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.

Interphase Cytogenetic Analysis of Micronucleated and Multinucleated Cells Supports the Premature Chromosome Condensation Hypothesis as the Mechanistic Origin of Chromothripsis.

The discovery of chromothripsis in cancer genomes challenges the long-standing concept of carcinogenesis as the result of progressive genetic events. Despite recent advances in describing chromothripsis, its mechanistic origin remains elusive. The prevailing conception is that it arises from a massive accumulation of fragmented DNA inside micronuclei (MN), whose defective nuclear envelope ruptures or leads to aberrant DNA replication, before main nuclei enter mitosis. An alternative hypothesis is that the premature chromosome condensation (PCC) dynamics in asynchronous micronucleated cells underlie chromosome shattering in a single catastrophic event, a hallmark of chromothripsis. Specifically, when main nuclei enter mitosis, premature chromatin condensation provokes the shattering of chromosomes entrapped inside MN, if they are still undergoing DNA replication. To test this hypothesis, the agent RO-3306, a selective ATP-competitive inhibitor of CDK1 that promotes cell cycle arrest at the G2/M boundary, was used in this study to control the degree of cell cycle asynchrony between main nuclei and MN. By delaying the entrance of main nuclei into mitosis, additional time was allowed for the completion of DNA replication and duplication of chromosomes inside MN. We performed interphase cytogenetic analysis using asynchronous micronucleated cells generated by exposure of human lymphocytes to γ-rays, and heterophasic multinucleated Chinese hamster ovary (CHO) cells generated by cell fusion procedures. Our results demonstrate that the PCC dynamics during asynchronous mitosis in micronucleated or multinucleated cells are an important determinant of chromosome shattering and may underlie the mechanistic origin of chromothripsis.

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.

Development of an automatable micro-PCC biodosimetry assay for rapid individualized risk assessment in large-scale radiological emergencies.

In radiation accidents and large-scale radiological emergencies, a fast and reliable triage of individuals according to their degree of exposure is important for accident management and identification of those who need medical assistance. In this work, the applicability of cell-fusion-mediated premature chromosome condensation (PCC) in G0-lymphocytes is examined for the development of a rapid, minimally invasive and automatable micro-PCC assay, which requires blood volumes of only 100 µl and can be performed in 96-well plates, towards risk assessments and categorization of individuals based on dose estimates. Chromosomal aberrations are visualized for dose-estimation analysis within two hours, without the need of blood culturing for two days, as required by conventional cytogenetics. The various steps of the standard-PCC procedure were adapted and, for the first time, lymphocytes in blood volumes of 100 µl were successfully fused with CHO-mitotics in 96-well plates of 2 ml/well. The plates are advantageous for high-throughput analysis since the various steps required are applied to all 96-wells simultaneously. Interestingly, the use of only 1.5 ml hypotonic and Carnoy's fixative per well offers high quality PCC-images, and the morphology of lymphocyte PCCs is identical to that obtained using the conventional PCC-assay, which requires much larger blood volumes and 15 ml tubes. For dose assessments, appropriate calibration curves were constructed and for PCC analysis specialized software (MetaSystems) was used. The micro-PCC assay can be combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes. This allows dose assessments on the basis of accurate scoring of dicentric and centric ring chromosomes in G0-lymphocyte PCCs, which is particularly helpful when further evaluation into treatment-level categories of exposed individuals is needed. The micro-PCC assay has significant advantages for early triage biodosimetry when compared to other cytogenetic biodosimetry assays. It is rapid, cost-effective, and could pave the way to its subsequent automation.

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.

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.