Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Proceed order?

Export
  • 1
    Keywords: validation ; DNA ; cytogenetics ; PROBES ; molecular
    Abstract: 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 similar to 6 kb plasmid onto an unusually small, similar to 55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-kappa B2 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.
    Type of Publication: Journal article published
    PubMed ID: 19108707
    Signatur Availability
    BibTip Others were also interested in ...
  • 2
    Keywords: CELLS ; GROWTH ; GROWTH-FACTOR ; INHIBITOR ; CELL ; Germany ; human ; POPULATION ; GENE-EXPRESSION ; DIFFERENTIATION ; MECHANISM ; CARCINOGENESIS ; TISSUES ; SKIN ; BIOLOGY ; CYCLE ; fibroblasts ; IN-SITU ; REVERSE-TRANSCRIPTASE ; PROMOTER ; AGE ; LENGTH ; NETHERLANDS ; REPLICATION ; HEMATOPOIETIC STEM-CELLS ; histone deacetylase inhibitor ; HaCaT ; organotypic culture ; molecular biology ; SKIN KERATINOCYTES ; telomere length ; STRAINS ; HTERT GENE ; HISTONE ACETYLATION ; regeneration ; GROWTH-FACTORS ; CULTURES ; Age-dependent telomere attrition ; COCULTURES ; HTERT ; In situ telomere length analysis ; Melanocyte ; Telomere length heterogeneity
    Abstract: 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 similar to 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 similar to 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. (C) 2009 Published by Elsevier B.V
    Type of Publication: Journal article published
    PubMed ID: 19419690
    Signatur Availability
    BibTip Others were also interested in ...
  • 3
    Keywords: ASSOCIATION, BIOLOGY, CELL, CELLS, CHROMATIN, COMPLEX, COMPLEXES, DIFFUSION, DNA-REPLICATION, DYNAMI
    Abstract: 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
    Type of Publication: Journal article published
    PubMed ID: 19211845
    Signatur Availability
    BibTip Others were also interested in ...
  • 4
    Keywords: IONIZING-RADIATION ; radiotherapy ; IN-SITU HYBRIDIZATION ; COMET ASSAY ; LYMPHOCYTES ; FISH ; RANDOMIZED-TRIAL ; radiosensitivity ; RECTAL-CANCER ; CHROMOSOME-ABERRATIONS
    Abstract: In radiotherapy the normal tissue reaction is often a limiting factor for radiation treatment. Still there is no screening method, which predicts normal tissue reaction on radiotherapy, especially in comparison to tumor tissue, and therefore allows tailoring of the radiation dose to each patient. Here, we present a case of severe radiation-related side effects. We applied classical cytogenetic techniques (Giemsa-banding and staining of centromeric regions), the comet assay as well as multicolor fluorescence in situ hybridization on peripheral blood lymphocytes of this patient in order to determine the radio-sensitivity on the DNA level and to correlate these findings with the clinical outcome. Our investigations revealed abnormalities on chromosome 9, deficiencies in the DNA-repair capacity after radiation exposure and a high number of radiation induced chromosomal aberrations. A detected high amount of residual damage two or three hours after radiation exposure and repair as well as the high number of chromosomal aberrations (ChAs) suggests a correlation between repair capacity and radiation induced ChAs. We concluded that the detected abnormalities might serve as a genetic basis for the radio-sensitive phenotype of this patient. Taken together this report strengthens the idea that intensive DNA genomic analysis of individual patients can serve as the basis for more favourable treatment of cancer patients.
    Type of Publication: Journal article published
    Signatur Availability
    BibTip Others were also interested in ...
  • 5
    Keywords: CELLS ; EXPRESSION ; IN-VITRO ; CELL ; Germany ; DISTINCT ; PROTEIN ; PROTEINS ; CARCINOGENESIS ; KERATINOCYTES ; SKIN ; cell cycle ; CELL-CYCLE ; CYCLE ; chromosome ; NUCLEI ; FORM ; MOUSE ; Western-blot ; INSTABILITY ; LOCALIZATION ; NUCLEUS ; LENGTH ; western blot ; C-MYC ; INTERPHASE ; INTERPHASE CELLS ; MITOSIS ; GENOMIC INSTABILITY ; HaCaT ; INTERPHASE NUCLEI ; CLUSTER ; CHROMOSOMES ; DNA-SEQUENCES ; PATTERN ; VARIANT ; END ; FISH-protein double labeling ; HUMAN FIBROBLASTS ; MAMMALIAN TELOMERES ; regulation ; telomere aggregates ; telomere repeat binding factor 2 ; three-dimensional nuclear organization ; TRANSITION
    Abstract: 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
    Type of Publication: Journal article published
    PubMed ID: 15679112
    Signatur Availability
    BibTip Others were also interested in ...
  • 6
    Keywords: COMPARATIVE GENOMIC HYBRIDIZATION ; IN-SITU HYBRIDIZATION ; TRANSLOCATION ; CHRONIC MYELOGENOUS LEUKEMIA ; CHRONIC MYELOID-LEUKEMIA ; COPY NUMBER CHANGES ; SOLID TUMORS ; CYTOGENETIC ANALYSIS ; blast crisis ; C-ABL-PROTEINS
    Abstract: 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.
    Type of Publication: Journal article published
    Signatur Availability
    BibTip Others were also interested in ...
  • 7
    Keywords: IN-VITRO ; CANCER-CELLS ; HUMAN TUMOR-CELLS ; senescence ; ATM ; DNA-DAMAGE RESPONSE ; TRF2 ; SMALL-MOLECULE ; CHROMOSOMAL REARRANGEMENTS ; TELOMERE CAPPING ALTERATION
    Abstract: 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.
    Type of Publication: Journal article published
    PubMed ID: 23188717
    Signatur Availability
    BibTip Others were also interested in ...
  • 8
    Keywords: CANCER ; MICROSCOPY ; INFORMATION ; QUANTIFICATION ; POPULATION ; NUCLEI ; ACQUISITION ; NUMBER ; IN-SITU HYBRIDIZATION ; FISH ; LENGTH ; CHILDREN ; FLUORESCENCE ; INTERPHASE NUCLEI ; flow-FISH ; SOCIETY ; senescence ; telomere ; automatic ; HUMAN-CELLS ; ALT
    Abstract: 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. (c) 2005 international Society for Analytical Cytology
    Type of Publication: Journal article published
    PubMed ID: 16228977
    Signatur Availability
    BibTip Others were also interested in ...
  • 9
    Keywords: CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; Germany ; human ; GENE ; HYBRIDIZATION ; TISSUE ; LINES ; PATIENT ; MARKER ; TISSUES ; ANTIGEN ; SKIN ; T cells ; T-CELLS ; CELL-LINES ; DOWN-REGULATION ; SIGNAL ; IN-SITU ; DESIGN ; MUTATION ; CELL-LINE ; LINE ; ABERRATIONS ; HETEROZYGOSITY ; MELANOMA ; REGION ; MUTATIONS ; MONOCLONAL-ANTIBODIES ; MHC CLASS-I ; PHENOTYPE ; IMMUNE ESCAPE ; TUMOR ESCAPE ; in situ hybridization ; MALIGNANT-CELLS ; RE ; LEVEL ; EVENTS ; LOSSES ; CD8(+) T cell ; B2M GENE ; MEDIATED LYSIS ; PROCESSING MACHINERY
    Abstract: 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 beta 2-microglobulin (beta 2m) gene. We asked whether abnormalities of chromosome 15, harboring the beta 2m gene on 15q21, in addition to beta 2m gene mutations, are causative for the HILA class I-negative phenotype of melanoma cells. Experimental Design: To answer this, we established primary cell lines from the beta 2m-negative metastatic melanoma tissues of four different patients and analyzed them for beta 2m 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 beta 2m 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) beta 2m gene mutations
    Type of Publication: Journal article published
    PubMed ID: 16740750
    Signatur Availability
    BibTip Others were also interested in ...
  • 10
    Keywords: ENERGIES ; SIMULATIONS ; IN-VITRO ; BLOOD ; COMBINATION ; human ; VITRO ; imaging ; EXPOSURE ; HYBRIDIZATION ; transcription ; NUCLEAR-MEDICINE ; radiation ; DNA ; SIMULATION ; BIOLOGY ; MEMBER ; MEMBERS ; chromosome ; NUCLEI ; IN-SITU ; ENERGY ; NUMBER ; ABERRATIONS ; LYMPHOCYTES ; NUCLEUS ; MAMMALIAN-CELLS ; FLUORESCENCE ; PERIPHERAL-BLOOD ; INTERPHASE ; nuclear medicine ; CLUSTERS ; MITOSIS ; Monte Carlo ; MONTE-CARLO ; CLUSTER ; CHROMOSOMES ; clustering ; DNA-CONTENT ; ENERGY-TRANSFER ; HUMAN-LYMPHOCYTES ; in situ hybridization ; INDUCED STRUCTURAL-ABERRATIONS ; INTERPHASE NUCLEUS ; JUNCTIONS ; MFISH ; ORDER ; radiology
    Abstract: Purpose: Analysing chromosome aberrations induced by low linear energy transfer (LET) radiation in order to characterize systematic spatial clustering among the 22 human autosomes in human lymphocytes and to compare their relative participation in interchanges. Materials and methods: A multicolour fluorescence in situ hybridization (mFISH) data set, specifying colour junctions in metaphases of human peripheral blood lymphocytes 72 h after in vitro exposure to low LET radiation, was analysed separately and in combination with previously published results. Monte Carlo computer simulations and mathematical modelling guided data analysis. Results and conclusions: Statistical tests on aberration data confirmed two clusters of chromosomes, {1, 16, 17, 19, 22} and {13, 14, 15, 21, 22}, as having their members being on average closer to each other than randomness would predict. The first set has been reported previously to be near the centre of the interphase nucleus and to be formed mainly by gene-rich chromosomes, while the second set comprises the nucleolus chromosomes. The results suggest a possible interplay between chromosome positioning and transcription. A number of other clusters suggested in the literature were not confirmed and considerable randomness of chromosome-chromosome juxtapositions was present. In addition, and consistent with previous results, it was found that chromosome participation in interchanges is approximately proportional to the two-thirds power of the DNA content
    Type of Publication: Journal article published
    PubMed ID: 15360089
    Signatur Availability
    BibTip Others were also interested in ...
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...