Multiwell-based G0-PCC assay for radiation biodosimetry.

In major radiological events, rapid assays to detect ionizing radiation exposure are crucial for effective medical interventions. The purpose of these assays is twofold: to categorize affected individuals into groups for initial treatments, and to provide definitive dose estimates for continued care and epidemiology. However, existing high-throughput cytogenetic biodosimetry assays take about 3 days to yield results, which delays critical interventions. We have developed a multiwell-based variant of the chemical-induced G0-phase Premature Chromosome Condensation Assay that delivers same-day results. Our findings revealed that using a concentration of phosphatase inhibitor lower than recommended significantly increases the yield of cells with highly condensed chromosomes. These chromosomes exhibited increased fragmentation in a dose-dependent manner, enabling to quantify radiation damage using a custom Deep Learning algorithm. This algorithm demonstrated reasonable performance in categorizing doses into distinct treatment groups (84% and 80% accuracy for three and four iso-treatment dose bins, respectively) and showed reliability in determining the actual doses received (correlation coefficient of 0.879). This method is amendable to full automation and has the potential to address the need for same-day, high-throughput cytogenetic test for both dose categorization and dose reconstruction in large-scale radiation emergencies.

G0-PCC-FISH derived multi-parametric biodosimetry methodology for accidental high dose and partial body exposures.

High dose radiation exposures are rare. However, medical management of such incidents is crucial due to mortality and tissue injury risks. Rapid radiation biodosimetry of high dose accidental exposures is highly challenging, considering that they usually involve non uniform fields leading to partial body exposures. The gold standard, dicentric assay and other conventional methods have limited application in such scenarios. As an alternative, we propose Premature Chromosome Condensation combined with Fluorescent In-situ Hybridization (G0-PCC-FISH) as a promising tool for partial body exposure biodosimetry. In the present study, partial body exposures were simulated ex-vivo by mixing of uniformly exposed blood with unexposed blood in varying proportions. After G0-PCC-FISH, Dolphin's approach with background correction was used to provide partial body exposure dose estimates and these were compared with those obtained from conventional dicentric assay and G0-PCC-Fragment assay (conventional G0-PCC). Dispersion analysis of aberrations from partial body exposures was carried out and compared with that of whole-body exposures. The latter was inferred from a multi-donor, wide dose range calibration curve, a-priori established for whole-body exposures. With the dispersion analysis, novel multi-parametric methodology for discerning the partial body exposure from whole body exposure and accurate dose estimation has been formulated and elucidated with the help of an example. Dose and proportion dependent reduction in sensitivity and dose estimation accuracy was observed for Dicentric assay, but not in the two PCC methods. G0-PCC-FISH was found to be most accurate for the dose estimation. G0-PCC-FISH has potential to overcome the shortcomings of current available methods and can provide rapid, accurate dose estimation of partial body and high dose accidental exposures. Biological dose estimation can be useful to predict progression of disease manifestation and can help in pre-planning of appropriate & timely medical intervention.

Applicability of Scoring Calyculin A-Induced Premature Chromosome Condensation Objects for Dose Assessment Including for Radiotherapy Patients.

As an extension to a previous study, a linear calibration curve covering doses from 0 to 10 Gy was constructed and evaluated in the present study using calyculin A-induced premature chromosome condensation (PCC) by scoring excess PCC objects. The main aim of this study was to assess the applicability of this PCC assay for doses below 2 Gy that are critical for triage categorization. Two separate blind tests involving a total of 6 doses were carried out; 4 out of 6 dose estimates were within the 95% confidence limits (95% CL) with the other 2 just outside. In addition, blood samples from five cancer patients undergoing external beam radiotherapy (RT) were also analyzed, and the results showed whole-body dose estimates statistically comparable to the dicentric chromosome assay (DCA) results. This is the first time that calyculin A-induced PCC was used to analyze clinical samples by scoring excess objects. Although dose estimates for the pre-RT patient samples were found to be significantly higher than the mean value for the healthy donors and were also significantly higher than those obtained using DCA, all these pre-treatment patients fell into the same category as those who may have received a low dose (<1 Gy) and do not require immediate medical care during emergency triage. Additionally, for radiological accidents with unknown exposure scenario, PCC objects and rings can be scored in parallel for the assessment of both low- and high-dose exposures. In conclusion, scoring excess objects using calyculin A-induced PCC is confirmed to be another potential biodosimetry tool in radiological emergency particularly in mass casualty scenarios, even though the data need to be interpreted with caution when cancer patients are among the casualties.

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures.

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (60Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters ß/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

Chemical-Induced Premature Chromosome Condensation Protocol.

Chromosome analysis is one of most fundamental techniques for cytogenetic studies. Chromosomes are conventionally prepared from mitotic cells arrested by colcemid block protocol. Premature chromosome condensation (PCC) technique is an alternative to obtain chromosomes. It was more than half century ago that the first observation of PCC phenomena reported. Since then, cell-fusion-mediated PCC method has been developed and introduced in many fields of chromosome analysis. More than quarter century ago, novel PCC technique using chemical drug has been developed. Afterwards, this simple and efficient drug-induced PCC technique becomes a standard protocol for preparing chromosomes. Thus, it seems to be the good time to introduce PCC technique protocol for the artisans in the field of cytogenetic studies.

Visualizing Active Replication Regions in S-Phase Chromosomes.

A basic question of cell biology is how DNA folds to chromosome. A number of recently accumulated evidences have suggested that folding of chromosome proceeds tightly coupled with DNA replication progresses. Drug-induced PCC is a useful tool for visualization of the interphase nuclei, in particular, S-phase, as S-phase prematurely condensed chromosomes (S-phase PCC). Active replicating DNA is labeled directly with Cy3-dUTP by bead loading method, and then S-phase nuclei is immediately condensed prematurely by calyculin A to obtain S-phase PCC. Active replicating regions on S-PCC are observed under a scanning confocal microscope. Cy3-dUTP-labeled S-phase PCCs clearly reveal the drastic transitional change of chromosome formation through S-phase, starting from a "cloudy nebula" to numerous numbers of "beads on a string" and finally to "striped arrays of banding structured chromosome" known as G- or R-banding pattern. The number, distribution, and shape of replication foci were also measured in individual subphase of S-phase; maximally ~1400 foci of 0.35 µm average radius size were scored at the beginning of S-phase, and the number is reduced to ~100 at the end of S-phase. Drug-induced PCC clearly provided the new insight that eukaryote DNA replication is tightly coupled with the chromosome condensation/compaction for construction of eukaryote higher-ordered chromosome structure.

Chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose ionizing radiation.

The premature chromosome condensation (PCC) assay is considered as complementary bio-dosimetry tool for chromosome aberration assay and the PCC assay can be used to estimate high dose exposure. Though the PCC ring is considered as prospective biomarker, chromosome length ratio (ratio of longest and shortest chromosome length in PCC spreads) of chemically induced PCC is shown to be very good indicator of ionizing radiation. In view of this, an in-vitro study has been performed using PCC assay to suggest chromosome length ratio (LR) as potential bio-dosimeter induced by high dose ionizing radiation. Blood samples were collected from healthy subjects (n = 3) after prior consent and irradiated to ten different doses ranging between 0 and 20 Gy using 6 MV LINAC X-rays with dose rate of 5.6 Gy/min. Irradiated lymphocytes were cultured and calyculin induced PCC spreads were prepared. PCC spreads were captured using image analysis system and chromosome lengths were measured using open-source ImageJ software. For each dose, LR for 50 chromosome spreads were computed and mean LR value was calculated. LR varies between 6.0 ± 0.08 and 23.6 ± 0.55 for the dose range between 2 and 20 Gy. The dose response curve for LR was observed to be linear with y = 1.02x + 3.36, R2 = 0.97. Linear dose response relationship obtained in the present study confirms the prospective use of LR measurement. This study is first of its kind to examine chromosome length ratio as a biomarker of DNA damage in cells exposed to high dose X-ray exposure.

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Hydroxyurea and Caffeine Impact pRb-like Protein-Dependent Chromatin Architecture Profiles in Interphase Cells of Vicia faba.

The survival of cells depends on their ability to replicate correctly genetic material. Cells exposed to replication stress can experience a number of problems that may lead to deregulated proliferation, the development of cancer, and/or programmed cell death. In this article, we have induced prolonged replication arrest via hydroxyurea (HU) treatment and also premature chromosome condensation (PCC) by co-treatment with HU and caffeine (CF) in the root meristem cells of Vicia faba. We have analyzed the changes in the activities of retinoblastoma-like protein (RbS807/811ph). Results obtained from the immunocytochemical detection of RbS807/811ph allowed us to distinguish five unique activity profiles of pRb. We have also performed detailed 3D modeling using Blender 2.9.1., based on the original data and some final conclusions. 3D models helped us to visualize better the events occurring within the nuclei and acted as a high-resolution aid for presenting the results. We have found that, despite the decrease in pRb activity, its activity profiles were mostly intact and clearly recognizable, with some local alterations that may correspond to the increased demand in transcriptional activity. Our findings suggest that Vicia faba's ability to withstand harsh environments may come from its well-developed and highly effective response to replication stress.

Investigation of DNA Damage and Cell-Cycle Distribution in Human Peripheral Blood Lymphocytes under Exposure to High Doses of Proton Radiotherapy.

This study systematically investigates how a single high-dose therapeutic proton beam versus X-rays influences cell-cycle phase distribution and DNA damage in human peripheral blood lymphocytes (HPBLs). Blood samples from ten volunteers (both male and female) were irradiated with doses of 8.00, 13.64, 15.00, and 20.00 Gy of 250 kV X-rays or 60 MeV protons. The dose-effect relations were calculated and distributed by plotting the frequencies of DNA damage of excess Premature Chromosome Condensation (PCC) fragments and rings in the G2/M phase, obtained via chemical induction with calyculin A. The Papworth's u test was used to evaluate the distribution of DNA damage. The study shows that high doses of protons induce HPBL DNA damage in the G2/M phase differently than X-rays do. The results indicate a different distribution of DNA damage following high doses of irradiation with protons versus photons between donors, types of radiation, and doses. The proliferation index confirms the impact of high doses of mitosis and the influence of radiotherapy type on the different HPBL response. The results illuminate the cellular and molecular mechanisms that underlie differences in the distribution of DNA damage and cell-cycle phases; these findings may yield an improvement in the efficacy of the radiotherapies used.

Kinetics of DNA Repair in Vicia faba Meristem Regeneration Following Replication Stress.

The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even "chaotic" events are dealt with in a proper degree of order.

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.

Chromosomal aberration dynamics through the cell cycle.

DNA double-strand breaks are the crucial lesions underlying the formation of chromosomal aberrations, their formation and kinetics have been extensively studied, although dynamics of the repair process has not been fully understood. By using a combination of different cytogenetic techniques to analyze cells in G0, G2 and M phase, in the present study we perform a follow up study of the dynamics of different radiation induced chromosomal aberrations. Data here presented show that in G0 phase chromosome fragments lacking telomere signals (incomplete chromosome elements, ICE) show a slow repair, but when repair occurs tend to reconstitute the original chromosomes, and those that do not repair seem to be selected by interphase cell death and cell cycle checkpoints. In contrast, complete chromosome aberrations, as dicentrics, show a very fast formation kinetics. Similar frequencies of dicentrics were observed in G0, G2 and M cells, indicating that this chromosome-type of aberration can progress through the cell cycle without negative selection. Our study reinforce the hypothesis that ICE are strongly negatively selected from G2 to M phase. However, the G2/M checkpoint seems to be not involved in this selection. The ICE frequencies observed after G2/M abrogation by caffeine are similar to the ones without abrogation, and clearly lower to the ones observed in G2.

Premature chromosome condensation assay to study influence of high-level natural radiation on the initial DNA double strand break repair in human G0 lymphocytes.

The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G0 lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.

Effects of culturing technique on human peripheral blood lymphocytes response to proton and X-ray radiation.

Purpose: The main aim of this study was to comparatively investigate the effects of culturing methods on the response of human peripheral blood lymphocytes to irradiation exposure.Materials and methods: Whole blood and isolated lymphocytes were ex vivo exposed to two radiation sources (60 MeV proton or 250 kV X-ray radiation) with different doses (0.3, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 Gy), and genotoxic markers were subsequently assayed. The observed effects were compared as dose-response relationships using two end points (CBMN and PCC tests) and different biomarkers (NDI, PCC index, MNi frequency and excess PCC fragments).Results and conclusions: The results showed different effects of the culturing techniques on the response of human peripheral blood lymphocytes to radiation. The MNi frequency and excess PCC fragments were significantly higher when lymphocytes were cultured after being isolated. After irradiation, no differences were seen in the NDI between the lymphocytes of the two culturing techniques; however, there were differences in the PPC index. When planning or performing cytogenetic studies, the possibility of such effects and their potential to impact the variability of the results of human biomonitoring studies should be considered important and taken into account.

An alternative approach for the induction of premature chromosome condensation in human peripheral blood lymphocytes using mitotic Akodon cells.

Purpose: The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic Akodon cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique.Materials and methods: Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic Akodon cells.Results: The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for Akodon-induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation.Conclusion: The mitotic Akodon cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.

Assessment of the nuclear medicine personnel occupational exposure to radioiodine.

PURPOSE: To physically and cytogenetically screen medical personnel of Department of Endocrinology and Nuclear Medicine, Holy Cross Cancer Center, Kielce, Poland (DENM) who are occupationally exposed to 131I. MATERIALS AND METHODS: The exposure was monitored by whole-body and finger ring dosimeters. The thyroid iodine intake was measured by a whole-body spectrometer equipped with two semiconductor gamma radiation detectors. A cytokinesis-block micronucleus assay and the premature chromosome condensation technique were used to assess the aberration score. Cytogenetic analyses were carried out on a group of 29 workers and were compared to 32 controls (healthy donors), matched for gender and age. RESULTS: On average, the exposed group showed a significantly higher frequency of genetic damage and a higher proliferation index compared to the control group. Smoking status, age and duration of exposure influenced the observed effects in both groups. No differences in measured biomarkers were observed after stratification of the exposed group into two subgroups based on the measured 131I activity below and above 6 Bq. CONCLUSION: The findings suggest that radiation protection principles based on whole-body and finger ring dosimetry, supported by activity measurements with a whole-body spectrometer, may be insufficient to monitor the absorbed dose estimation of the nuclear medicine staff who are occupationally exposed to 131I. Furthermore, their future health risks are influenced by confounders. Direct assessments comparing physical and biological dose estimations on the larger group are needed to accurately monitor occupational radiation exposure.

γ-H2AX Foci Persistence at Chromosome Break Suggests Slow and Faithful Repair Phases Restoring Chromosome Integrity.

Many toxic agents can cause DNA double strand breaks (DSBs), which are in most cases quickly repaired by the cellular machinery. Using ionising radiation, we explored the kinetics of DNA lesion signaling and structural chromosome aberration formation at the intra- and inter-chromosomal level. Using a novel approach, the classic Premature Chromosome Condensation (PCC) was combined with γ-H2AX immunofluorescence staining in order to unravel the kinetics of DNA damage signalisation and chromosome repair. We identified an early mechanism of DNA DSB joining that occurs within the first three hours post-irradiation, when dicentric chromosomes and chromosome exchanges are formed. The slower and significant decrease of "deleted chromosomes" and 1 acentric telomere fragments observed until 24 h post-irradiation, leads to the conclusion that a second and error-free repair mechanism occurs. In parallel, we revealed remaining signalling of γ-H2AX foci at the site of chromosome fusion long after the chromosome rearrangement formation. Moreover there is important signalling of foci on the site of telomere and sub-telomere sequences suggesting either a different function of γ-H2AX signalling in these regions or an extreme sensibility of the telomere sequences to DNA damage that remains unrepaired 24 h post-irradiation. In conclusion, chromosome repair happens in two steps, including a last and hardly detectable one because of restoration of the chromosome integrity.

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.

G1 Premature Chromosome Condensation (PCC) Assay.

Premature chromosome condensation (PCC) is a sensitive and unique way to detect interphase chromosome damage and its recovery in mammalian cells irradiated with ionizing radiation. In this chapter, we describe G1 PCC assay with which one can measure immediate chromosome breaks in G1 type chromosomes and their repair/rejoining. In order to induce G1 PCC, one needs to fuse mitotic cells with G1 cells to be tested. There are two methods to fuse cells; one is to use Sendai virus or its equivalent, and another method needs polyethylene glycol (PEG) as a fusing agent. The date obtained with PCC assay can bridge the gap between radiation-induced DNA damage (mainly double strand breaks) and chromosome aberrations observable at metaphase stage.