratories of the different RENEB countries and to assess the capacity of response in the event of radiological or nuclear accident involving mass casualties. The survey focused on several main areas: laboratory's general information, country and staff involved in biological and physical dosimetry; retrospective assays used, the number of assays available per laboratory and other information related to biodosimetry and emergency preparedness. Following technical intercomparisons amongst RENEB members, an update of the survey was performed one year later concerning the staff and the available assays. CONCLUSIONS: The analysis of RENEB questionnaires allowed a detailed assessment of existing capacity of the RENEB network to respond to nuclear and radiological emergencies. This highlighted the key importance of international cooperation in order to guarantee an effective and timely response in the event of radiological or nuclear accidents involving a considerable number of casualties. The deployment of the scientific and technical capabilities existing within the RENEB network members seems mandatory, to help other countries with less or no capacity for biological or physical dosimetry, or countries overwhelmed in case of a radiological or nuclear accident involving a large number of individuals.

RENEB accident simulation exercise.

PURPOSE: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. MATERIALS AND METHODS: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. RESULTS: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). CONCLUSIONS: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.

The harmonization process to set up and maintain an operational biological and physical retrospective dosimetry network: QA QM applied to the RENEB network.

PURPOSE: The European Network of Biological and Physical Retrospective Dosimetry 'RENEB' has contributed to European radiation emergency preparedness. To give homogeneous dose estimation results, RENEB partners must harmonize their processes. MATERIALS AND METHODS: A first inter-comparison focused on biological and physical dosimetry was used to detect the outliers in terms of dose estimation. Subsequently, trainings were organized to improve both tools dose estimation. A second inter-comparison was performed to validate training efficiency. Simultaneously, based on ISO standards, a QA&QM manual on all dosimetry assays was produced which states a common basis and harmonized procedures for each assay. The evaluation of the agreement of RENEB partners to follow the QA&QM manual was performed through a questionnaire. The integration of new members into the network was carried out in the same way, whatever the assays. RESULTS: The training courses on biological and physical dosimetry were judged to be successful because most of the RENEB members' dose estimates improved in the second inter-comparison. The QA&QM manual describes the consensus for the minimum requirements and the performance criteria for both dosimetry assays. The questionnaire revealed that the whole network capacity currently can manage between 15 and 3800 samples once. CONCLUSION: The methodology used to harmonize all dosimetry practice within the network RENEB was highly successful. The network is operational to manage a mass casualty radiation accident for immediate dose assessment.

Web based scoring is useful for validation and harmonisation of scoring criteria within RENEB.

PURPOSE: To establish a training data set of digital images and to investigate the scoring criteria and dose assessment of the dicentric assay within the European network of biodosimetry (RENEB), a web based scoring inter-comparison was undertaken by 17 RENEB partners. MATERIALS AND METHODS: Two sets of 50 high resolution images were uploaded onto the RENEB website. One set included metaphases after a moderate exposure (1.3 Gy) and the other set consisted of metaphases after a high dose exposure (3.5 Gy). The laboratories used their own calibration curves for estimating doses based on observed aberration frequencies. RESULTS: The dose estimations and 95% confidence limits were compared to the actual doses and the corresponding z-values were satisfactory for the majority; only the dose estimations from two laboratories were too low or too high. The coefficients of variation were 17.6% for the moderate and 11.2% for the high dose. Metaphases with controversial results could be identified for training purposes. CONCLUSIONS: Overall, the web based scoring of the two galleries by the 17 laboratories produced very good results. Application of web based scoring for the dicentric assay may therefore be a relevant strategy for an operational biodosimetry assistance network.

RENEB - Running the European Network of biological dosimetry and physical retrospective dosimetry.

PURPOSE: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. RESULTS: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. CONCLUSIONS: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay).

PURPOSE: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. MATERIALS AND METHODS: Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. RESULTS: Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. CONCLUSIONS: Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.

Biodosimetry for High-Dose Exposures Based on Dicentric Analysis in Lymphocytes Released from the G2-Block by Caffeine.

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridization technique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.

Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells.

Ionizing radiation (IR) induces double strand breaks (DSBs) in cellular DNA, which if not repaired correctly can cause chromosome translocations leading to cell death or cancer. Incorrect joining of DNA ends generating chromosome translocations can be catalyzed either by the dominant DNA-PKcs-dependent, classical non-homologous end-joining (c-NHEJ), or by an alternative end-joining (alt-EJ) process, functioning as backup to abrogated c-NHEJ, or homologous recombination repair. Alt-EJ operates with slower kinetics as compared to c-NHEJ and generates larger alterations at the junctions; it is also considered crucial to chromosome translocation-formation. A recent report posits that this view only holds for rodent cells and that in human cells c-NHEJ is the main mechanism of chromosome translocation formation. Since this report uses designer nucleases that induce DSBs with unique characteristics in specific genomic locations and PCR to detect translocations, we revisit the issue using stochastically distributed DSBs induced in the human genome by IR during the G2-phase of the cell cycle. For visualization and analysis of chromosome translocations, which manifest as chromatid translocations in cells irradiated in G2, we employ classical cytogenetics. In wild-type cells, we observe a significant contribution of alt-EJ to translocation formation, as demonstrated by a yield-reduction after treatment with inhibitors of Parp, or of DNA ligases 1 and 3 (Lig1, Lig3). Notably, a nearly fourfold increase in translocation formation is seen in c-NHEJ mutants with defects in DNA ligase 4 (Lig4) that remain largely sensitive to inhibitors of Parp, and of Lig1/Lig3. We conclude that similar to rodent cells, chromosome translocation formation from randomly induced DSBs in human cells largely relies on alt-EJ. We discuss DSB localization in the genome, characteristics of the DSB and the cell cycle as potential causes of the divergent results generated with IR and designer nucleases.

Low concentrations of caffeine induce asymmetric cell division as observed in vitro by means of the CBMN-assay and iFISH.

The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism unrelated to the formation of chromatid breaks. The cytogenetic approach used, combining CBMN with iFISH, is proposed as a valuable tool to test chemically induced asymmetric cell divisions.

Triage biodosimetry using centromeric/telomeric PNA probes and Giemsa staining to score dicentrics or excess fragments in non-stimulated lymphocyte prematurely condensed chromosomes.

The frequency of dicentric chromosomes in human peripheral blood lymphocytes at metaphase is considered as the "gold-standard" method for biological dosimetry and, presently, it is the most widely used for dose assessment. Yet, it needs lymphocyte stimulation and a 2-day culture, failing the requirement of rapid dose estimation, which is a high priority in radiation emergency medicine and triage biodosimetry. In the present work, we assess the applicability of cell fusion mediated premature chromosome condensation (PCC) methodology, which enables the analysis of radiation-induced chromosomal aberrations directly in non-stimulated G0-lymphocytes, without the 2-day culture delay. Despite its advantages, quantification of an exposure by means of the PCC-method is not currently widely used, mainly because Giemsa-staining of interphase G0-lymphocyte chromosomes facilitates the analysis of fragments and rings, but not of dicentrics. To overcome this shortcoming, the PCC-method is combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric peptide nucleic acid (PNA)-probes. This new approach enables an accurate analysis of dicentric and centric ring chromosomes, which are formed within 8h post irradiation and will, therefore, be present in the blood sample by the time it arrives for dose estimation. For triage biodosimetry, a dose response curve for up to 10Gy was constructed and compared to that obtained using conventional metaphase analysis with Giemsa or centromeric/telomeric PNA-probes in metaphase. Since FISH is labor intensive, a simple PCC-method scoring Giemsa-stained fragments in excess of 46 was also assessed as an even more rapid approach for triage biodosimetry. First, we studied the rejoining kinetics of fragments and constructed a dose-response curve for 24h repair time. Then, its applicability was assessed for four different doses and compared with the PCC-method using centromeric/telomeric PNA-probes, through the evaluation of speed of analysis and minimum number of cells required for dose estimation and categorization of exposed individuals.

Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome condensation induces mechanical stress and triggers shattering and chromothripsis in chromosomes or chromosome arms still undergoing DNA replication or repair in micronuclei or asynchronous multinucleate cells, when primary nuclei enter mitosis.

Detection and automated scoring of dicentric chromosomes in nonstimulated lymphocyte prematurely condensed chromosomes after telomere and centromere staining.

PURPOSE: To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose-response curve and automation of the process. METHODS AND MATERIALS: Blood samples from healthy donors were exposed to (60)Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. RESULTS: We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose-response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. CONCLUSION: The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.

Non-targeted radiation effects in vivo: a critical glance of the future in radiobiology.

Radiation-induced bystander effects (RIBE), demonstrate the induction of biological non-targeted effects in cells which have not directly hit by radiation or by free radicals produced by ionization events. Although RIBE have been demonstrated using a variety of biological endpoints the mechanism(s) of this phenomenon still remain unclear. The controversial results of the in vitro RIBE and the evidence of non-targeted effects in various in vivo systems are discussed. The experimental evidence on RIBE, indicate that a more analytical and mechanistic in depth approach is needed to secure an answer to one of the most intriguing questions in radiobiology.

Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining.

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors.