The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres.

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

Mechanism of the antiproliferative activity of some naphthalene diimide G-quadruplex ligands.

G-quadruplexes are higher-order nucleic acid structures that can form in G-rich telomeres and promoter regions of oncogenes. Telomeric quadruplex stabilization by small molecules can lead to telomere uncapping, followed by DNA damage response and senescence, as well as chromosomal fusions leading to deregulation of mitosis, followed by apoptosis and downregulation of oncogene expression. We report here on investigations into the mechanism of action of tetra-substituted naphthalene diimide ligands on the basis of cell biologic data together with a National Cancer Institute COMPARE study. We conclude that four principal mechanisms of action are implicated for these compounds: 1) telomere uncapping with subsequent DNA damage response and senescence; 2) inhibition of transcription/translation of oncogenes; 3) genomic instability through telomeric DNA end fusions, resulting in mitotic catastrophe and apoptosis; and 4) induction of chromosomal instability by telomere aggregate formation.

On the Power of Additional and Complex Chromosomal Aberrations in CML.

Unregulated proliferation of mainly myeloid bone marrow cells and genetic changes in the hematopoietic stem cell system are important features in Chronic Myeloid Leukemia (CML). In clinical diagnosis of CML, classical banding techniques, fluorescence in situ hybridization (FISH) probing for the Philadelphia chromosome (Ph) or polymerase chain reaction amplifying the fusion products of the BCR-ABL fusion are state of the art techniques. Nevertheless, the genome of CML patients harbors many more cytogenetic changes. These might be hidden in subpopulations due to clonal events or involved in extremely complex aberrations. To identify these additional changes, several cytogenetic and molecular genetic techniques could be applied. Nevertheless, it has been proposed that identifying these aberrations is time consuming and costly and since they cannot be converted into a benefit for the patients, the necessity to perform these investigations has been questioned. In the times where highly specialized medicine is advancing into several areas of cancer, this attitude needs to be reassessed. Therefore, we looked at the usefulness of a combination of different techniques to unravel the genetic changes in CML patients and to identify new chromosomal aberrations, which potentially can be correlated to different stages of the disease and the strength of therapy resistance. We are convinced that the combination of these techniques could be extremely useful in unraveling even the most complex karyotypes and in dissecting different clones contributing to the disease. We propose that by doing so, this would improve CML diagnostic and prognostic findings, especially with regard to CML resistance mechanisms and new therapeutic strategies.

Tissue context-activated telomerase in human epidermis correlates with little age-dependent telomere loss.

Telomerase- and telomere length regulation in normal human tissues is still poorly understood. We show here that telomerase is expressed in the epidermis in situ independent of age but was repressed upon the passaging of keratinocytes in monolayer culture. However, when keratinocytes were grown in organotypic cultures (OTCs), telomerase was re-established, indicating that telomerase activity is not merely proliferation-associated but is regulated in a tissue context-dependent manner in human keratinocytes. While not inducible by growth factors, treatment with the histone deacetylation inhibitor FK228 restored telomerase activity in keratinocytes grown in monolayer cultures. Accordingly, CHIP analyses demonstrated an acetylated, active hTERT promoter in the epidermis in situ and in the epidermis of OTCs but a deacetylated, silenced hTERT promoter with subsequent propagation in monolayer culture suggesting that histone acetylation is part of the regulatory program to guarantee hTERT expression/telomerase activity in the epidermis. In agreement with the loss of telomerase activity, telomeres shortened during continuous propagation in monolayer culture by an average of approximately 70 base pairs (bp) per population doubling (pd). However, telomere erosion varied strongly between different keratinocyte strains and even between individual cells within the same culture, thereby arguing against a defined rate of telomere loss per replication cycle. In the epidermis in situ, as determined from early-passage keratinocytes and tissue sections from different age donors, we calculated a telomere loss of only approximately 25 bp per year. Since we determined the same rate for the non-regenerating melanocytes and dermal fibroblasts, our data suggest that in human epidermis telomerase is a protective mechanism against excessive telomere loss during the life-long regeneration.

Chromosomes are predominantly located randomly with respect to each other in interphase human cells.

To test quantitatively whether there are systematic chromosome-chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome-chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.

The coincidence of chromosome 15 aberrations and beta2-microglobulin gene mutations is causative for the total loss of human leukocyte antigen class I expression in melanoma.

PURPOSE: Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8+ T cells, is known to be caused by mutations in the beta2-microglobulin (beta2m) gene. We asked whether abnormalities of chromosome 15, harboring the beta2m gene on 15q21, in addition to beta2m gene mutations, are causative for the HLA class I-negative phenotype of melanoma cells. EXPERIMENTAL DESIGN: To answer this, we established primary cell lines from the beta2m-negative metastatic melanoma tissues of four different patients and analyzed them for beta2m gene mutations and chromosome 15 aberrations, the latter by loss of heterozygosity analysis, fluorescence in situ hybridization (FISH), and multicolor FISH. RESULTS: Mutations at the beta2m gene level were detected in all cell lines. The loss of heterozygosity analysis of microsatellite markers located on chromosome 15 in three of the four cell lines pointed to an extensive loss of chromosome 15 material. Subsequent molecular cytogenetic analysis revealed the coexistence of apparently normal and rearranged versions of chromosome 15 in three cell lines whereas the fourth cell line solely showed rearranged versions. Two of the four cell lines exhibited a special type of intrachromosomal rearrangement characterized by FISH signals specific for the subtelomeric region of 15q at both ends of the chromosome and one centromeric signal in between. CONCLUSIONS: Our data indicate that the complete loss of HLA class I expression in melanoma cells is due to the coincidence of the following mutational events: (a) chromosome 15 instability associated with an extensive loss of genetic material and (b) beta2m gene mutations.

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells.

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.

FISH in cancer diagnosis and prognostication: from cause to course of disease.

The last 20 years have witnessed an astounding evolution of cytogenetic approaches to cancer diagnosis and prognostication. Molecular techniques and, in particular, nonisotopically-labeled nucleic acid probes and fluorescence in situ hybridization (FISH)-based techniques have replaced the costly and potentially dangerous radioactive techniques used in research and the clinical detection of genetic alterations in tumor cells. Fluorescent DNA probes also enabled the screening for very subtle chromosomal changes. Clinical laboratories now choose from a growing number of FISH-based cytogenetic tests to support physician's diagnoses of the causes and the course of a disease. Depending on the specimen, state-of-the-art FISH techniques allow the localization and scoring of 10-24 different targets and overcome previous problems associated with target colocalization and detection system bandwidth. FISH-based analyses have been applied very successfully to the analysis of single cells and have demonstrated the existence of cell clones of different chromosomal make-up within human tumors. This information provides disease-specific information to the attending physician and should enable the design of patient-specific protocols for disease intervention.

Establishment and characterization of a highly tumourigenic and cancer stem cell enriched pancreatic cancer cell line as a well defined model system.

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.

'Chromosomal Rainbows' Detect Oncogenic Rearrangements of Signaling Molecules in Thyroid Tumors.

Altered signal transduction can be considered a hallmark of many solid tumors. In thyroid cancers the receptor tyrosine kinase (rtk) genes NTRK1 (Online Mendelian Inheritance in Man = OMIM *191315, also known as 'TRKA'), RET ('Rearranged during Transfection protooncogene', OMIM *164761) and MET (OMIM *164860) have been reported as activated, rearranged or overexpressed. In many cases, a combination of cytogenetic and molecular techniques allows elucidation of cellular changes that initiate tumor development and progression. While the mechanisms leading to overexpression of the rtk MET gene remain largely unknown, a variety of chromosomal rearrangements of the RET or NTKR1 gene could be demonstrated in thyroid cancer. Abnormal expressions in these tumors seem to follow a similar pattern: the rearrangement translocates the 3'- end of the rtk gene including the entire catalytic domain to an expressed gene leading to a chimeric RNA and protein with kinase activity. Our research was prompted by an increasing number of reports describing translocations involving ret and previously unknown translocation partners.We developed a high resolution technique based on fluorescence in situ hybridization (FISH) to allow rapid screening for cytogenetic rearrangements which complements conventional chromosome banding analysis. Our technique applies simultaneous hybridization of numerous probes labeled with different reporter molecules which are distributed along the target chromosome allowing the detection of cytogenetic changes at near megabasepair (Mbp) resolution. Here, we report our results using a probe set specific for human chromosome 10, which is altered in a significant portion of human thyroid cancers (TC's). While rendering accurate information about the cytogenetic location of rearranged elements, our multi-locus, multi-color analysis was developed primarily to overcome limitations of whole chromosome painting (WCP) and chromosome banding techniques for fine mapping of breakpoints in papillary thyroid cancer (PTC).

Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping.

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase-negative human cell line.

Telomerase-negative tumor cells maintain their telomeres via an alternative lengthening of telomeres (ALT) mechanism. This process involves the association of telomeres with promyelocytic leukemia nuclear bodies (PML-NBs). Here, the mobility of both telomeres and PML-NBs as well as their interactions were studied in human U2OS osteosarcoma cells, in which the ALT pathway is active. A U2OS cell line was constructed that had lac operator repeats stably integrated adjacent to the telomeres of chromosomes 6q, 11p, and 12q. By fluorescence microscopy of autofluorescent LacI repressor bound to the lacO arrays the telomere mobility during interphase was traced and correlated with the telomere repeat length. A confined diffusion model was derived that describes telomere dynamics in the nucleus on the time scale from seconds to hours. Two telomere groups were identified that differed with respect to the nuclear space accessible to them. Furthermore, translocations of PML-NBs relative to telomeres and their complexes with telomeres were evaluated. Based on these studies, a model is proposed in which the shortening of telomeres results in an increased mobility that could facilitate the formation of complexes between telomeres and PML-NBs.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA.

BACKGROUND: Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance. RESULTS: We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-kappaB2 locus. CONCLUSION: The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Automatic telomere length measurements in interphase nuclei by IQ-FISH.

To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations.