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. INTRODUCTIONTelomeres are specialized chromatin structures at the end of linear chromosomes, in which repetitive DNA sequences (5Ј-TTAGGG-3Ј in vertebrates) associate into a nucleoprotein complex (de Lange et al., 2006). This complex-the "telosome"-protects the chromosome ends from degradation and genomic rearrangement and includes the "shelterin" proteins TRF1, TRF2, and POT1 that directly recognize the TTAGGG repeat sequence (de Lange, 2005;Bertuch and Lundblad, 2006;Croy and Wuttke, 2006;Blasco, 2007). Because of incomplete DNA synthesis at the chromosome ends, 50 -200 base pairs of telomeric DNA are lost during each replication cycle (Harley et al., 1990;Martens et al., 2000). After ϳ60 -80 cell divisions telomere repeats are shortened from a typical initial length of 10 -15 kb in human cells to ϳ5 kb and below, which triggers cell senescence or apoptosis (Harley et al., 1990;Martens et al., 2000;Blasco, 2007). Accordingly, tumor cells need to compensate the loss of their telomere repeats in order to sustain an unlimited proliferative potential. In most cases this is accomplished by reactivating telomerase, a reverse transcriptase that synthesizes telomeric repeats at the chromosome ends (Greider and Blackburn, 1985;Chan and Blackburn, 2004;Collado et al., 2007;Johnson and Broccoli, 2007). However, a fraction of ϳ10 -15% of tumors is able to maintain their telomeres in the absence of telomerase activity. This process has been designated as alternative lengthening of telomeres (ALT; Bryan et al., 1995;Neumann and Reddel, 2006;Johnson and Broccoli, 2007). In yeast and mammals, it has been shown that the ALT mechanism involves homologous recombination events between telomere repeats (Dunham et al., 2000;Kass-Eisler and Greider, 2000;Lundblad, 2002;Muntoni and Reddel, 2005). It is characte...

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.

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.

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.

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. Key terms: automatic; quantification; telomere; FISH; microscopy; fluorescence; ALT; senescence Monitoring of telomere length regulation has become an important aspect of stem-cell research, studies on cellular senescence, and cancer research. A number of techniques have been developed to study telomere length in a quantitative manner. The standard procedure is Southern blot hybridization (1). The quantitative fluorescence in situ hybridization (Q-FISH) on metaphase chromosomes allows the quantification of the telomeric FISH signals on individual chromosomes (2-4). Since fluorescence intensity of the telomeric signals was found to be proportional to the size of telomeric repeats, Q-FISH is now widely used (2). For measuring telomere length in interphase nuclei, flow cytometry or fluorescence microscopy can be applied (5-7). Nonadhesive hematopoietic cells seem to be better suited for flow cytometric analyses than solid tumor cells (8,9). Therefore, for the analysis of nonhematopoietic cells, interphase FISH is the method of choice. However, the fluorescence microscopical method to quantify telomere length in interphase nuclei has so far only been performed manually on a restricted number of cells (6,7). To combine the positive aspects of flow cytometric measurements with the ability to quantify individual nuclei by fluorescence microscopical examination, we took advantage of fluorescence-based automatic microscope (FLAME). Thus, all the advantages of interphase measurements, i.e., the analysis of individual cells and the applicability to nonproliferating cells, can be combined with the analyses of statistically relevant cell populations. To develop a reliable method to automatically quantify telomere length in interphase nuclei, we applied a two-color hybridization assay and measured the fluorescence signals with FLAME. We then described the parameters essential for intra-and interexperimental comparisons. We performed intensity measurements in interphase nuclei and compared the results of single channel measurements of the target probe with the results obtained after introducing an internal reference and performing double channel measurements. We validated the quantitative-FISH on interphase nuclei (IQ-FISH) method by measuring telomere lengths of differe...

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.

Purpose:Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8 + Tcells, is known to be caused by mutations in the h2-microglobulin (b2m) gene. We asked whether abnormalities of chromosome 15, harboring the b2m gene on 15q21, in addition to b2m 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 h2m-negative metastatic melanoma tissues of four different patients and analyzed them for b2m 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 b2m 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) b2m gene mutations.Melanoma cells can be effectively eradicated in vivo by the cytotoxic activity of HLA class I-restricted tumor antigenspecific CD8 + T cells as recently shown in clinical trials of adoptive T-cell transfer (1, 2). However, these studies also showed that adoptively transferred T cells infiltrating the metastasis not only act as cytotoxic immune effectors but also as immune editors (3, 4) selecting for tumor cell variants that escape immune surveillance by down-regulation of antigen expression (1). Besides antigen down-regulation, other properties of melanoma cells that interfere with T-cell effector function have been described, such as release of immunosuppressive cytokines (5, 6), release of Fas ligand-bearing microvesicles (7), and alterations in the surface presentation of HLA class I molecules (8-10).HLA class I molecules are heterodimeric noncovalently associated complexes consisting of the constant h2-microglobulin (h2m) light chain and the variable HLA heavy a chain. Irreversible alterations in the HLA class I phenotype of melanoma cells, such as loss of a single HLA allele, an HLA locus, or an HLA haplotype, are generally caused by

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