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Journal of cellular biochemistry, 120, 4804–4812
April, 2019

Optimization of prostate cancer cell detection using multiplex tyramide signal amplification.

Roy, Sounak, Axelrod, Haley D, Valkenburg, Kenneth C, Amend, Sarah, Pienta, Kenneth J

Approximately 29 000 men die of prostate cancer (PCa) each year in the United States, and 90% to 100% of them are due to incurable bone metastasis. It is difficult to determine (1) when PCa disseminates in the natural history of the disease; (2) where cancer cell disseminates before becoming overt metastatic lesions; and (3) which tumors are aggressive and which are indolent. Tumor tissue and liquid (blood and bone marrow) biopsies provide important information to answer these questions, but significant limitations exist for immunostaining strategies that assess protein expression in these tissues. Classic immunohistochemistry (IHC) assays can typically assess expression of one or two proteins per tissue section. We have developed a novel immunofluorescence staining protocol to detect a panel of seven proteins on PCa tissue from primary tumor biopsies and metastatic lesion autopsy tissue, as well as cancer cells from liquid biopsies. We used a tyramide-based system to amplify the true signal and optimized the protocol to reduce background signal, thereby boosting the signal-to-noise ratio. Any protein-specific antibody in this protocol can be exchanged for a different validated antibody. This protocol therefore, represents a highly informative and flexible assay that can be used to provide important information about cancer tissue for the purpose of improving detection, diagnosis, and treatment.

Digital object identifier (DOI): 10.1002/jcb.28016

Biochemical and biophysical research communications, 511, 658–664
April, 2019

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells.

Benlamara, Sarah, Aubry, Laetitia, Fabregue, Julien, Bénit, Paule, Rustin, Pierre, Rak, Malgorzata

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

Digital object identifier (DOI): 10.1016/j.bbrc.2019.02.033

Radiation protection dosimetry, 182, 139–145
December, 2018

DEVELOPMENT OF A MINIATURIZED VERSION OF DICENTRIC CHROMOSOME ASSAY TOOL FOR RADIOLOGICAL TRIAGE.

Balajee, Adayabalam S, Smith, Tammy, Ryan, Terri, Escalona, Maria, Dainiak, Nicholas

Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of dicentric chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.

Digital object identifier (DOI): 10.1093/rpd/ncy127

The FEBS journal, 285, 3769–3785
October, 2018

Naphthalene diimide-derivatives G-quadruplex ligands induce cell proliferation inhibition, mild telomeric dysfunction and cell cycle perturbation in U251MG glioma cells.

Muoio, Daniela, Berardinelli, Francesco, Leone, Stefano, Coluzzi, Elisa, di Masi, Alessandra, Doria, Filippo, Freccero, Mauro, Sgura, Antonella, Folini, Marco, Antoccia, Antonio

In the present paper, the biological effects of three different naphthalene diimides (NDIs) G-quadruplex (G4) ligands (H-NDI-Tyr, H-NDI-NMe2, and tetra-NDI-NMe2) were comparatively evaluated to those exerted by RHPS4, a well-characterized telomeric G4-ligand, in an in vitro model of glioblastoma. Data indicated that NDIs were very effective in blocking cell proliferation at nanomolar concentrations, although displaying a lower specificity for telomere targeting compared to RHPS4. In addition, differently from RHPS4, NDIs failed to enhance the effect of ionizing radiation, thus suggesting that additional targets other than telomeres could be involved in the strong NDI-mediated anti-proliferative effects. In order to test telomeric off-target action of NDIs, a panel of genes involved in tumor progression, DNA repair, telomere maintenance, and cell-cycle regulation were evaluated at transcriptional and translational level. Specifically, the compounds were able to cause a marked reduction of TERT and BCL2 amounts as well as to favor the accumulation of proteins involved in cell cycle control. A detailed cytofluorimetric analysis of cell cycle progression by means of bromodeoxyuridine (BrdU) incorporation and staining of phospho-histone H3 indicated that NDIs greatly reduce the progression through S-phase and lead to G1 accumulation of BrdU-positive cells. Taken together, these data indicated that, besides effects on telomeres and oncogenes such as Tert and Bcl2, nanomolar concentrations of NDIs determined a sustained block of cell proliferation by slowing down cell cycle progression during S-phase. In conclusion, our data indicate that NDIs G4-ligands are powerful antiproliferative agents, which act through mechanisms that ultimately lead to altered cell-cycle control.

Digital object identifier (DOI): 10.1111/febs.14628

Cancers, 10
October, 2018

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

M'kacher, Radhia, Frenzel, Monika, Al Jawhari, Mustafa, Junker, Steffen, Cuceu, Corina, Morat, Luc, Bauchet, Anne-Laure, Stimmer, Lev, Lenain, Aude, Dechamps, Nathalie, Hempel, William M, Pottier, Geraldine, Heidingsfelder, Leonhard, Laplagne, Eric, Borie, Claire, Oudrhiri, Noufissa, Jouni, Dima, Bennaceur-Griscelli, Annelise, Colicchio, Bruno, Dieterlen, Alain, Girinsky, Theodore, Boisgard, Raphael, Bourhis, Jean, Bosq, Jacques, Mehrling, Thomas, Jeandidier, Eric, Carde, Patrice

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Digital object identifier (DOI): 10.3390/cancers10110414

Cancers, 10
July, 2018

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability , [email protected], .

Cuceu, Corina, Colicchio, Bruno, Jeandidier, Eric, Junker, Steffen, Plassa, François, Shim, Grace, Mika, Justyna, Frenzel, Monika, Al Jawhari, Mustafa, Hempel, William M, O'Brien, Grainne, Lenain, Aude, Morat, Luc, Girinsky, Theodore, Dieterlen, Alain, Polanska, Joanna, Badie, Christophe, Carde, Patrice, M'Kacher, Radhia

: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). : We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. : In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. : In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

Digital object identifier (DOI): 10.3390/cancers10070233

Scientific reports, 8, 2286
February, 2018

DNA damage in leukocytes after internal ex-vivo irradiation of blood with the α-emitter Ra-223.

Schumann, Sarah, Eberlein, Uta, Muhtadi, Razan, Lassmann, Michael, Scherthan, Harry

Irradiation with high linear energy transfer α-emitters, like the clinically used Ra-223 dichloride, severely damages cells and induces complex DNA damage including closely spaced double-strand breaks (DSBs). As the hematopoietic system is an organ-at-risk for the treatment, knowledge about Ra-223-induced DNA damage in blood leukocytes is highly desirable. Therefore, 36 blood samples from six healthy volunteers were exposed ex-vivo (in solution) to different concentrations of Ra-223. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the decay, ranging from 0 to 142 mGy. γ-H2AX + 53BP1 co-staining and analysis was performed in leukocytes isolated from the irradiated blood samples. For DNA damage quantification, leukocyte samples were screened for occurrence of α-induced DNA damage tracks and small γ-H2AX + 53BP1 DSB foci. This revealed a linear relationship between the frequency of α-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX + 53BP1 DSB foci indicative of β-irradiation was similar to baseline values, being in agreement with a negligible β-contribution (3.7%) to the total absorbed dose to the blood. Our calibration curve will contribute to the biodosimetry of Ra-223-treated patients and early after incorporation of α-emitters.

Digital object identifier (DOI): 10.1038/s41598-018-20364-7

Investigative ophthalmology & visual science, 59, 561–571
January, 2018

A Novel C-Terminal Mutation in Gsdma3 (C+/H-) Leads to Alopecia and Corneal Inflammatory Response in Mice.

Swirski, Sebastian, Röger, Carsten, Pienkowska-Schelling, Aldona, Ihlenburg, Cynthia, Fischer, Gösta, May, Oliver, Vorm, Mariann, Owczarek-Lipska, Marta, Neidhardt, John

Mutations in the gene encoding Gasdermin A3 (Gsdma3) have been described to cause severe skin phenotypes, including loss of sebaceous glands and alopecia, in mice. We discovered a novel C-terminal mutation in Gsdma3 in a new mouse line and characterized a less frequently reported corneal phenotype, likely caused by degeneration of Meibomian glands of the inner eyelid. We used histologic methods to evaluate the effects of the C+/H- mutation on sebaceous gland and skin morphology as well as Meibomian glands of the inner eyelid and corneal tissue. Chromosomal aberrations were excluded by karyogram analyses. The mutation was identified by Sanger sequencing of candidate genes. Analyses of skin samples from affected mice confirmed the frequently reported phenotypes associated with mutations in Gsdma3: Degeneration of sebaceous glands and complete loss of pelage. Immunologic staining of corneal samples suggested an inflammatory response with signs of neovascularization in half of the affected older mice. While the corneal phenotype was observed at irregular time points, mainly after 6 months, its appearance coincided with a degeneration of Meibomian glands in the eyelids of affected animals. The mutation described herein is associated with inflammation and neovascularization of corneal tissue. Simultaneous degeneration of Meibomian glands in affected animals suggested a change in tear-film composition as the underlying cause for the corneal phenotype. Our data further support that different pathogenic mechanisms underlie some of the reported mutations in Gsdma3.

Digital object identifier (DOI): 10.1167/iovs.17-22658

Scientific Reports, 8(1), 1141
2018

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness

Cirrone, GAP, Manti, L, Margarone, D, Petringa, G, Giuffrida, L, Minopoli, A, Picciotto, A, Russo, G, Cammarata, F, Pisciotta, P, others

Protontherapy is hadrontherapy’s fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy’s superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy’s ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

Molecular cytogenetics, 11, 4
2018

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

Bloomfield, Mathew, Duesberg, Peter

Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression onfirming classical predictions based on DNA content

Digital object identifier (DOI): 10.1186/s13039-017-0350-4

Scientific reports, 7, 3291
June, 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

Animal science journal = Nihon chikusan Gakkaiho, 88, 27–32
January, 2017

Comparative genomic hybridization in detection of DNA changes in canine lymphomas.

Drážovská, Monika, Šiviková, Katarína, Dianovský, Ján, Horňák, Miroslav

In this study, chromosomal imbalances in tumor tissues (lymphomas) and nucleotide changes in tumor suppressor TP53 were studied in a Bernese Mountain dog bitch and a cross breed bitch. Using comparative genomic hybridization, numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumor growth: in the cross breed bitch, a deletion on the chromosome 9, and duplications on chromosomes 5, 8 and 17 have been found. In the Bernese Mountain Dog bitch, losses on chromosomes 1, 5, 8, 12, 18, 22, 27, 29 and gains on chromosomes 1, 2, 9, 11, 15, 16, 18, 20, 23, 24, 25, 28, 29, 30, 34, 36, 37 and 38 were identified. With the sequencing of the TP53 gene, one silent mutation, transition A/G at position 138 in exon 5 was detected, without changing the amino acid.

Digital object identifier (DOI): 10.1111/asj.12582

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

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

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

Oncotarget, 7(9), 10182–10192
March, 2016

Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system.

Morishita, Maki, Muramatsu, Tomoki, Suto, Yumiko, Hirai, Momoki, Konishi, Teruaki, Hayashi, Shin, Shigemizu, Daichi, Tsunoda, Tatsuhiko, Moriyama, Keiji, Inazawa, Johji

Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus.

Digital object identifier (DOI): 10.18632/oncotarget.7186

Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 57, 173–179
February, 2016

DNA Damage in Peripheral Blood Lymphocytes of Thyroid Cancer Patients After Radioiodine Therapy.

Eberlein, Uta, Scherthan, Harry, Bluemel, Christina, Peper, Michel, Lapa, Constantin, Buck, Andreas Konrad, Port, Matthias, Lassmann, Michael

The aim of the study was to investigate DNA double-strand break (DSB) formation and its correlation to the absorbed dose to the blood in patients with surgically treated differentiated thyroid cancer undergoing their first radioiodine therapy for remnant ablation. Twenty patients were included in this study. At least 7 peripheral blood samples were obtained before and between 0.5 and 120 h after administration of radioiodine. From the time-activity curves of the blood and the whole body, residence times for the blood self-irradiation and the irradiation from the whole body were determined. Peripheral blood lymphocytes were isolated, ethanol-fixed, and subjected to immunofluorescence staining for colocalizing γ-H2AX/53BP1 DSB-marking foci. The average number of DSB foci per cell per patient sample was analyzed as a function of the absorbed dose to the blood and compared with an in vitro calibration curve for (131)I and (177)Lu established previously in our institution. The average number of radiation-induced foci (RIF) per cell increased over the first 3 h after radionuclide administration and decreased thereafter. A linear fit from 0 to 2 h as a function of the absorbed dose to the blood agreed with our in vitro calibration curve. At later time points, RIF numbers diminished, along with dropping dose rates, indicating progression of DNA repair. Individual patient data were characterized by a linear dose-dependent increase and a biexponential response function describing a fast and a slow repair component. With the experimental results and model calculations presented in this work, a dose-response relationship is demonstrated, and an analytic function describing the time course of the in vivo damage response after internal irradiation of patients with (131)I is established.

Digital object identifier (DOI): 10.2967/jnumed.115.164814

Reprod Domest Anim, 51(1), 171–174
February, 2016

A Non-Reciprocal Autosomal Translocation 64,XX, t(4;10)(q21;p15) in an Arabian Mare with Repeated Early Embryonic Loss.

Ghosh, S., Das, P. J., Avila, F., Thwaits, B. K., Chowdhary, B. P., Raudsepp, T.

Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a phenotypically normal 3-year-old Arabian mare that had repeated resorption of conceptuses prior to day 45 of gestation and was diagnosed with REEL. Conventional and molecular cytogenetic analyses revealed that the mare had normal chromosome number 64,XX but carried a non-mosaic and non-reciprocal autosomal translocation t(4;10)(q21;p15). This is a novel translocation described in horses with REEL and the first such report in Arabians. Previous cases of REEL due to autosomal translocations have exclusively involved Thoroughbreds. The findings underscore the importance of routine cytogenetic screening of breeding animals.

Digital object identifier (DOI): 10.1111/rda.12636

Gastroenterol Res Pract, 2016, 6089658
2016

The Role of Chromosomal Instability and Epigenetics in Colorectal Cancers Lacking beta-Catenin/TCF Regulated Transcription.

Abdel-Rahman, Wael M., Lotsari-Salomaa, Johanna E., Kaur, Sippy, Niskakoski, Anni, Knuutila, Sakari, Järvinen, Heikki, Mecklin, Jukka-Pekka, Peltomäki, Päivi

All colorectal cancer cell lines except RKO displayed active β-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.

Digital object identifier (DOI): 10.1155/2016/6089658

Nat Commun, 7, 10529
2016

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

Lescale, Chloé, Abramowski, Vincent, Bedora-Faure, Marie, Murigneux, Valentine, Vera, Gabriella, Roth, David B., Revy, Patrick, de Villartay, Jean-Pierre, Deriano, Ludovic

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Digital object identifier (DOI): 10.1038/ncomms10529