<p>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.</p>

ABSTRACT: Malignant melanomas are characterized by increased karyotypic complexity, extended aneuploidy and heteroploidy. We report a melanoma metastasis to the peritoneal cavity with an exceptionally stable, abnormal pseudodiploid karyotype as verified by G-Banding, subtelomeric, centromeric and quantitative Fluorescence in Situ Hybridization (FISH). Interestingly this tumor had no detectable telomerase activity as indicated by the Telomere Repeat Amplification Protocol. Telomeric Flow-FISH and quantitative telomeric FISH on mitotic preparations showed that malignant cells had relatively short telomeres. Microsatellite instability was ruled out by the allelic pattern of two major mononucleotide repeats. Our data suggest that a combination of melanoma specific genomic imbalances were sufficient and enough for this fatal tumor progression, that was not accompanied by genomic instability, telomerase activity, or the engagement of the alternative recombinatorial telomere lengthening pathway.

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.

<p>BACKGROUND: Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C approximately 7,900 Mbp) with prevalence of DNA repeats (&gt; 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation. RESULTS: We demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using HindIII and EcoRI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning. CONCLUSION: We present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye.</p>

<p>Prostate cancer is the most commonly diagnosed cancer among men in the United States. Recently, fusion of <em>TMPRSS2</em> with ETS family oncogenic transcription factors has been identified as a common molecular alteration in prostate cancer, where most often the rearrangement places <em>ERG</em> under the androgen-regulated transcriptional control of <em>TMPRSS2</em>. Here, we carried out rapid amplification of cDNA ends (RACE) on a prostate cancer specimen carrying an atypical aberration discovered by array-based comparative genomic hybridization (array CGH), suggesting an alternative fusion partner of <em>ERG</em>. We identified novel transcribed sequences fused to <em>ERG</em>, mapping 4 kb upstream of the <em>TMPRSS2</em> start site. The sequences derive from an apparent second <em>TMPRSS2</em> isoform, which we found also expressed in some prostate tumors, suggesting similar androgen-regulated control. In a reverse transcription-polymerase chain reaction (RT-PCR)-based survey of 63 prostate tumor specimens (54 primary and nine lymph node metastases), 44 (70%) cases expressed either the known or novel variant <em>TMPRSS2</em>-<em>ERG</em> fusion, 28 (44%) expressed both, 10 (16%) expressed only the known, and notably six (10%) expressed only the variant isoform fusion. In this specimen set, the presence of a <em>TMPRSS2</em>-<em>ERG</em> fusion showed no statistical association with tumor stage, Gleason grade or recurrence-free survival. Nonetheless, the discovery of a novel variant <em>TMPRSS2</em> isoform-<em>ERG</em> fusion adds to the characterization of ETS-family rearrangements in prostate cancer, and has important implications for the accurate molecular diagnosis of <em>TMPRSS2</em>-<em>ETS</em> fusions.</p>

MDM2 (HDM2) is a ubiquitin ligase that can target the p53 tumor suppressor protein for degradation. The RING domain is essential for the E3 activity of MDM2, and we show here that the extreme C-terminal tail of MDM2 is also critical for efficient E3 activity. Loss of E3 function in MDM2 mutants deleted of the C-terminal tail correlated with a failure of these mutants to oligomerize with MDM2, or with the related protein MDMX (HDMX). However, MDM2 containing point mutations within the C-terminus that inactivated E3 function retained the ability to oligomerize with the wild-type MDM2 RING domain and MDMX, and our results indicate that oligomers containing both wild-type MDM2 and a C-terminal mutant protein retain E3 function both in auto-degradation and degradation of p53. Interestingly, the E3 activity of C-terminal point mutants of MDM2 can also be supported by interaction with wild-type MDMX, suggesting that MDMX can directly contribute to E3 function.

Recently, we identified recurrent gene fusions involving the 5' untranslated region of the androgen-regulated gene TMPRSS2 and the ETS (E26 transformation-specific) family genes ERG, ETV1 or ETV4 in most prostate cancers. Whereas TMPRSS2-ERG fusions are predominant, fewer TMPRSS2-ETV1 cases have been identified than expected on the basis of the frequency of high (outlier) expression of ETV1 (refs 3-13). Here we explore the mechanism of ETV1 outlier expression in human prostate tumours and prostate cancer cell lines. We identified previously unknown 5' fusion partners in prostate tumours with ETV1 outlier expression, including untranslated regions from a prostate-specific androgen-induced gene (SLC45A3) and an endogenous retroviral element (HERV-K_22q11.23), a prostate-specific androgen-repressed gene (C15orf21), and a strongly expressed housekeeping gene (HNRPA2B1). To study aberrant activation of ETV1, we identified two prostate cancer cell lines, LNCaP and MDA-PCa 2B, that had ETV1 outlier expression. Through distinct mechanisms, the entire ETV1 locus (7p21) is rearranged to a 1.5-megabase prostate-specific region at 14q13.3-14q21.1 in both LNCaP cells (cryptic insertion) and MDA-PCa 2B cells (balanced translocation). Because the common factor of these rearrangements is aberrant ETV1 overexpression, we recapitulated this event in vitro and in vivo, demonstrating that ETV1 overexpression in benign prostate cells and in the mouse prostate confers neoplastic phenotypes. Identification of distinct classes of ETS gene rearrangements demonstrates that dormant oncogenes can be activated in prostate cancer by juxtaposition to tissue-specific or ubiquitously active genomic loci. Subversion of active genomic regulatory elements may serve as a more generalized mechanism for carcinoma development. Furthermore, the identification of androgen-repressed and insensitive 5' fusion partners may have implications for the anti-androgen treatment of advanced prostate cancer.

The order of appearance of different genetic aberrations during the shift from diploidy/near-diploidy to aneuploidy in colorectal cancers is not yet clear. We studied genetic alterations in flow cytometrically-sorted DNA diploid and corresponding aneuploid epithelial cell populations from each of 20 colorectal tumors using comparative genomic hybridization, FISH, and PCR. Analysis of the 19 cases in which aberrations were found in the flow-sorted diploid population indicated that large-scale aneuploidization in colorectal cancer was preceded by amplification of oncogene(s) localized to chromosome 20q13.2 and by KRAS mutations, but not by TP53 deletions or losses of large chromosomal regions such as 4q, 8p and 18q.

We analyzed complex chromosomal aberrations in 37 adult patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) using classical cytogenetic method, FISH with locus-specific probes, multicolor FISH (mFISH) and multicolor banding (mBAND). Unbalanced structural aberrations, leading to a gain or loss of chromosomal material, were frequently observed in bone marrow cells. In 30 patients (81.1%) loss or rearrangement of chromosome 5, 7 and/or 11 was found. The most frequent numerical change was trisomy 8 as expected (detected in six patients-16.2%) and the most frequent breakpoints 5q13, 5q33, 7q31, 10p12, 11q23, 12p13, 17p11 and 21q22 were determined.

In bone marrow cells of 33 patients with myelodysplastic syndrome and acute myeloid leukemia, structural rearrangements of chromosome 7 were found with conventional G-banding: 8 with deletions 7q and 25 with translocations. In 29 of the patients, complex karyotypes were confirmed using multicolor fluorescence in situ hybridization (mFISH). Commercial probes (Abbot Molecular) were used for 7q22, 7q31, and 7q35, the regions most frequently deleted in myeloid malignancies. In three cases without deletions, high-resolution multicolor banding (mBAND) for chromosome 7 revealed other aberrations. Five groups of chromosomal rearrangements were established: (a) deletion 7q as a sole aberration (2 cases), (b) deletion 7q and complex karyotypes (6 cases), (c) combined translocations and deletions of 7q (17 cases), (d) combined translocation and deletion 7p (5 cases), and (e) translocation of chromosomes 7 without deletion 7p or 7q (3 cases). Deletions of all three FISH-screened regions were the most frequent, with heterogeneous breakpoints. The region 7p13.2 approximately p15.2 was most commonly deleted. Most of the deletions were cryptic, not detectable with conventional cytogenetics. Aberrations of chromosome 7 are associated with a very poor outcome; survival time in our cohort was short (median 7 months).

<p>High-resolution multicolor banding (mBAND) analysis was applied to precisely fine-map the genomic extent of 7q deletions in a series of 26 marginal zone lymphoma patients displaying the abnormality on conventional karyotypes. Using this approach, the breakpoints and the extent of deletions revealed by conventional banding techniques had to be re-defined in 70% of cases. Although no common minimal region of deletion was delineated, mBAND demonstrated the involvement of the 7q32 region in more than 90% of cases. In addition, unsuspected translocations and intrachromosomal changes could be identified in four cases. Taken together, these data demonstrate that mBAND represents an alternative cytogenetic tool in the comprehensive analysis of chromosome aberrations in hematologic malignancies, allowing rapid screening and precise delineation of structural rearrangements of a defined chromosome. This also confirms the localization in the vicinity of band 7q32 of putative candidate gene(s) involved in the pathogenic development of the disease.</p>

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

We have tested whether a direct correlation of sequence information and staining properties of chromosomes is possible and whether this combined information can be used to precisely map any position on the chromosome. Despite huge differences of compaction between the naked DNA and the DNA packed in chromosomes we found a striking correlation when visualizing the GGCC density on both levels. Software was developed that allows one to superimpose chromosomal fluorescence intensity profiles generated by chromolysin A3 (CMA3) staining with GGCC density extracted from the Ensembl database. Thus, any position along the chromosome can be defined in megabase pairs (Mb) besides the cytoband information, enabling direct alignment of chromosomal information with the sequence data. The mapping tool was validated using 13 different BAC clones, resulting in a mean difference from Ensembl data of 2 Mb (ranging from 0.79 to 3.57 Mb). Our results indicate that the sequence density information and information gained with sequence-specific fluorochromes are superimposable. Thus, the visualized GGCC motif density along the chromosome (sequence bands) provides a unique platform for comparing different types of genomic information.

Primary pulmonary lymphoma is rare. The most common histological type is the bronchus-associated lymphoid tissue lymphoma. This type of lymphoma has an indolent course and excellent response to therapy. One-third of all cases are diagnosed incidentally. However, due to the rarity of this disease, little is known about its natural history in terms of dissemination and evolution. Herein, we report the unusual case of a 61-year-old man who refused treatment after being diagnosed with bronchus-associated lymphoid tissue lymphoma and died 2 years later from massive lung infiltration without dissemination to other organs.

In bone marrow cells of 33 patients with myelodysplastic syndrome and acute myeloid leukemia, structural rearrangements of chromosome 7 were found with conventional G-banding: 8 with deletions 7q and 25 with translocations. In 29 of the patients, complex karyotypes were confirmed using multicolor fluorescence in situ hybridization (mFISH). Commercial probes (Abbot Molecular) were used for 7q22, 7q31, and 7q35, the regions most frequently deleted in myeloid malignancies. In three cases without deletions, high-resolution multicolor banding (mBAND) for chromosome 7 revealed other aberrations. Five groups of chromosomal rearrangements were established: (a) deletion 7q as a sole aberration (2 cases), (b) deletion 7q and complex karyotypes (6 cases), (c) combined translocations and deletions of 7q (17 cases), (d) combined translocation and deletion 7p (5 cases), and (e) translocation of chromosomes 7 without deletion 7p or 7q (3 cases). Deletions of all three FISH-screened regions were the most frequent, with heterogeneous breakpoints. The region 7p13.2 approximately p15.2 was most commonly deleted. Most of the deletions were cryptic, not detectable with conventional cytogenetics. Aberrations of chromosome 7 are associated with a very poor outcome; survival time in our cohort was short (median 7 months).

A substantial number of patients with early-stage colorectal cancer relapse from metastatic disease. Identification of these patients by genetic profiling of their primary tumours may allow more informed follow-up and tailored administration of adjuvant therapy. Primary tumours from 70 patients with early-stage and largely microsatellite-stable colorectal cancer were profiled using metaphase-based comparative genomic hybridization (CGH) and the aberrations confirmed independently in a subset of patients using microarray-based CGH. Of the 70 cancers, 61 were amenable to CGH, and follow-up data was available from 56 patients. Genomic aberrations were correlated with patients' survival using univariate, multivariate and Kaplan-Meier survival curves. Metastatic primary tumours exhibited more complex genomic aberrations than non-metastatic primary tumours. Loss of chromosome 4p was an independent prognostic factor in early-stage colorectal cancer using multivariate analysis (Hazard ratio, 9.6; 95% CI, 3.3-28; P = 0.0001). Loss of both chromosome arms 8p and 18q had a statistically significant negative effect on disease-free survival. Moreover, primary tumours with loss of both chromosomes 4 and 14q bestowed poorer prognosis than tumours with loss of any one of the two chromosomes (P<0.0001). Genetic profiling of primary tumours of patients with early-stage colorectal cancer is of significant value in identifying the subset of patients who may relapse with metastatic disease. Accordingly, the molecular genetic features of primary tumours should be considered in the mainstream management of patients with this specific stage of the disease.

The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci5, 6. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that usually presents as a solitary nodule on sunexposed areas, develops within 6-8 weeks and spontaneously regresses after 3-6 months. KAs share features such as infiltration and cytological atypia with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether or not KA is a variant of SCC. To date no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. We screened fresh frozen material from 132 KAs and 37 SCCs for gross chromosomal aberrations by using comparative genomic hybridization (CGH). Forty-nine KAs (37.1%) and 31 SCCs (83.7%) showed genomic aberrations, indicating a higher degree of chromosomal instability in SCCs. Gains of chromosomal material from 1p, 14q, 16q, 20q, and losses from 4p were seen significantly more frequently in SCCs compared with KAs (P-values 0.0033, 0.0198, 0.0301, 0.0017, and 0.0070), whereas loss from 9p was seen significantly more frequently in KAs (P-value 0.0434). The patterns of recurrent aberrations were also different in the two types of neoplasms, pointing to different genetic mechanisms involved in their developments.

This study investigated the effects of exposing human embryonic stem cells (hESC) to 4oC and 25oC for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25oC and 4oC for 24h was 77.3 ± 4.8 % and 64.4 ± 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25oC and 4oC was 71.0 ± 0.5 % and 69.0 ± 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37oC. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure.

The Moloney murine leukemia virus-transformed suspension cell line WMP2 is derived from wild mice (Mus musculus) of the WMP/WMP strain. These mice carry nine pairs of metacentric Robertsonian translocation chromosomes. As the chromosomes of the wild-type mouse are all acrocentric, metaphase spreads of the WMP2 cells seam to be highly suited for physical gene mapping. Here we studied the WMP2 line using spectral karyotyping (SKY) combined with new established mouse specific multicolor banding (mcb) probes for the chromosomes X, 3, 4, 6 and 18. SKY revealed that the WMP2 cell line developed further four derivative chromosomes. After application of mcb five previously unrecognizable intrachromosomal rearrangements with 9 breakpoints were detected for the studied chromosomes.