Purpose: Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G 0 -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 c-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 G 0 -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.
ARTICLE HISTORY
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.
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