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International journal of radiation biology, 93, 36--47
2017

RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay).

Depuydt, Julie, Baeyens, Ans, Barnard, Stephen, Beinke, Christina, Benedek, Anett, Beukes, Philip, Buraczewska, Iwona, Darroudi, Firouz, De Sanctis, Stefania, Dominguez, Inmaculada, Monteiro Gil, Octávia, Hadjidekova, Valeria, Kis, Enikő, Kulka, Ulrike, Lista, Florigio, Lumniczky, Katalin, M'kacher, Radhia, Moquet, Jayne, Obreja, Doina, Oestreicher, Ursula, Pajic, Jelena, Pastor, Nuria, Popova, Ljubomira, Regalbuto, Elisa, Ricoul, Michelle, Sabatier, Laure, Slabbert, Jacobus, Sommer, Sylwester, Testa, Antonella, Thierens, Hubert, Wojcik, Andrzej, Vral, Anne

In the framework of the 'Realizing the European Network of Biodosimetry' (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1206231

Oncotarget
2017

Low numbers of pre-leukemic fusion genes are frequently present in umbilical cord blood without affecting DNA damage response.

Kosik, Pavol, Skorvaga, Milan, Durdik, Matus, Jakl, Lukas, Nikitina, Ekaterina, Markova, Eva, Kozics, Katarina, Horvathova, Eva, Belyaev, Igor

Despite widely accepted notion that many childhood leukemias are likely developed from hematopoietic stem/progenitor cells (HSPC) with pre-leukemic fusion genes (PFG) formed in embryonic/fetal development, the data on PFG incidence in newborns are contradictive. To provide a better understanding of a prenatal origin of leukemia, umbilical cord blood from 500 newborns was screened for the presence of the most frequent PFG associated with pediatric B-cell acute lymphoblastic leukemia. This screening revealed relatively high incidence of ETV6-RUNX1, BCR-ABL1 (p190) and MLL-AF4 at very low frequencies, averaging ~14 copies per 100,000 cells. We assume that most of these PFG might originate relatively late in embryonic/fetal development and will be eliminated later during postnatal development. The obtained results suggested that higher PFG copy numbers originating in specific time windows of the hematopoietic stem cell hierarchy may define a better prognostic tool for the assessment of leukemogenic potential. We have observed no significant effect of low-copy PFG on radiation-induced DNA damage response, accumulation of endogenous DNA double-stranded breaks, and apoptosis in either lymphocytes or HSPC. Imaging flow cytometry showed lower level of γH2AX foci in HSPC in comparison to lymphocytes suggesting better protection of HSPC from DNA damage.

Digital object identifier (DOI): 10.18632/oncotarget.16211

Appl. Sci., 7, 330
2017

Large-Scale Permanent Slide Imaging and Image Analysis for Diatom Morphometrics

Michael Kloster, Oliver Esper, Gerhard Kauer, Bánk Beszteri

Light microscopy analysis of diatom frustules is widely used in basic and applied research, notably taxonomy, morphometrics, water quality monitoring and paleo-environmental studies. Although there is a need for automation in these applications, various developments in image processing and analysis methodology supporting these tasks have not become widespread in diatom-based analyses. We have addressed this issue by combining our automated diatom image analysis software SHERPA with a commercial slide-scanning microscope. The resulting workflow enables mass-analyses of a broad range of morphometric features from individual frustules mounted on permanent slides. Extensive automation and internal quality control of the results helps to minimize user intervention, but care was taken to allow the user to stay in control of the most critical steps (exact segmentation of valve outlines and selection of objects of interest) using interactive functions for reviewing and revising results. In this contribution, we describe our workflow and give an overview of factors critical for success, ranging from preparation and mounting through slide scanning and autofocus finding to final morphometric data extraction. To demonstrate the usability of our methods we finally provide an example application by analysing Fragilariopsis kerguelensis valves originating from a sediment core, which substantially extends the size range reported in the literature.

Digital object identifier (DOI): 10.3390

Nucleic acids research, 45, 1860--1871
2017

RMI1 and TOP3α limit meiotic CO formation through their C-terminal domains.

Séguéla-Arnaud, Mathilde, Choinard, Sandrine, Larchevêque, Cécile, Girard, Chloé, Froger, Nicole, Crismani, Wayne, Mercier, Raphael

At meiosis, hundreds of programmed DNA double-strand breaks (DSBs) form and are repaired by homologous recombination. From this large number of DSBs, only a subset yields crossovers (COs), with a minimum of one CO per chromosome pair. All DSBs must be repaired and every recombination intermediate must be resolved to avoid subsequent entanglement and chromosome breakage. The conserved BLM-TOP3α-RMI1 (BTR) complex acts on early and late meiotic recombination intermediates to both limit CO outcome and promote chromosome integrity. In Arabidopsis, the BLM homologues RECQ4A and RECQ4B act redundantly to prevent meiotic extra COs, but recombination intermediates are fully resolved in their absence. In contrast, TOP3α is needed for both processes. Here we show through the characterization of specific mutants that RMI1 is a major anti-CO factor, in addition to being essential to prevent chromosome breakage and entanglement. Further, our findings suggest a specific role of the C-terminal domains of RMI1 and TOP3α, that respectively contain an Oligo Binding domain (OB2) and ZINC finger motifs, in preventing extra-CO. We propose that these domains of TOP3α and RMI1 define a sub-domain of the BTR complex which is dispensable for the resolution of recombination intermediates but crucial to limit extra-COs.

Digital object identifier (DOI): 10.1093/nar/gkw1210

International journal of radiation biology, 93, 48--57
2017

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

Terzoudi, Georgia I, Pantelias, Gabriel, Darroudi, Firouz, Barszczewska, Katarzyna, Buraczewska, Iwona, Depuydt, Julie, Georgieva, Dimka, Hadjidekova, Valeria, Hatzi, Vasiliki I, Karachristou, Ioanna, Karakosta, Maria, Meschini, Roberta, M'Kacher, Radhia, Montoro, Alegria, Palitti, Fabrizio, Pantelias, Antonio, Pepe, Gaetano, Ricoul, Michelle, Sabatier, Laure, Sebastià, Natividad, Sommer, Sylwester, Vral, Anne, Zafiropoulos, Demetre, Wojcik, Andrzej

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. 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. 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. 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.

Digital object identifier (DOI): 10.1080/09553002.2016.1234725

Oncotarget, 8, 26269--26280
2017

Opposite effects of GCN5 and PCAF knockdowns on the alternative mechanism of telomere maintenance.

Jeitany, Maya, Bakhos-Douaihy, Dalal, Silvestre, David C, Pineda, Jose R, Ugolin, Nicolas, Moussa, Angela, Gauthier, Laurent R, Busso, Didier, Junier, Marie-Pierre, Chneiweiss, Hervé, Chevillard, Sylvie, Desmaze, Chantal, Boussin, François D

Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.

Digital object identifier (DOI): 10.18632/oncotarget.15447

International journal of radiation biology, 93, 58--64
2017

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

Moquet, Jayne, Barnard, Stephen, Staynova, Albena, Lindholm, Carita, Monteiro Gil, Octávia, Martins, Vanda, Rößler, Ute, Vral, Anne, Vandevoorde, Charlot, Wojewódzka, Maria, Rothkamm, Kai

Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1207822

PloS one, 12, e0178877
2017

Depletion of ATP and glucose in advanced human atherosclerotic plaques.

Ekstrand, Matias, Widell, Emma, Hammar, Anna, Akyürek, Levent M, Johansson, Martin, Fagerberg, Björn, Bergström, Göran, Levin, Malin C, Fogelstrand, Per, Borén, Jan, Levin, Max

Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques. Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques. Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression.

Digital object identifier (DOI): 10.1371/journal.pone.0178877

International journal of radiation biology, 93, 99--109
2017

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans - joint RENEB and EURADOS inter-laboratory comparisons.

Ainsbury, Elizabeth, Badie, Christophe, Barnard, Stephen, Manning, Grainne, Moquet, Jayne, Abend, Michael, Antunes, Ana Catarina, Barrios, Lleonard, Bassinet, Celine, Beinke, Christina, Bortolin, Emanuela, Bossin, Lily, Bricknell, Clare, Brzoska, Kamil, Buraczewska, Iwona, Castaño, Carlos Huertas, Čemusová, Zina, Christiansson, Maria, Cordero, Santiago Mateos, Cosler, Guillaume, Monaca, Sara Della, Desangles, François, Discher, Michael, Dominguez, Inmaculada, Doucha-Senf, Sven, Eakins, Jon, Fattibene, Paola, Filippi, Silvia, Frenzel, Monika, Georgieva, Dimka, Gregoire, Eric, Guogyte, Kamile, Hadjidekova, Valeria, Hadjiiska, Ljubomira, Hristova, Rositsa, Karakosta, Maria, Kis, Enikő, Kriehuber, Ralf, Lee, Jungil, Lloyd, David, Lumniczky, Katalin, Lyng, Fiona, Macaeva, Ellina, Majewski, Matthaeus, Vanda Martins, S, McKeever, Stephen W S, Meade, Aidan, Medipally, Dinesh, Meschini, Roberta, M'kacher, Radhia, Gil, Octávia Monteiro, Montero, Alegria, Moreno, Mercedes, Noditi, Mihaela, Oestreicher, Ursula, Oskamp, Dominik, Palitti, Fabrizio, Palma, Valentina, Pantelias, Gabriel, Pateux, Jerome, Patrono, Clarice, Pepe, Gaetano, Port, Matthias, Prieto, María Jesús, Quattrini, Maria Cristina, Quintens, Roel, Ricoul, Michelle, Roy, Laurence, Sabatier, Laure, Sebastià, Natividad, Sholom, Sergey, Sommer, Sylwester, Staynova, Albena, Strunz, Sonja, Terzoudi, Georgia, Testa, Antonella, Trompier, Francois, Valente, Marco, Hoey, Olivier Van, Veronese, Ivan, Wojcik, Andrzej, Woda, Clemens

RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Digital object identifier (DOI): 10.1080/09553002.2016.1206233

Scientific reports, 7, 3291
2017

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

Kaddour, Akram, Colicchio, Bruno, Buron, Diane, El Maalouf, Elie, Laplagne, Eric, Borie, Claire, Ricoul, Michelle, Lenain, Aude, Hempel, William M, Morat, Luc, Al Jawhari, Mustafa, Cuceu, Corina, Heidingsfelder, Leonhard, Jeandidier, Eric, Deschênes, Georges, Dieterlen, Alain, El May, Michèle, Girinsky, Theodore, Bennaceur-Griscelli, Annelise, Carde, Patrice, Sabatier, Laure, M'kacher, Radhia

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Digital object identifier (DOI): 10.1038/s41598-017-03198-7

Endocr Relat Cancer, 23(8), 635–650
August, 2016

Effect of low doses of estradiol and tamoxifen on breast cancer cell karyotypes.

Rondón-Lagos, Milena, Rangel, Nelson, Di Cantogno, Ludovica Verdun, Annaratone, Laura, Castellano, Isabella, Russo, Rosalia, Manetta, Tilde, Marchiò, Caterina, Sapino, Anna

Evidence supports a role of 17&-estradiol (E2) in carcinogenesis and the large majority of breast carcinomas are dependent on estrogen. The anti-estrogen tamoxifen (TAM) is widely used for both treatment and prevention of breast cancer; however, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. The nature of E2- or TAM-induced chromosomal damage has been explored using relatively high concentrations of these agents, and only some numerical aberrations and chromosomal breaks have been analyzed. This study aimed to determine the effects of low doses of E2 and TAM (10(&8 )mol L(&1) and 10(&6 )mol L(&1) respectively) on karyotypes of MCF7, T47D, BT474, and SKBR3 breast cancer cells by comparing the results of conventional karyotyping and multi-FISH painting with cell proliferation. Estrogen receptor (ER)-positive (+) cells showed an increase in cell proliferation after E2 treatment (MCF7, T47D, and BT474) and a decrease after TAM treatment (MCF7 and T47D), whereas in ER& cells (SKBR3), no alterations in cell proliferation were observed, except for a small increase at 96 h. Karyotypes of both ER+ and ER& breast cancer cells increased in complexity after treatments with E2 and TAM leading to specific chromosomal abnormalities, some of which were consistent throughout the treatment duration. This genotoxic effect was higher in HER2+ cells. The ER&/HER2+ SKBR3 cells were found to be sensitive to TAM, exhibiting an increase in chromosomal aberrations. These in vitro results provide insights into the potential role of low doses of E2 and TAM in inducing chromosomal rearrangements in breast cancer cells.

Digital object identifier (DOI): 10.1530/ERC-16-0078

Mol Med Rep, 14(1), 103–110
July, 2016

A semi‑automated FISH‑based micronucleus‑centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation.

Vral, Anne, Decorte, Veerle, Depuydt, Julie, Wambersie, André, Thierens, Hubert

The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.

Digital object identifier (DOI): 10.3892/mmr.2016.5265

Radiat Prot Dosimetry
July, 2016

A New Cytogenetic Biodosimetry Image Repository for the Dicentric Assay.

Romm, Horst, Beinke, Christina, Garcia, Omar, Di Giorgio, Marina, Gregoire, Eric, Livingston, Gordon, Lloyd, David, Martinez-Lopez, Wilner, Moquet, Jayne E., Sugarman, Stephen L., Wilkins, Ruth C., Ainsbury, Elizabeth A.

The BioDoseNet was founded by the World Health Organization as a global network of biodosimetry laboratories for building biodosimetry laboratory capacities in countries. The newly established BioDoseNet image repository is a databank of ~25 000 electronically captured images of metaphases from the dicentric assay, which have been previously analysed by international experts. The detailed scoring results and dose estimations have, in most cases, already been published. The compilation of these images into one image repository provides a valuable tool for training and research purposes in biological dosimetry. No special software is needed to view and score the image galleries. For those new to the dicentric assay, the BioDoseNet Image Repository provides an introduction to and training for the dicentric assay. It is an excellent instrument for intra-laboratory training purposes or inter-comparisons between laboratories, as recommended by the International Organization for Standardisation standards. In the event of a radiation accident, the repository can also increase the surge capacity and reduce the turnaround time for dose estimations. Finally, it provides a mechanism for the discussion of scoring discrepancies in difficult cases.

Digital object identifier (DOI): 10.1093/rpd/ncw158

Nucleic Acids Res
June, 2016

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Schipler, Agnes, Mladenova, Veronika, Soni, Aashish, Nikolov, Vladimir, Saha, Janapriya, Mladenov, Emil, Iliakis, George

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

Digital object identifier (DOI): 10.1093/nar/gkw487

J Radiat Res, 57(3), 220–226
June, 2016

Analysis of chromosome translocation frequency after a single CT scan in adults.

Abe, Yu, Miura, Tomisato, Yoshida, Mitsuaki A., Ujiie, Risa, Kurosu, Yumiko, Kato, Nagisa, Katafuchi, Atsushi, Tsuyama, Naohiro, Kawamura, Fumihiko, Ohba, Takashi, Inamasu, Tomoko, Shishido, Fumio, Noji, Hideyoshi, Ogawa, Kazuei, Yokouchi, Hiroshi, Kanazawa, Kenya, Ishida, Takashi, Muto, Satoshi, Ohsugi, Jun, Suzuki, Hiroyuki, Ishikawa, Tetsuo, Kamiya, Kenji, Sakai, Akira

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.

Digital object identifier (DOI): 10.1093/jrr/rrv090

Indian J Hematol Blood Transfus, 32(2), 154–161
June, 2016

Evaluation of ETV6/RUNX1 Fusion and Additional Abnormalities Involving ETV6 and/or RUNX1 Genes Using FISH Technique in Patients with Childhood Acute Lymphoblastic Leukemia.

Aydin, Cigdem, Cetin, Zafer, Manguoglu, Ayse Esra, Tayfun, Funda, Clark, Ozden Altiok, Kupesiz, Alphan, Akkaya, Bahar, Karauzum, Sibel Berker

Childhood acute lymphoblastic leukemia (ALL) is the most common type of childhood leukemia. Specifically, ALL is a malignant disorder of the lymphoid progenitor cells, with a peak incidence among children aged 2-5 years. The t(12;21)(p13;q22) translocation occurs in 25 \% of childhood B cell precursor ALL. In this study, bone marrow samples were obtained from 165 patients with childhood ALL. We analyzed the t(12;21) translocation and other related abnormalities using the fluorescent in situ hybridization (FISH) technique with the ETV6(TEL)/RUNX1(AML1) ES dual color translocation probe. Conventional cytogenetic analyses were also performed. ETV6 and RUNX1 related chromosomal abnormalities were found in 42 (25.5 \%) of the 165 patients with childhood ALL. Among these 42 patients, structural changes were detected in 33 (78.6 \%) and numerical abnormalities in 9 (21.4 \%). The frequency of FISH abnormalities in pediatric ALL cases were as follows: 8.5 \% for t(12;21)(p13;q22) ETV6/RUNX1 fusion, 6.0 \% for RUNX1 amplification, 3.0 \% for tetrasomy/trisomy 21, 1.8 \% for ETV6 deletion, 1.21 \% for ETV6 deletion with RUNX1 amplification, 1.21 \% for ETV6 amplification with RUNX1 amplification, 0.6 \% for polyploidy, 0.6 \% for RUNX1 deletion, and 0.6 \% for diminished ETV6 signal. The most common structural abnormality was the t(12;21) translocation, followed by RUNX1 amplification and ETV6 deletion, while the most commonly observed numerical abnormality was trisomy 21.

Digital object identifier (DOI): 10.1007/s12288-015-0557-7

Chromosome Res
May, 2016

Karyotype diversity suggests that Laonastes aenigmamus (Laotian rock rat) (Rodentia, Diatomyidae) is a multi-specific genus.

Richard, Florence, Gerbault-Seureau, Michèle, Douangboupha, Bounneuang, Keovichit, Kham, Hugot, Jean-Pierre, Dutrillaux, Bernard

Laonastes aenigmamus (Khanyou) is a recently described rodent species living in geographically separated limestone formations of the Khammuan Province in Lao PDR. Chromosomes of 21 specimens of L. aenigmamus were studied using chromosome banding as well as fluorescent in situ hybridization (FISH) techniques using human painting, telomere repeats, and 28S rDNA probes. Four different karyotypes were established. Study with human chromosome paints and FISH revealed that four large chromosomes were formed by multiple common tandem fusions, with persistence of some interstitial telomeres. The rearrangements separating the different karyotypes (I to IV) were also reconstructed. Various combinations of Robertsonian translocations or tandem fusions involving the same chromosomes differentiate these karyotypes. These rearrangements create a strong gametic barrier, which isolates specimens with karyotype II from the others. C-banding and FISH with telomere repeats also exhibit large and systematized differences between karyotype II and others. These data indicate an ancient reproductive separation and suggest that Laonastes is not a mono-specific genus.

Digital object identifier (DOI): 10.1007/s10577-016-9527-7

Radiat Environ Biophys
March, 2016

Chromosome aberrations in Japanese fishermen exposed to fallout radiation 420-1200~km distant from the nuclear explosion test site at Bikini Atoll: report 60~years after the incident.

Tanaka, Kimio, Ohtaki, Megu, Hoshi, Masaharu

During the period from March to May, 1954, the USA conducted six nuclear weapon tests at the "Bravo" detonation sites at the Bikini and Enewetak Atolls, Marshall Islands. At that time, the crew of tuna fishing boats and cargo ships that were operating approximately 150-1200 km away from the test sites were exposed to radioactive fallout. The crew of the fishing boats and those on cargo ships except the "5th Fukuryu-maru" did not undergo any health examinations at the time of the incident. In the present study, chromosome aberrations in peripheral blood lymphocytes were examined in detail by the G-banding method in 17 crew members from 8 fishing boats and 2 from one cargo ship, 60 years after the tests. None of the subjects examined had suffered from cancer. The percentages of both stable-type aberrations such as translocation, inversion and deletion, and unstable-type aberrations such as dicentric and centric ring in the study group were significantly higher (1.4- and 2.3-fold, respectively) than those in nine age-matched controls. In the exposed and control groups, the percentages of stable-type aberrations were 3.35 \% and 2.45 \%, respectively, and the numbers of dicentric and centric ring chromosomes per 100 cells were 0.35 and 0.15, respectively. Small clones were observed in three members of the exposed group. These results suggest that the crews were exposed to slightly higher levels of fallout than had hitherto been assumed.

Digital object identifier (DOI): 10.1007/s00411-016-0648-3

Tumour Biol, 37(3), 4041–4052
March, 2016

Establishment and characterization of a human intrahepatic cholangiocarcinoma cell line derived from an Italian patient.

Cavalloni, Giuliana, Peraldo-Neia, Caterina, Varamo, Chiara, Casorzo, Laura, Dell'Aglio, Carmine, Bernabei, Paola, Chiorino, Giovanna, Aglietta, Massimo, Leone, Francesco

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/-) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo.

Digital object identifier (DOI): 10.1007/s13277-015-4215-3