Silke Brüderlein

Genome-wide analysis of sixteen chordomas by comparative genomic hybridization and cytogenetics of the first human chordoma cell line, U-CH1.

Cytogenetic information on chordomas is rudimentary and restricted to GTG‐banding analysis of 26 cases worldwide. In this study, we present the chromosomal imbalances detected in a series of 16 chordomas (10 sacrococcyeal, five sphenooccipital, and one spinal) from 13 patients using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). On average, 3.2 losses and 4.2 gains were detected per tumor. The most common DNA copy number alterations were losses on chromosomal arms 3p (50%) and 1p (44%). Losses of 3p were detected in five of seven primary chordomas. Therefore, the loss of 3p might be an early event in chordoma genesis. The most common gains involved 7q (69%), 20 (50%), 5q (38%), and 12q (38%). Additionally, we raised the first human chordoma cell line, U‐CH1, from a recurrence of a sacral chordoma. U‐CH1 and its parent tumor had almost the same CGH profile. According to GTG‐banding and multicolor FISH, U‐CH1 has the following clonal chromosomal abnormalities: der(1)t(1;22), del(4), +del(5), +del(6), +7, del(9), del(10), +der(20)t(10;20), +21. Thus, the novel permanent human chordoma cell line U‐CH1 has chordoma‐typical cytogenetic aberrations. Our data suggest that tumor suppressor genes or mismatch repair genes (located at 1p31 and 3p14) and oncogenes (located in 7q36) might be involved in chordoma genesis.

Gain of chromosome arm 9p is characteristic of primary mediastinal b‐cell lymphoma (MBL): Comprehensive molecular cytogenetic analysis and presentation of a novel MBL cell line

Primary mediastinal B‐cell lymphoma (MBL) is an aggressive Non‐Hodgkins Lymphoma, which has been recognized as a distinct disease entity. We performed a comprehensive molecular cytogenetic study analyzing 43 MBLs. By comparative genomic hybridization (CGH), the most common aberrations were gains of chromosome arms 9p and Xq, which were present in 56% and 40% of cases, respectively. Based on the limited resolution of CGH, this technique may underestimate the real incidence of aberrations. Therefore, we also did an interphase cytogenetic study with eight DNA probes mapping to chromosome regions frequently altered in B‐cell lymphomas. With this approach, both 9p and Xq gains were found in more than 70% of cases (75% and 87%, respectively). The findings were compared with results obtained in 308 other B‐cell lymphomas. Gains in 9p were identified in only six of the 308 cases, and only one of these lymphomas with 9p gains was not primarily extranodal in origin (P < 10−20 for CGH data and P < 10−11 for fluorescence in situ hybridization data). We also present a novel MBL cell line, MedB‐1, which carries the genetic aberrations characteristic of this entity.

Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non‐neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U‐CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence‐like phenotype. The U‐CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]

Sex-specific telomere length profiles and age-dependent erosion dynamics of individual chromosome arms in humans

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Quantifying Telomere Lengths of Human Individual Chromosome Arms by Centromere-Calibrated Fluorescence in Situ Hybridization and Digital Imaging

mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro. Copyright

Low frequency of chromosomal imbalances in anaplastic ependymomas as detected by comparative genomic hybridization.

During aging, telomeres are gradually shortened, eventually leading to cellular senescence. By T/C-FISH (telomere/centromere-FISH), we investigated human telomere length differences on single chromosome arms of 205 individuals in different age groups and sexes. For all chromosome arms, we found a linear correlation between telomere length and donor age. Generally, males had shorter telomeres and higher attrition rates. Every chromosome arm had its individual age-specific telomere length and erosion pattern, resulting in an unexpected heterogeneity in chromosome- specific regression lines. This differential erosion pattern, however, does not seem to be accidental, since we found a correlation between average telomere length of single chromosome arms in newborns and their annual attrition rate. Apart from the above-mentioned sex-specific discrepancies, chromosome arm-specific telomere lengths were strikingly similar in men and women. This implies a mechanism that arm specifically regulates the telomere length independent of gender, thus leading to interchromosomal telomere variations.

Biallelic deletion within 16p13.13 including SOCS-1 in Karpas1106P mediastinal B-cell lymphoma line is associated with delayed degradation of JAK2 protein.

Telomere length analysis has aroused considerable interest in biology and oncology. However, most published data are pan-genomic Southern-blot-based estimates. We developed T/C-FISH (telomere/centromere-FISH), allowing precise measurement of individual telomeres at every single chromosome arm. Metaphase preparations are co-hybridized with peptide nucleic acid probes for telomeric sequences and the chromosome 2 centromere serving as internal reference. Metaphase images are captured and karyotyped using dedicated software. A software module determines the absolute integrated fluorescence intensities of the p- and q-telomeres of each chromosome and the reference signal. Normalized data are derived by calculating the ratio of absolute telomere and reference signal intensities, and descriptive statistics are calculated. T/C-FISH detects even small differences in telomere length. Using T/C-FISH we have discovered an epigenetic process occurring in the human male postzygote or early embryo: in umbilical cord blood lymphocytes, telomeres on male Xqs are around 1100 bp shorter than female Xqs.

LiSa‐2, a novel human liposarcoma cell line with a high capacity for terminal adipose differentiation

We screened 26 ependymomas in 22 patients (7 WHO grade I, myxopapillary, myE; 6 WHO grade II, E; 13 WHO grade III, anaplastic, aE) using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). 25 out of 26 tumors showed chromosomal imbalances on CGH analysis. The chromosomal region most frequently affected by losses of genomic material clustered on 13q (9/26). 6/7 myE showed a loss on 13q14‐q31. Other chromosomes affected by genomic losses were 6q (5/26), 4q (5/26), 10 (5/26), and 2q (4/26). The most consistent chromosomal abnormality in ependymomas so far reported, is monosomy 22 or structural abnormality 22q, identified in approximately one third of Giemsabanded cases with abnormal karyotypes. Using FISH, loss or monosomy 22q was detected in small subpopulations of tumor cells in 36% of cases. The most frequent gains involved chromosome arms 17 (8/26), 9q (7/26), 20q (7/26), and 22q (6/26). Gains on 1q were found exclusively in pediatric ependymomas (5/10). Using FISH, MYCN proto‐oncogene DNA amplifications mapped to 2p23‐p24 were found in 2 spinal ependymomas of adults. On average, myE demonstrated 9.14, E 5.33, and aE 1.77 gains and/or losses on different chromosomes per tumor using CGH. Thus, and quite paradoxically, in ependymomas, a high frequency of imbalanced chromosomal regions as revealed by CGH does not indicate a high WHO grade of the tumor but is more frequent in grade I tumors.

Chromosomal changes during progression of transitional cell carcinoma of the bladder and delineation of the amplified interval on chromosome arm 8q

Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling‐1 (SOCS‐1). We have previously shown that SOCS‐1 is biallelically mutated in the primary mediastinal B‐cell lymphoma (PMBL) cell line MedB‐1, resulting in impaired JAK2 degradation and sustained phospho‐JAK2 action. SOCS‐1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS‐1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB‐1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB‐1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS‐1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL.

Hyperinsulinemia Induces Insulin Resistance on Glucose and Lipid Metabolism in a Human Adipocytic Cell Line: Paracrine Interaction with Myocytes

LiSa‐2 is a stable cell line derived from a poorly differentiated, pleomorphic liposarcoma. In serum‐containing medium, LiSa‐2 cells are fibroblastoid and rapidly dividing. In a serum‐free, chemically defined culture medium containing physiological concentrations of insulin, triiodothyronine and cortisol, LiSa‐2 cells divide slower and, extensively storing fat, acquire adipocyte morphology. In contrast to fibroblastoid LiSa‐2 cells, these adipocyte‐like LiSa‐2 cells highly express transcripts for peroxisome proliferator‐activated receptor‐γ, lipoprotein lipase, fatty acid synthetase, hormone‐sensitive lipase, adipocyte most abundant gene transcript‐1, glycerol‐3‐phosphate‐dehydrogenase and the insulin‐sensitive glucose transporter‐4, all of which are specific for differentiated adipocytes. However, leptin mRNA expression was demonstrated only after preventing DNA methylation by incorporation of 5‐aza‐deoxycytidine into cellular DNA. Functionally, adipocyte‐like LiSa‐2 cells show increased insulin‐dependent glucose uptake and lipid synthesis and are sensitive to lipolytic agents. This cell line may serve as an in vitro model for studying the regulation of human liposarcoma differentiation and for screening drugs for induction of differentiation‐associated growth arrest in liposarcomas. Int. J. Cancer 88:889–894, 2000.