Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.     

The characterisation of the lymphoma cell line U937, using comparative genomic hybridisation and multi-plex FISH.

The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed.     

Chromosomal DNA balance in human stem cell line 4BL

Ploidy of a chromosome set and some regular structural aberrations in the new human 4BL cell line by passage 205 have been characterized in the previous cytogenetic studies. The purpose of this study was to investigate, using the array CGH and FISH methods, the nature of regular monosomies in particular homologous pairs. Structural aberrations were detected in all the chromosome pairs distinguished as monosomies according to classical cytogenetic analyses. The most notable alterations have been detected in chromosomes 2, 4, 10, 13, and 17. Massive genetic material losses were a probable cause for the monosomy of chromosomes 4, 10, 13, and 17. The monosomy of the second pair of chromosomes was caused by a substantial transformation in one of the homologs typified as multiple duplications and the formation of a derivative—der(2)t(2;?)(q21;?). The application of array CGH aided us in identifying the regions of structural aberrations in chromosomes 2, 4, 10, 13, and 17, that allowed a more accurate identification with the use of the multicolor FISH method. The obtained results confirm the hypothesis concerning a coordinated emergence of deletions and duplications and their stabilizing effect on transformed chromosomes.     

DNA Copy Number Aberrations,and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes

Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents.     

Chromosomal study of polycythemia during different stages of the disease

A cytogenetic analysis of blood and bone marrow cells of 15 polycythemia vera patients was carried out at different stages of disease during the G-banding technique. Chromosome aberrations of single character were noted before treatment only in one case, i.e. with the patient at stage II of disease. Cell clones with marker chromosomes were revealed in 6 of 9 patients examined in the course of treatment at stages II and III. The cytogenetic analysis was applied to the terminal stage of polycythemia (blast crisis) in one case, when 3 aberrant clones with multiple quantitative and structural chromosome rearrangements were discovered in blood cell cultures with and without PHA. No preferential involvement of definite chromosomes in aberrations was noticed in all the cases examined, no deletion of the 20q --chromosome being discovered. The role of the treatment in the induction of chromosome aberrations is discussed in addition to its dependence on the stage of disease. It is possible that all the clones of pathological character may appear during the long-termed course of polycythemia in patients treated at more serious stages of the disease.     

Natural killer cell malignancies: clinicopathologic and molecular features

Malignancies of natural killer (NK) cells have increasingly been recognized as distinct clinicopathological entities. The tumor cells are characterized by an immunophenotype of CD2+, surface CD3-, cytoplasmic CD3epsilon+, and CD56+. The T cell receptor gene is in germline configuration, and a consistent association with Epstein-Barr virus is demonstrable. Pathologically, the tumor cells show variable cytological appearances, with frequent angioinvasion and angiocentricity associated with zonal necrosis. Clinically, most cases affect the nasal cavity or other parts of the upper aerodigestive tract, and are referred to as nasal NK cell lymphoma. A minority involve extranasal sites such as the skin, gastrointestinal tract and testis, and are often referred to as extranasal NK cell lymphoma. A particularly aggressive form presents fulminantly as disseminated disease, sometimes with a leukemic phase, and is referred to as aggressive NK cell lymphoma/leukemia. Cytogenetic and molecular analysis have shown DNA losses at chromosomes 6q, 11q, 13q and 17p to be recurrent aberrations in NK cell malignancies. Frequent DNA gains are also found in chromosomes 1p, 6p, 11q, 12q, 17q, 19p, 20q, and Xp. These regions of DNA losses and gains should be targets for further investigation in order to understand the molecular pathogenesis of this lymphoma. Finally, optimal treatment modalities need to be determined, as all subtypes of NK cell malignancies are associated with a poor prognosis.     

Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas

Summary We have investigated cytogenetically a total of 35 solitary lipomas, 10 of which have been reported previously. Of the 25 tumours presented herein for the first time, clonal chromosome aberrations were detected in 17. The remaining eight had normal karyotypes, although two of them had nonclonal aberrations in about one quarter of the cells. Based on the cytogenetic findings in all 35 lipomas, four major subgroups can be distinguished. These are characterized by: (I) hyperdiploid karyotypes including one or more supernumerary ring chromosomes (5 cases); (II) diploid karyotypes with mostly balanced rearrangements involving 12q13-14 (13 cases), including the rearrangement t(3;12) (q27-28;q13-14) in 4 cases; (III) hypodiploid or diploid karyotypes with other aberrations than ring chromosomes or rearrangements of 12q13-14 (8 cases); and (IV) normal karyotypes (9 cases).     

M-FISH applications in clinical genetics

Until recently, presence of de novo marker or derivative chromosomes was quite problematic for genetic counseling especially in prenatal diagnosis, because characterization of marker and derivative chromosomes by conventional cytogenetic techniques was nearly impossible. However, recently developed molecular cytogenetic technique named Multicolor Fluorescence in Situ Hybridization (M-FISH) which paints all human chromosomes in 24 different colors allows us to characterize marker and derivative chromosomes in a single hybridization. In this study, we applied M-FISH to determine the origin of 3 marker and 3 derivative chromosomes. Marker chromosomes were found to originate from chromosome 15 in two postnatal and one prenatal case. Of these, one of the postnatal cases displayed clinical findings of inv dup (115) syndrome and the other of infertility, and the prenatal case went through amniocentesis due to the triple test results. Karyotypes of the patients with derivative chromosomes were designated as 46,XY,der (21)t(1;21)(q32;p11), 46,XX,der(8)t(8;9)(p23;p22) and 46,XX,der(18)t(18;20)(q32;p11.2) according to cytogenetic and M-FISH studies. All of the M-FISH results were confirmed with locus specific or whole chromosome painting probes. The case with der (8)t(8;9) had trisomy 9(p22-pter) and monosomy 8(p23-pter) due to this derivative chromosome. The case with der(18)t(18;20) had trisomy 20(p11.2-pter) and monosomy 18(q32-qter). Parental origins of the derivative chromosomes were analyzed using microsatellite markers located in the trisomic chromosomal segments. Patients' clinical findings were compared with the literature.     

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding,CGH, M-FISH,and chromosome- and locus-specific FISH

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.     

Multicolor banding technique, spectral color banding (SCAN): new development and applications

Conventional banding techniques can characterize chromosomal aberrations associated with tumors and congenital diseases with considerable precision. However, chromosomal aberrations that have been overlooked or are difficult to analyze even by skilled cytogeneticists were also often noted. Following the introduction of multicolor karyotyping such as spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH), it is possible to identify this kind of cryptic or complex aberration comprehensively by a single analysis. To date, multicolor karyotyping techniques have been established as useful tools for cytogenetic analysis. However, since this technique depends on whole chromosome painting probes, it involves limitations in that the origin of aberrant segments can be identified only in units of chromosomes. To overcome these limitations, we have recently developed spectral color banding (SCAN) as a new multicolor banding technique based on the SKY methodology. This new technique may be deemed as an ideal chromosome banding technique since it allows representation of a multicolor banding pattern matching the corresponding G-banding pattern. We applied this technique to the analysis of chromosomal aberrations in tumors that had not been fully characterized by G-banding or SKY and found it capable of (1) detecting intrachromosomal aberrations; (2) identifying the origin of aberrant segments in units of bands; and (3) precisely determining the breakpoints of complex rearrangements. We also demonstrated that SCAN is expected to allow cytogenetic analysis with a constant adequate resolution close to the 400-band level regardless of the degree of chromosome condensation. As compared to the conventional SKY analysis, SCAN has remarkably higher accuracy for a particular chromosome, allowing analysis in units of bands instead of in units of chromosomes and is hence promising as a means of cytogenetic analysis.     

Biologic,Immunocytochemical, and Cytogenetic Characterization of Two New Human Melanoma Cell Lines: IIB-MEL-LES and IIB-MEL-IAN

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD₃ and GD₂ gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10,20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.     

Long-term cultivation of plasma cell leukemia cells and autologous lymphoblasts (LCL) in vitro: a comparative study.

Two long-term cell lines were established in vitro from the peripheral blood of a patient with plasma cell leukemia: one line with plasma cell proliferation, the other with lymphoblastoid cell proliferation (LCL). The 9-month-old plasma cell line showed the typical morphology of plasmoblasts. The cells neither had B- nor T-lymphocyte characterisitics, were EBV negative, and showed aneuploidy with various marker chromosomes, including the 14 q+ marker. The cytogenetic findings indicate a monoclonal proliferation of the plasmacells. No tumor growth in thymusless nude mice could be induced upon intracranial inoculation with these cells. In contrast, the autologous LCL, cultured after addition of exogenous EBV, showed the characteristic markers of lymphoblastoid cells, with the typical morphology of pear- and handmirror-shaped lymphoblasts, growing in clumps. They had C3- and Fc-receptors, surface-Ig, E-rosette-negativity, a diploid karyotype, and EBV dependent macromolecule synthesis. They lymphoblastoid cells produced intracranial tumors in nude mice in 8 out of 8 attempts.     

Incidence of numerical chromosome aberrations in meningioma tumors as revealed by fluorescence in situ hybridization using 10 chromosome-specific probes

OBJECTIVE: Although information on the cytogenetic characteristics of meningioma tumors has accumulated progressively over the past few decades, information on the genetic heterogeneity of meningiomas is still scanty. The aim of the present study was to analyze by interphase fluorescence in situ hybridization (FISH) the incidence of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y in a group of 70 consecutive meningioma tumors. Another goal was to establish the potential associations among the altered chromosomes, as a way to assess both intertumoral and intratumoral heterogeneity. METHODS: For the purpose of the study, 70 patients diagnosed with meningioma were analyzed. Interphase FISH for the detection of numerical abnormalities for chromosomes 1, 9, 10, 11, 14, 15, 17, 22, X, and Y was applied to fresh tumor samples from each of the patients studied. RESULTS: The overall incidence of numerical abnormalities was 76%. Chromosome Y in males and chromosome 22 in the whole series were the most common abnormalities (46% and 61%, respectively). Despite the finding that monosomy of chromosome 22/22q(-) deletions are the most frequent individual abnormality (53%), we have observed that chromosome gains are significantly more common than chromosome losses (60% versus 40%). Chromosome gains corresponded to abnormalities of chromosomes 1 (27%), 9 (25%), 10 (23%), 11 (22%), 14 (33%), 15 (22%), 17 (23%), and X in females (35%) and males (23%) whereas chromosome losses apart from chromosome 22 frequently involved chromosomes 14 (19%), X in males (23%), and Y in males (32%). Although an association was found among most gained chromosomes on one side and chromosome losses on the other side, different association patterns were observed. Furthermore, in the latter group, monosomy 22/22q(-) was associated with monosomy X in females and monosomy 14/14q(-) was associated with nulisomy Y in males. In addition, chromosome losses usually involved a large proportion of the tumor cells whereas chromosome gains were restricted to small tumor cell clones, including tetraploid cells. CONCLUSIONS: Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, as assessed by interphase FISH.     

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Summary Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21–q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2,12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.     

Genomic imbalances in esophageal squamous cell carcinoma identified by molecular cytogenetic techniques

This review summarizes the chromosomal changes detected by molecular cytogenetic approaches in esophageal squamous cell carcinoma (ESCC), the ninth most common malignancy in the world. Whole genome analyses of ESCC cell lines and tumors indicated that the most frequent genomic gains occurred at 1, 2q, 3q, 5p, 6p, 7, 8q, 9q, 11q, 12p, 14q, 15q, 16, 17, 18p, 19q, 20q, 22q and X, with focal amplifications at 1q32, 2p16-22, 3q25-28, 5p13-15.3, 7p12-22, 7q21-22, 8q23-24.2, 9q34, 10q21, 11p11.2, 11q13, 13q32, 14q13-14, 14q21, 14q31-32, 15q22-26, 17p11.2, 18p11.2-11.3 and 20p11.2. Recurrent losses involved 3p, 4, 5q, 6q, 7q, 8p, 9, 10p, 12p, 13, 14p, 15p, 18, 19p, 20, 22, Xp and Y. Gains at 5p and 7q, and deletions at 4p, 9p, and 11q were significant prognostic factors for patients with ESCC. Gains at 6p and 20p, and losses at 10p and 10q were the most significant imbalances, both in primary carcinoma and in metastases, which suggested that these regions may harbor oncogenes and tumor suppressor genes. Gains at 12p and losses at 3p may be associated with poor relapse-free survival. The clinical applicability of these changes as markers for the diagnosis and prognosis of ESCC, or as molecular targets for personalized therapy should be evaluated.     

Genetic alterations in pulmonary epithelioid hemangioendothelioma and epithelioid angiosarcoma

Epithelioid hemangioendothelioma (EHE) is a low-to-intermediate-grade vascular tumor that occurs in many organs, and epithelioid angiosarcoma (EA) is a subtype of angiosarcoma that is associated with high-grade malignancy. These two types of tumors have different forms of biological behavior. Pulmonary epithelioid hemangioendothelioma (PEH) and epithelioid angiosarcoma (PEA) are both very rare, and genetic studies on them are extremely limited. We examined and compared the cytogenetic characteristics of these two types of lung tumors in two patients utilizing the Array-Comparative Genomic Hybridization (Array-CGH) method. Considerable differences in the cytogenetic characteristics were observed between the two types of tumors. Small fragment gains (<10 MB) were dominant in PEH, whereas large fragment gains and deletions (>10 MB) were dominant in PEA. Some large fragment alterations, such as gains in chromosomes 19q and 19p, and deletions in chromosomes 9p and 13q, involved over half of a chromosome arm. PEH and PEA showed great cytogenetic differences; therefore, further genetic studies on these two types of tumors are warranted.     

Chromosome aberrations induced by high-LET carbon ions in radiosensitive and radioresistant tumour cells

Chromosome aberration formation was analysed in two human tumour cell lines displaying different radiosensitivity. Aberrations involving chromosomes 2, 4, and 5 were studied in one radioresistant cell line (WiDr) and in one radiosensitive cell line (MCF-7). Chromosome aberrations were studied by application of single-colour FISH. We studied the effects of monoenergetic 100 MeV/u carbon ions and carbon ions from extended Bragg peak. Chromosome aberrations induced by carbon ions were compared with aberrations induced by standard 200 kV X-rays. In both tumour cell lines, carbon ions induced aberrations more effectively than X-rays. The radioresistance and radiosensitivity of the corresponding cell lines, as observed for X-rays, were also found after carbon ion irradiation. In both cell lines, the typical effects of ion irradiation were an increased proportion of cells containing complex aberrations, and an increased complexity of these complex exchanges. However, comparable effects were induced in MCF-7 cells by a much lower dose than in WiDr cells. Insertions were also induced more efficiently in MCF-7 cells than in WiDr cells.     

Establishment of a cell line derived from a canine prostate carcinoma with a highly rearranged karyotype

Akin to the situation in humans, dogs are frequently affected by tumors of the prostate. The malignancies share many similarities between both species, for example, median age at the onset of the disease and metastatic behavior. In human prostatic tumor samples, investigations of prepared metaphase spreads showed a variety of chromosomal aberrations, with trisomies of chromosomes 7, 8, and 17 as the leading cytogenetic abnormalities. In this article we present one case of a canine adenocarcinoma of the prostate, including clinical examination and establishment of a cell line from a tumor sample obtained from the affected 10-year-old male Briard. Searching for similarities between both species in respect to chromosomal changes within the tumor samples, we investigated prepared metaphases of the canine cell line cytogenetically. These investigations presented a highly rearranged karyotype showing a large biarmed marker consisting of material from chromosomes 1 and 2 in addition to centromeric fusions between dog chromosomes 1 and 5 that both could be identified in every metaphase investigated, while centric fusions of chromosomes 4 and 5 occurred in up to 50% of the metaphases. The cell line grew very well and showed evidence of being spontaneously immortalized when it crossed the 20th passage.     

Chromosomal aberrations involving telomeres in BRCA1 deficient human and mouse cell lines

Cells defective in BRCA1 show genomic instability as evidenced by increased radiosensitivity, the presence of chromosomal abnormalities and the loss of heterozygosity at many loci. Reported chromosomal abnormalities in BRCA1 deficient cells include dicentric chromosomes. Dicentric chromosomes, in some cases, may arise as a result of end-to-end chromosome fusions, which represent signatures of telomere dysfunction. In this study we examined BRCA1 deficient human and mouse cells for the presence of chromosomal aberrations indicative of telomere dysfunction. We identified a lymphoblastoid cell line, GM14090, established from a BRCA1 carrier that showed elevated levels of dicentric chromosomes. Molecular cytogenetic analysis revealed that these dicentric chromosomes result from end-to-end chromosome fusions. The frequency of end-to-end chromosome fusions did not change after exposure of GM14090 cells to bleomycin but we observed elevated levels of chromosomal abnormalities involving interactions between DNA double strand breaks and uncapped telomeres in this cell line. We observed similar chromosomal abnormalities involving telomeres in the breast cancer cell line, HCC1937, homozygous for BRCA1 mutation. Finally, we analyzed mouse embryonic stem cells lacking functional Brca1 and observed the presence of telomere dysfunction following exposure of these cells to bleomycin. Our results reveal cytogenetic evidence of telomere dysfunction in BRCA1 deficient cells.