Publications

Filter by Keyword

Filter by Product


Methods Mol Biol, 730, 203--218
2011

The use of M-FISH and M-BAND to define chromosome abnormalities.

Ruth N. Mackinnon, Ilse Chudoba

Multicolour fluorescence in situ hybridisation (M-FISH) and multicolour banding (M-BAND) are advanced chromosome painting techniques combining multiple chromosome- or region-specific paints in one step. M-FISH identifies all chromosomes or chromosome arms at once, whereas M-BAND identifies the different regions of a single chromosome. The use of either or both can improve the accuracy of karyotyping and help identify cryptic chromosome rearrangements. These probes are prepared by pooling multiple chromosome- or chromosome region-specific DNA libraries, each labelled with a unique combination of fluorochromes. Commercial probes are available, avoiding the need for probe preparation. In the protocol described here, a commercial probe is used. Well-spread metaphases are prepared according to standard techniques, followed by alkaline denaturation and application of the denatured probe. After an incubation period, the slides are washed. A fluorescence microscope with filter sets specific to the fluorescent labels is used for analysis, together with specialised image analysis software. The software interprets the combination of fluorochromes to identify each chromosome and produce a false colour image specific for each chromosome or region. The single colour galleries - which show the hybridisation patterns of the individual fluorochromes - are useful to help interpret and confirm the false colour images produced by the software, including ambiguous signals.

Mol Cytogenet, 4(1), 8
2011

A rare case of t(11;22) in a mantle cell lymphoma like B-cell neoplasiaresulting in a fusion of IGL and CCND1: case report.

Cristiano Krings Rocha, Inka Praulich, Iris Gehrke, Michael Hallek, Karl-Anton Kreuzer

ABSTRACT: The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease.Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas.

Mol Cytogenet, 4, 16
2011

Biclonal myelodysplastic syndrome involving six chromosomes and monoallelicloss of RB1 - A rare case.

Walid Al-Achkar, Abdulsamad Wafa, Elisabeth Klein, Abdulmunim Aljapawe

ABSTRACT:Myelodysplastic syndrome (MDS) represents a group of clonal hematological disorders characterized by progressive cytopenia, and reflects to defects in erythroid, myeloid and megakaryocytic maturation. MDS is more frequently observed in older aged patients with cytogenetic abnormalities like monosomy of chromosome(s) 5 and/or 7. In 50\% of de novo MDS cases, chromosomal aberrations are found and rearrangements involving the retinoblastoma (RB1) gene in 13q14 are found.Here, we are presenting a case report of a rare biclonal MDS with a karyotype of 45, XY,-4, der(6)t(4;6)(p15.1;p21.3), der(8)t(4;8)(q31.2;q22), t(13;16)(q21.3;p11.2)11/45, XY, der(7)t(7;13)(p22.2~22.3;q21.3),-13 9. The patient was diagnosed according to WHO classification as refractory anemia with excess of blasts (RAEB-II).Immunophenotyping was positive for CD11b, CD11c, CD10, CD13, CD15, CD16 and CD33.We report, a novel and cytogenetically rare case of a biclonal MDS with complex chromosomal aberrations and deletion of RB1-gene in both clones. These findings are associated with a poor prognosis as the patient died 3 months after diagnosis.

BMC Biotechnol, 11, 121
2011

Combining M-FISH and Quantum Dot technology for fast chromosomalassignment of transgenic insertions.

Mohammed Yusuf, David L V Bauer, Daniel M Lipinski, Robert E MacLaren, Richard Wade-Martins, Kalim U Mir, Emanuela V Volpi

Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH) is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified.Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH), for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD) conjugate for the transgene detection.Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.

Leuk Res, 34(8), 1002--1006
August, 2010

Recurrent involvement of heterochromatic regions in multiple myeloma-amulticolor FISH study.

Kathrin Lange, Dorothea Gadzicki, Brigitte Schlegelberger, Gudrun G{\"o}hring

Chromosome aberrations are important prognostic markers in multiple myeloma (MM), but their identification may be hampered by complexity of the karyotypes. Using multicolor fluorescence in situ hybridization (mFISH), we found cryptic aberrations in 7 of 10 patients with a complex karyotype. Moreover, in addition to typical aberrations involving 1q, 13q, 14q and 17p and structural aberrations in chromosomes 1, 6, 9 and 19, (iso)dicentric chromosomes and whole-arm translocations were detected. These chromosome aberrations were generated by breaks in heterochromatic regions indicating an increased breakage of these regions, which may predispose to the generation of chromosome aberrations in multiple myeloma.

Mutat Res, 701(1), 52--59
August, 2010

Complex exchanges are responsible for the increased effectivenessof C-ions compared to X-rays at the first post-irradiation mitosis.

Ryonfa Lee, Sylwester Sommer, Carola Hartel, Elena Nasonova, Marco Durante, Sylvia Ritter

The purpose of the present study was to investigate as to what extent differences in the linear energy transfer (LET) are reflected at the chromosomal level. For this study human lymphocytes were exposed to 9.5 MeV/u C-ions (1 or 2 Gy, LET=175 keV/microm) or X-rays (1-6 Gy), harvested at 48, 72 or 96 h post-irradiation and aberrations were scored in first cycle metaphases using 24 color fluorescence in situ hybridization (mFISH). Additionally, in selected samples aberrations were measured in prematurely condensed G2-phase cells. Analysis of the time-course of aberrations in first cycle metaphases showed a stable yield of simple and complex exchanges after X-ray irradiation. In contrast, after C-ion exposure the yields profoundly increased with harvesting time complicating the estimation of the frequency of aberrations produced by high LET particles within the entire cell population. This is especially true for the yield of complex exchanges. Complex aberrations dominate the aberration spectrum produced by C-ions. Their fraction was about 50\% for the two measured doses. In contrast, isodoses of X-rays induced smaller proportions of complex aberrations (i.e. 5\% and 15\%, respectively). For both radiation qualities the fraction of complexes did not change with harvesting time. As expected from the different dose deposition of high and low LET radiation, complex exchanges produced by high LET C-ions involved more breaks and more chromosomes than those induced by isodoses of X-rays. Noteworthy, C-ions but not X-rays induced a small number of complex chromatid-isochromatid exchanges that are not expected for cells exposed in the G0-phase. The results obtained so far for cells arrested in G2-phase confirm these patterns. Altogether our data show that the increased effectiveness of C-ions for the induction of aberrations in first cycle cells is determined by complex exchanges, whereas for simple exchanges the relative biological effectiveness is about one.

Radiat Res, 174(1), 14--19
July, 2010

Inversions in chromosome 10 of human thyroid cells induced by acceleratedheavy ions.

D. Pignalosa, S. Ritter, M. Durante

Papillary thyroid carcinoma (PTC) is a known radiation-induced tumor. Rearrangements in human chromosome 10 and in particular intrachromosomal exchanges are often associated with PTC formation. In this study we measured intrachromosomal exchanges in human thyroid follicular cells exposed to sparsely or densely ionizing radiation. Assuming that inversions in chromosome 10 are a biomarker of PTC risk, we estimated the relative biological effectiveness (RBE) of heavy ions using a molecular marker in vitro. The analysis of chromosomal aberrations was performed with the mBAND technique, which allows detection of both inter- and intrachromosomal exchanges. Our results do not show any significant increase in the yield of intrachanges in samples exposed to heavy ions compared to X rays. Within the constraints imposed by the experimental model we used, we conclude that heavy ions would not necessarily be more effective than X rays in the induction of thyroid cancer.

Cancer Genet Cytogenet, 200(2), 79--99
July, 2010

Transgenic oncogenes induce oncogene-independent cancers with individualkaryotypes and phenotypes.

Andreas Klein, Nan Li, Joshua M Nicholson, Amanda A McCormack, Adolf Graessmann, Peter Duesberg

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.

Radiat Res, 174(1), 20--26
July, 2010

Influence of nuclear geometry on the formation of genetic rearrangementsin human cells.

M. Durante, D. Pignalosa, J. A. Jansen, X. F. Walboomers, S. Ritter

Interphase chromosomes are divided into discrete domains, with limited overlapping and movement. We explored the role of nuclear topology in the formation of chromosome aberrations by irradiating normal human fibroblasts with high-energy heavy ions from different directions. Cells with elliptical nuclei were grown in an aligned manner onto micrometer grooved culturing substrates to have a predetermined orientation with respect to the accelerated iron ions. Particles were directed either perpendicular to the cell layer or along the major or minor axis of the nucleus. Analysis of chromosome aberrations by mFISH showed that, at the same radiation dose, the yield of chromosomal damage and its complexity are largely modified by the irradiation geometry. The results demonstrate that the architecture of the cell nucleus determines the formation of chromosomal rearrangements.

Mutat Res
March, 2010

mBAND analysis of chromosome aberrations in human epithelial cellsinduced by gamma-rays and secondary neutrons of low dose rate.

M. Hada, B. Gersey, P. B. Saganti, R. Wilkins, F. A. Cucinotta, H. Wu

Human risks from chronic exposures to both low- and high-LET radiation are of intensive research interest in recent years. In the present study, human epithelial cells were exposed in vitro to gamma-rays at a dose rate of 17mGy/h or secondary neutrons of 25mGy/h. The secondary neutrons have a broad energy spectrum that simulates the Earth's atmosphere at high altitude, as well as the environment inside spacecrafts like the Russian MIR station and the International Space Station (ISS). Chromosome aberrations in the exposed cells were analyzed using the multicolor banding in situ hybridization (mBAND) technique with chromosome 3 painted in 23 colored bands that allows identification of both inter- and intrachromosome exchanges including inversions. Comparison of present dose responses between gamma-rays and neutron irradiations for the fraction of cells with damaged chromosome 3 yielded a relative biological effectiveness (RBE) value of 26+/-4 for the secondary neutrons. Our results also revealed that secondary neutrons of low dose rate induced a higher fraction of intrachromosome exchanges than gamma-rays, but the fractions of inversions observed between these two radiation types were indistinguishable. Similar to the previous findings after acute radiation exposures, most of the inversions observed in the present study were accompanied by other aberrations. The fractions of complex type aberrations and of unrejoined chromosomal breakages were also found to be higher in the neutron-exposed cells than after gamma-rays. We further analyzed the location of the breaks involved in chromosome aberrations along chromosome 3, and observed hot spots after gamma-ray, but not neutron, exposures.

Radiother Oncol, 95, 73-78
2010

Chromosomal aberrations in peripheral blood lymphocytes of prostatecancer patients treated with IMRT and carbon ions.

Carola Hartel, Anna Nikoghosyan, Marco Durante, Sylwester Sommer, Elena Nasonova, Claudia Fournier, Ryonfa Lee, J{\"u}rgen Debus, Daniela Schulz-Ertner, Sylvia Ritter

BACKGROUND AND PURPOSE: To investigate the cytogenetic damage in blood lymphocytes of patients treated for prostate cancer with different radiation qualities and target volumes. MATERIALS AND METHODS: Twenty patients receiving carbon-ion boost irradiation followed by IMRT or IMRT alone for the treatment of prostate cancer entered the study. Cytogenetic damage induced in peripheral blood lymphocytes of these patients was investigated at different times during the radiotherapy course using Giemsa staining and mFISH. A blood sample from each patient was taken before initiation of radiation therapy and irradiated in vitro to test for individual radiosensitivity. In addition, in vitro dose-effect curves for the induction of chromosomal exchanges by X-rays and carbon ions of different energies were measured. RESULTS: The yield of chromosome aberrations increased during the therapy course, and the frequency was lower in patients irradiated with carbon ions as compared to patients treated with IMRT with similar target volumes. A higher frequency of aberrations was measured by increasing the target volume. In vitro, high-LET carbon ions were more effective than X-rays in inducing aberrations and yielded a higher fraction of complex exchanges. The yield of complex aberrations observed in vivo was very low. CONCLUSION: The investigation showed no higher aberration yield induced by treatment with a carbon-ion boost. In contrast, the reduced integral dose to the normal tissue is reflected in a lower chromosomal aberration yield when a carbon-ion boost is used instead of IMRT alone. No cytogenetic #signature# of exposure to densely ionizing carbon ions could be detected in vivo.

Int J Radiat Biol, 85(11), 1051--1059
November, 2009

Response of human hematopoietic stem and progenitor cells to energeticcarbon ions.

Daniela Becker, Thilo Els{\"a}sser, Torsten Tonn, Erhard Seifried, Marco Durante, Sylvia Ritter, Claudia Fournier

To characterise the radiation response of human hematopoietic stem and progenitor cells (HSPC) with respect to X and carbon ion irradiation.HSPC from peripheral blood of healthy donors treated with granulocyte-colony stimulating factor (G-CSF) were enriched for the transmembrane glycoprotein CD34 (cluster of differentiation) and irradiated with X rays or carbon ions (29 keV/microm monoenergetic beam and 60-85 keV/microm spread-out Bragg peak), mimicking radiotherapy conditions. Apoptotic cell death, cell cycle progression and the frequency of chromosomal aberrations were determined.After radiation exposure no inhibition in the progression of the cell cycle was detected. However, an enhanced frequency of apoptotic cells and an increase in aberrant cells were observed, both effects being more pronounced for carbon ions than X rays, resulting in a relative biological effectiveness (RBE) of 1.4-1.7. The fraction of complex-type aberrations was higher following carbon ion exposure.RBE values of carbon ions are low, as expected for radiosensitive cells. The observed frequencies of apoptotic cells and chromosome aberrations in HSPC are similar to those reported for human peripheral blood lymphocytes suggesting that at least with respect to apoptosis and chromosomal aberrations mature lymphocytes reflect the respective radiation responses of their proliferating progenitors.

Cancer Genet Cytogenet, 193(2), 123--126
September, 2009

A case of childhood acute myeloid leukemia AML (M5) with a neocentricchromosome neo(1)(qter-->q23 approximately 24::q23 approximately24-->q43-->neo-->q43-->qter) and tetrasomy of chromosomes 8 and 21.

de Figueiredo, Amanda Faria, Hasmik Mkrtchyan, Thomas Liehr, Eliane Maria Soares Ventura, de Jesus Marques-Salles, Terezinha, Neide Santos, Raul Corr{\^e}a Ribeiro, Eliana Abdelhay, Maria Luiza Macedo Silva

Hyperdiploidy is rarely observed in childhood acute myeloid leukemia (AML). Described here is the case of a 2(1/2)-year-old girl with AML-M5 and 51 chromosomes characterized by double tetrasomy of chromosomes 8 and 21 and also a neocentric derivative chromosome neo(1)(qter-->q23 approximately 24::q23 approximately 24-->q43-->neo-->q43-->qter). Little is known about the prognostic significance of these chromosomal abnormalities in childhood AML. In the actual case, complete remission was achieved after chemotherapy, which continued for 7 months. No acquired neocentric chromosome 1 has been described previously, even though neocentromere formation has been reported for other chromosomes in neoplasms.

Cancer Genet. Cytogenet., 193, 44- 53
2009

Gene amplification in myeloid leukemias elucidated by fluorescence in situ hybridization.

K.C. Rayeroux, L.J. Campbell

Gene amplification in hematologic malignancies is uncommon. When karyotyping leukemia cells, gene amplification is generally seen as double-minute (dmin) chromosomes and homogeneously staining regions (hsr). One of the more commonly amplified regions is MYC at 8q24.21, but amplification of MLL at 11q23 and regions on 9p, 19q, and elsewhere on 11q have been reported. Increased copy number of these genes has been associated with poor prognosis. Over an 11-year period, we identified 31 cases of possible gene amplification, 27 of which had enough sample material for further investigations. A total of 17 cases had dmin only, 13 cases had hsr only, and 1 case had both dmin and hsr in the karyotype. Fluorescence in situ hybridization (FISH) analysis identified amplification of MYC in 12 cases, all on dmin, and amplification of MLL in eight cases, all on hsr. Regions other than MYC and MLL were amplified in eight cases and, using multicolor FISH and multicolor banding, we identified a number of novel regions of amplification: 13q11 approximately q12.1, 15q26.1 approximately q26.3, and 17q12. We also identified one case where two different chromosomal regions were simultaneously amplified in the same cell line.

Mol Cytogenet, 2, 7
2009

Unbalanced chromosome 1 abnormalities leading to partial trisomy1q in four infants with Down syndrome and acute megakaryocytic leukemia.

Maria Luiza Macedo Silva, do Socorro Pombo-de-Oliveira, Maria, Susana C Raimondi, Hasmik Mkrtchyan, Eliana Abdelhay, de Figueiredo, Amanda Faria, de Souza, Mariana Tavares, Daniela Ribeiro Ney Garcia, de Ventura, Eliane Maria Soares, de Sousa, Adriana Martins, Thomas Liehr

ABSTRACT: BACKGROUND: Children with Down syndrome (DS) have an increased risk of childhood acute leukemia, especially acute megakaryoblastic leukemia (AMKL) also called acute myeloid leukemia (AML) type M7. Here four yet unreported infants with such malignancies are reported. RESULTS: An unbalanced translocation involving chromosome 1 was identified by GTG banding in all cases. These were characterized in more detail by molecular cytogenetic approaches. Additional molecular analysis revealed in three of the four cases mutations in exon 2 of the GATA binding protein 1 (globin transcription factor 1), located in Xp11.23. CONCLUSION: Our results corroborate that abnormalities of chromosome 1 are common in DS-associated AMKL. Whether this chromosomal region contains gene(s) involved in hematopoietic malignant transformation remains to be determined.

Cancer Genet Cytogenet, 182(1), 56--60
April, 2008

Banding and molecular cytogenetic studies detected a CBFB-MYH11 fusion gene that appeared as abnormal chromosomes 1 and 16 in a baby with acute myeloid leukemia FAB M4-Eo.

Maria Luiza Macedo Silva, Susana C Raimondi, Eliana Abdelhay, Madeleine Gross, Hasmik Mkrtchyan, de Figueiredo, Amanda Faria, Raul C Ribeiro, de Jesus Marques-Salles, Terezinha, Elaine S Sobral, Marcelo Poirot Gerardin Land, Thomas Liehr

The acute myeloid leukemia (AML) subtype M4Eo occurs in 5\% of all AML cases and is usually associated with either an inv(16)(p13.1q22) or a t(16;16)(p13.1;q22) chromosomal abnormality. At the molecular level, these abnormalities generate a CBFB-MYH11 fusion gene. Patients with this genetic alteration are usually assigned to a low-risk group and thus receive standard chemotherapy. AML-M4Eo is rarely found in infants. We describe clinical, conventional banding, and molecular cytogenetic data for a 12-month-old baby with AML-M4Eo and a chimeric CBFB-MYH11 fusion gene masked by a novel rearrangement between chromosomes 1 and 16. This rearrangement characterizes a new type of inv(16)(p13.1q22) masked by a chromosome translocation.

Digital object identifier (DOI): 10.1016/j.cancergencyto.2007.12.014

Radiation Research, 170, 458- 466
2008

Chromosome inter- and intrachanges detected by arm-specific DNA probes in the progeny of human lymphocytes exposed to energetic heavy ions.

D. Pignalosa, A. Bertucci, G. Gialanella, G. Grossi, L. Manti, M. Pugliese, P. Scampoli, M. Durante

We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or gamma rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to gamma rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

Med Hypotheses, 71(1), 126--140
2008

Defining the steps that lead to cancer: replicative telomere erosion,aneuploidy and an epigenetic maturation arrest of tissue stem cells.

Reinhard Stindl

Recently, an influential sequencing study found that more than 1700 genes had non-silent mutations in either a breast or colorectal cancer, out of just 11 breast and 11 colorectal tumor samples. This is not surprising given the fact that genomic instability is the hallmark of cancer cells. The plethora of genomic alterations found in every carcinoma does not obey the 'law of genotype-phenotype correlation', since the same histological subtype of cancer harbors different gene mutations and chromosomal aberrations in every patient. In an attempt to make sense out of the observed genetic and chromosomal chaos in cancer, I propose a cascade model. According to this model, tissue regeneration depends on the proliferation and serial activation of stem cells. Replicative telomere erosion limits the proliferative life span of adult stem cells and results in the Hayflick limit (M1). However, local tissue exhaustion or old age might promote the activation of M1-deficient tissue stem cells. Extended proliferation of these cells leads to telomere-driven chromosomal instability and aneuploidy (abnormal balance of chromosomes and/or chromosome material). Several of the aforementioned steps have been already described in the literature. However, in contrast to common theories, it is proposed here that the genomic damage blocks the epigenetic differentiation switch. As a result of aneuploidy, differentiation-specific genes cannot be activated by modification of methylation patterns. Consequently, the phenotype of cancer tissue is largely determined by the epigenetic maturation arrest of tissue stem cells, which in addition enables a fraction of cancer cells to proliferate, invade and metastasize, as normal adult stem cells do. The new model combines genetic and epigenetic alterations of cancer cells in one causative cascade and offers an explanation for why identical histologic cancer types harbor a confusing variety of chromosomal and gene aberrations. The Viennese Cascade, as presented here, may end the debate on if and how 'tumor-unspecific' aneuploidy leads to cancer.

Mol Cancer, 7, 76
2008

Human ESCs predisposition to karyotypic instability: Is a matterof culture adaptation or differential vulnerability among hESC linesdue to inherent properties?

Puri Catalina, Rosa Montes, Gertru Ligero, Laura Sanchez, de la Cueva, Teresa, Clara Bueno, Paola E Leone, Pablo Menendez

BACKGROUND: The use of human embryonic stem cells (hESCs) in research is increasing and hESCs hold the promise for many biological, clinical and toxicological studies. Human ESCs are expected to be chromosomally stable since karyotypic changes represent a pitfall for potential future applications. Recently, several studies have analysed the genomic stability of several hESC lines maintained after prolonged in vitro culture but controversial data has been reported. Here, we prompted to compare the chromosomal stability of three hESC lines maintained in the same laboratory using identical culture conditions and passaging methods. RESULTS: Molecular cytogenetic analyses performed in three different hESC lines maintained in parallel in identical culture conditions revealed significant differences among them in regard to their chromosomal integrity. In feeders, the HS181, SHEF-1 and SHEF-3 hESC lines were chromosomally stable up to 185 passages using either mechanical or enzymatic dissection methods. Despite the three hESC lines were maintained under identical conditions, each hESC line behaved differently upon being transferred to a feeder-free culture system. The two younger hESC lines, HS181 (71 passages) and SHEF-3 (51 passages) became chromosomally unstable shortly after being cultured in feeder-free conditions. The HS181 line gained a chromosome 12 by passage 17 and a marker by passage 21, characterized as a gain of chromosome 20 by SKY. Importantly, the mosaicism for trisomy 12 gradually increased up to 89\% by passage 30, suggesting that this karyotypic abnormality provides a selective advantage. Similarly, the SHEF-3 line also acquired a trisomy of chromosome 14 as early as passage 10. However, this karyotypic aberration did not confer selective advantage to the genetically abnormal cells within the bulk culture and the level of mosaicism for the trisomy 14 remained overtime between 15\%-36\%. Strikingly, however, a much older hESC line, SHEF-1, which was maintained for 185 passages in feeders did not undergo any numerical or structural chromosomal change after 30 passages in feeder-free culture and over 215 passages in total. CONCLUSION: These results support the concept that feeder-free conditions may partially contribute to hESC chromosomal changes but also confirm the hypothesis that regardless of the culture conditions, culture duration or splitting methods, some hESC lines are inherently more prone than others to karyotypic instability.

Cytogenet. Genome Res., 121, 79- 87
2008

Elevated chromosome translocation frequencies in New Zealand nuclear test veterans.

M.A. Wahab, E.M. Nickless, R. Najar-M'Kacher, C. Parmentier, J.V. Podd, R.E. Rowland

In 1957/58 the British Government conducted a series of nuclear tests in the mid-Pacific codenamed Operation Grapple, which involved several naval vessels from Britain and New Zealand. Two New Zealand frigates with 551 personnel onboard were stationed at various distances between 20 and 150 nautical miles from ground zero. In the present study we applied the cytomolecular technique mFISH (multicolour fluorescent in situ hybridisation) to investigate a potential link between chromosome abnormalities and possible past radiation exposure in New Zealand nuclear test veterans who participated in Operation Grapple. Compared to age matched controls, the veterans showed significantly higher (P < 0.0001) frequencies of chromosomal abnormalities (275 translocations and 12 dicentrics in 9,360 cells vs. 96 translocations and 1 dicentric in 9,548 cells in the controls), in addition to a significant excess of CCRs (complex chromosomal rearrangements) in the veterans. A Kolmogorov-Smirnoff test showed that the distributions of translocations for the two groups were significantly different.