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Biomedical subjects

U M Martens

Publications and source records attributed to U M Martens.

15 recordsLinked to original sources
PubMed2006

Telomere dysfunction and loss of p53 cooperate in defective mitotic segregation of chromosomes in cancer cells.

Aneuploidy is a fundamental principle of many cancer cells and is mostly related to defects in mitotic segregation of chromosomes. Many solid tumors as well as some preneoplastic lesions have been shown to contain polyploid chromosome numbers. The exact mechanisms behind whole-genome duplications are not known but have been linked to compromised mitotic checkpoint genes. We now report that the telomere checkpoint plays a key role for polyploidy in colon cancer cells. Telomerase suppression by a dominant-negative mutant of hTERT and consecutive telomere dysfunction in wild-type HCT116 colon cancer cells resulted in only minor stable chromosomal alterations. However, higher ploidy levels with up to 350 chromosomes were found when the cell-cycle checkpoint proteins p53 or p21 were absent. These findings indicate that telomere dysfunction in the absence of cell-cycle control may explain the high frequency of alterations in chromosome numbers found in many solid tumors.

Chromosome Segregation↗
PubMed2003

Quantitative analysis of mixed chimerism following allogeneic bone marrow transplantation using telomere flow-FISH.

Assessment of mixed chimerism is of particular interest for allogeneic bone marrow or peripheral blood stem cell transplantation with reduced-intensity conditioning in order to study contribution of donor-type and host-type lymphohematopoiesis. Because the length of telomere repeat sequences is frequently shorter in leukemic compared to normal hematopoietic cells, this telomere repeat polymorphism might be a useful marker to analyze mixed chimerism in selected patients with short telomeres. Recently, fluorescence in situ hybridization and flow cytometry (flow-FISH) have been shown to be valuable tools to analyze the mean telomere length in hematopoietic cells. Here, we demonstrate in a case study on a patient with chronic lymphocytic leukemia (CLL) that telomere flow-FISH can in principle be exploited to quantitate the amount of donor- and host-type cells for chimerism analysis based on distinct histogram distributions which reflect cell populations with different telomere length.

Bone Marrow Transplantation↗
PubMed2003

Lack of telomerase activity in human mesenchymal stem cells.

Telomerase activity and telomere maintenance have been associated with immortality in tumor and embryonic stem cells. Whereas most normal somatic cells are telomerase negative, low levels of this enzyme have been found in adult stem cells from the skin, gut and the hematopoietic system. Here, we show that telomerase activity is not detectable in human mesenchymal stem cells (hMSCs), which have the phenotype SH2+, SH3+, SH4+, CD29+, CD44+, CD14-, CD34- and CD45-, and have the capacity to differentiate into adipocytes, chondrocytes and osteoblasts. These data suggest that hMSCs have a different telomere biology compared to other adult stem cells. Alternatively, true mesenchymal stem cells might be a very rare subpopulation that have a detection level that is below the sensitivity of the TRAP assay.

Antigens, CD↗
PubMed2002

Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors.

Oncoretroviral vectors have been successfully used in gene therapy trials, yet low transduction rates and loss of transgene expression are still major obstacles for their application. To overcome these problems we modified the widely used Moloney murine leukemia virus-derived retroviral vector pMX by replacing the 3'LTR with the spleen focus-forming virus LTR and inserting the woodchuck hepatitis B virus post-translational regulatory element. To compare requirements crucial for efficient transgene expression, we generated the hybrid retroviral vectors pMOWS and pOWS that harbor the complete murine embryonic stem cell virus (MESV)-leader sequence or a shortened MESV-leader not comprising primer binding site (PBS) and splice donor (SD). Applying these retroviral vectors significantly augmented transgene expression in hematopoietic cell lines and progenitor cells. For transduction of murine embryonic stem (ES) cells the retroviral vector pMOWS that harbors the MESV-PBS and -SD was superior resulting in 65% green fluorescent protein (GFP) expressing ES cells. Surprisingly, in murine and human primitive hematopoietic progenitor cells (HPC), the highest efficiency of up to 66% GFP expressing cells was achieved with pOWS, a retroviral vector that retains the negative regulatory element coinciding with the MoMuLV-PBS. In summary our hybrid retroviral vectors facilitate significantly improved transgene expression in multipotent cells and thus possess great potential for reconstituting genes in primary cells of disease models, as well as for gene therapy.

Animals↗
PubMed2001

A highly selective telomerase inhibitor limiting human cancer cell proliferation.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Cell Division↗
PubMed2000

Accumulation of short telomeres in human fibroblasts prior to replicative senescence.

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.

Cell Division↗
PubMed2000

Measurement of telomere length in haematopoietic cells using in situ hybridization techniques.

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with; age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (approximately 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.

Adult↗
PubMed1999

Telomere length measurements using digital fluorescence microscopy.

BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.

Algorithms↗
PubMed1999

Absence or low number of telomere repeats at junctions of dicentric chromosomes.

Human ovarian surface epithelial (HOSE) cells transfected with the E6 and E7 oncogenes of the human papilloma virus (PV) do not express measurable telomerase activity. Relative to untransfected control cells, HOSE-PV cells have an extended in vitro lifespan characterized by a very high frequency of telomeric associations (TAs) of chromosomes. In order to study the role of telomere shortening in the formation of TAs, we studied the telomere length in 120 dicentric chromosomes in HOSE-PV cells by using quantitative fluorescence in situ hybridization. Forty percent of the dicentric chromosomes had no fluorescence signal at the junction site, and in the remainder the fluorescence at the junction was less than at corresponding unjoined ends. These observations support a critical role of telomere shortening in the development of TAs and the subsequent genetic instability observed in a majority of tumor cells.

Cells, Cultured↗
PubMed1998

Short telomeres on human chromosome 17p.

Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could mediate important biological effects as was shown in yeast, in which loss of a single telomere results in cell-cycle arrest and chromosome loss. We now demonstrate by quantitative fluorescence in situ hybridization (Q-FISH; ref. 7) that the number of T2AG3 repeats on specific chromosome arms is very similar in different tissues from the same donor and varies only to some extent between donors. In all sixteen individuals studied, telomeres on chromosome 17p were shorter than the median telomere length--a finding confirmed by analysis of terminal restriction fragments from sorted chromosomes. These observations provide evidence of chromosome-specific factors regulating the number of T2AG3 repeats in individual telomeres and raise the possibility that the relatively short telomeres on chromosome 17p contribute to the frequent loss of 17p alleles in human cancers.

Adult↗
PubMed1997

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats.

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Animals↗
PubMedDate not supplied

The implication of telomerase activity and telomere stability for replicative aging and cellular immortality (Review).

Telomerase and telomeres have been shown to be involved in the control of cell proliferation, the regulation of cell senescence and the unlimited proliferation capacity of malignant cells. Human telomeres are specialized chromosomal end structures composed of TTAGGG repeats. They function to protect chromosomes from degradation, fusion and recombination. Since the termini of linear molecules are replicated only in the 5'-3' direction by conventional DNA polymerases and require an RNA primer to initiate DNA synthesis, the removal of the RNA primer results in DNA loss with each cell division. To date, telomere shortening has been observed in most dividing somatic cells, eventually leading to cell senescence when critically short telomeres are reached. Telomerase has been identified as a ribonucleoprotein enzyme that can synthesize telomeric repeats onto chromosomes. Borderline telomerase activity has been detected in human primitive hematopoietic cells and in stimulated lymphocytes which increased with cytokine induced ex vivo expansion. However, in most other normal somatic cells, telomerase has not been detected, and consequently telomere shortening can be anticipated after a limited number of population doublings. In contrast, spontaneously immortalized tumor cell lines and the majority of malignant tumors demonstrate high telomerase activity, stable telomere length and unlimited proliferative potential. Mechanisms for telomerase and telomere length regulation are under extensive investigation. These have included the cloning of the RNA component and telomerase associated proteins, antisense experiments that have demonstrated progressive telomere length shortening in the absence of telomerase, and the identification of telomere binding proteins which may regulate telomerase by creating a negative feedback signal. This review aims to summarize important results in the rapidly moving field of telomeres and telomerase.

Base Sequence↗