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P M Lansdorp

Publications and source records attributed to P M Lansdorp.

At least 37 records · Page 2Linked to original sources
PubMed2000

Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry.

Chronic myeloid leukemia (CML) is a clonal, multilineage myeloproliferative disorder characterized by the Philadelphia chromosome (Ph) and a marked expansion of myeloid cells. Previous studies have indicated that the telomere length in blood cells may indicate their replicative history. However, the large variation in telomere length between individuals complicates the use of this parameter in CML and other hematologic disorders. To circumvent this problem, we compared the telomere length in peripheral blood or bone marrow cells with purified normal (Ph(-)) T lymphocytes from the same CML patient using fluorescence in situ hybridization and flow cytometry. Overall telomere fluorescence was significantly reduced in Ph(+) cells from patients with CML compared to blood leukocytes from normal individuals (P < 0.001) or normal (Ph(-)) T lymphocytes from the same individuals (n = 51, P < 0.001). Cells from patients in accelerated phase or blast phase (AP/BP) showed significantly shorter average telomere length than cells from patients in chronic phase (CP, P = 0.02) or cytogenetic remission (CR, P = 0.03). Patients in CP who subsequently developed BP within 2 years had significantly shorter telomeres than those who did not develop BP for at least 2 years (P < 0.05). Accelerated replication-dependent telomere shortening in Ph(+ )versus Ph(-) leukocytes supports previous evidence that Ph(+) stem cells cycle more actively than their counterparts in normal individuals. Our data further suggest that telomere shortening may serve as a surrogate marker of disease progression in patients with CP CML, supporting a mechanistic link between CML stem cell turnover, genetic instability, and malignant evolution in this disease. (Blood. 2000;95:1883-1890) (Blood. 2000;95:1883-1890)

Adolescent↗
PubMed2000

Oligoclonal expansions in the CD8(+)CD28(-) T cells largely explain the shorter telomeres detected in this subset: analysis by flow FISH.

We have previously reported that CD8(+)CD28(-) T cells have relatively shorter telomeres compared with CD8(+)CD28(+) T cells. Oligoclonal expansion is a common feature of CD8(+) T cells in human peripheral blood, and these expansions predominantly occur in the CD57(+)/CD28(-) population. We studied the telomere length in subsets of CD8(+) T cells using quantitative fluorescence in situ hybridization and flow cytometry (flow FISH). Our results confirm that CD8(+)CD28(-) T cells have shorter telomeres as compared with their CD28(+) counterpart cells. In addition, the oligoclonally expanded cells within the CD8(+)CD28(-) T cell subset generally have even shorter telomeres than the CD28(-) subset as a whole. We conclude that the presence of clonal expansions in the CD8(+)CD28(-) T cell population largely explain the shorter telomeres in this subset. These clonally expanded CD8(+)CD28(-) T cells generally have characteristics of terminally differentiated effector cells. Nevertheless, there is considerable individual variation in the degree of telomere shortening in these cells, which may reflect host genetic factors as well as the type and timing of the antigenic exposure.

CD28 Antigens↗
PubMed2000

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

Animals↗
PubMed2000

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Animals↗
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

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

Adolescent↗
PubMed1999

Asymmetric cell divisions in hematopoietic stem cells.

In order to study cell kinetics involved in long-term hematopoiesis, we studied single sorted candidate hematopoietic stem cells (HSC) from fetal liver cultured in the presence of a mixture of stimulatory cytokines. After 8-10 days in culture, the number of cells varied from less than a hundred to more than ten thousand cells. Single cells in slowly growing colonies were recloned upon reaching a 100-200-cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which the proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous generation of heterogeneity in cell cycle properties among the clonal progeny of HSC appears a relevant mechanism to maintain long-term maintenance of hematopoiesis in vitro and in vivo.

Adult↗
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↗
PubMed1999

Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability.

In most eukaryotes, poly(ADP-ribose) polymerase (PARP) recognizes DNA strand interruptions generated in vivo. DNA binding by PARP triggers primarily its own modification by the sequential addition of ADP-ribose units to form polymers; this modification, in turn, causes the release of PARP from DNA ends. Studies on the effects of the disruption of the gene encoding PARP (Adprt1, formerly Adprp) in mice have demonstrated roles for PARP in recovery from DNA damage and in suppressing recombination processes involving DNA ends. Telomeres are the natural termini of chromosomes and are, therefore, potential targets of PARP. Here, by the use of two different techniques, we show that mice lacking PARP display telomere shortening compared with wild-type mice. Telomere shortening is seen in different genetic backgrounds and in different tissues, both from embryos and adult mice. In vitro telomerase activity, however, is not altered in Adprt1-/- mouse fibroblasts. Furthermore, cytogenetic analysis of mouse embryonic fibroblasts reveals that lack of PARP is associated with severe chromosomal instability, characterized by increased frequencies of chromosome fusions and aneuploidy. The absence of PARP does not affect the presence of single-strand overhangs, naturally present at the ends of telomeres. This study therefore reveals an unanticipated role for PARP in telomere length regulation and provides insights into its functions in maintaining genomic integrity.

Aneuploidy↗
PubMed1999

Accelerated telomere shortening in the human inactive X chromosome.

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.

Acetylation↗
PubMed1999

An 18q- syndrome breakpoint resides between the duplicated serpins SCCA1 and SCCA2 and arises via a cryptic rearrangement with satellite III DNA.

The 18q-syndrome is representative of a group of terminal deficiency or macrodeletion syndromes characterized by mental retardation and congenital malformations. To gain insight into the mechanism of chromosomal loss and stabilization in these disorders, we cloned a putative terminal deletion breakpoint from an 18q-syndrome patient. The 18q21.3 breakpoint occurred between two nearly identical serine protease inhibitor (serpin) genes, SCCA1 and SCCA2. Although cytogenetic studies suggested that this chromosomal aberration was formed by a simple terminal deletion, DNA sequence analysis, pulsed-field gel electrophoresis and fluorescence in situ hybridization showed that the breakpoint was contiguous with a 35 bp filler sequence followed by a satellite III DNA-containing telomeric fragment of 475-1000 kb. This type of satellite III DNA sequence was not detected on the normal chromosome 18, but was highly homologous with types of satellite III DNA sequences normally located on the short arms (p11) of the acrocentric chromosomes and other heterochromatic regions. This DNA sequence analysis suggested that the terminal deficiency in this 18q-syndrome patient arose via illegitimate (non-homologous) recombination. Moreover, these data raise the possibility that a subset of chromosomal aberrations appearing cytogenetically and molecularly as simple terminal truncations or deletions are caused by small (<1000 kb) cryptic rearrangements.

Abnormalities, Multiple↗
PubMed1998

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization.

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

Cell Division↗
PubMed1998

Asymmetric cell divisions sustain long-term hematopoiesis from single-sorted human fetal liver cells.

Hematopoietic stem cells (HSCs) in adult marrow are believed to be derived from fetal liver precursors. To study cell kinetics involved in long-term hematopoiesis, we studied single-sorted candidate HSCs from fetal liver that were cultured in the presence of a mixture of stimulatory cytokines. After 8-10 d, the number of cells in primary cultures varied from <100 to >10,000 cells. Single cells in slow growing colonies were recloned upon reaching a 100-200 cell stage. Strikingly, the number of cells in subclones varied widely again. These results are indicative of asymmetric divisions in primitive hematopoietic cells in which proliferative potential and cell cycle properties are unevenly distributed among daughter cells. The continuous generation of functional heterogeneity among the clonal progeny of HSCs is in support of intrinsic control of stem cell fate and provides a model for the long-term maintenance of hematopoiesis in vitro and in vivo.

ADP-ribosyl Cyclase↗
PubMed1998

Induction of telomerase activity by in vivo X-irradiation of mouse splenocytes and its possible role in chromosome healing.

Telomeres serve as protective caps for the chromosome ends. They are one of the functional elements required for the stable transmission of eukaryotic chromosomes. Telomerase, a ribonucleoprotein, stabilises the telomere length by adding telomere repeats on to chromosome ends. Telomeres and telomerase can play a role in the formation of chromosome aberrations and especially in healing of the chromosome or chromatid breaks produced by radiation-induced DNA damage. Telomerase-independent processes also appear to be capable of capping broken chromosome ends. We have studied the expression of telomerase, telomere status and chromosome rearrangements in mouse splenocytes following different doses (0.5, 1.0, 2.0 or 3.0 Gy) of X-irradiation in vivo up to 224 days post-exposure. A dose-dependent increase in telomerase activity up to 2 Gy X-ray exposure was observed immediately after irradiation. The increased enzyme activity was detected even up to day 224 post-irradiation, the last time point studied, especially at higher doses (2 Gy and 3 Gy). A significant difference in average telomere length, measured by quantitative fluorescence in situ hybridisation (Q-FISH) on metaphase chromosomes, noticed immediately after irradiation indicates terminal deletion or altered telomere chromatin. However, telomere length was not statistically significant from the control at the later time points studied. Presence of telomere repeats at the chromosomal breakage sites revealed by FISH with peptide nucleic acid (PNA) telomeric probe indicates a possible role of telomerase-dependent or independent processes in chromosome healing and telomere capture in mammalian cells. We found that approximately 25 to 50% of the newly formed telomeres at the breakage sites are in the range of 200 bp to 1 kb, which might suggest that these repeats could have been added by telomerase which showed a corresponding increase following irradiation.

Animals↗
PubMed1998

Telomere length regulation in mice is linked to a novel chromosome locus.

Little is known about the mechanisms that regulate species-specific telomere length, particularly in mammalian species. The genetic regulation of telomere length was therefore investigated by using two inter-fertile species of mice, which differ in their telomere length. Mus musculus (telomere length >25 kb) and Mus spretus (telomere length 5-15 kb) were used to generate F1 crosses and reciprocal backcrosses, which were then analyzed for regulation of telomere length. This analysis indicated that a dominant and trans-acting mechanism exists capable of extensive elongation of telomeres in somatic cells after fusion of parental germline cells with discrepant telomere lengths. A genome wide screen of interspecific crosses, using M. spretus as the recurrent parent, identified a 5-centimorgan region on distal chromosome 2 that predominantly controls the observed species-specific telomere length regulation. This locus is distinct from candidate genes encoding known telomere-binding proteins or telomerase components. These results demonstrate that an unidentified gene(s) mapped to distal chromosome 2 regulates telomere length in the mouse.

Animals↗
PubMed1998

Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation.

Telomerase, a ribonucleoprotein enzyme, has been detected in immortalised cells and in majority of human cancers. Numerical and structural chromosome aberrations are commonly observed in tumour cell lines. To study the expression of telomerase and its influence on the formation of chromosomal aberrations, human colon carcinoma cell line (SW480) and human nonpolyposis colorectal carcinoma (HNPCC) cell lines (NA50600, NA59 and NA61) were exposed to 2 or 4 Gy X-rays. Increased telomerase activity was observed in all these cell lines at 24 h postirradiation and a 3 to 7 fold increase was seen at 4 Gy dose as detected by Telomere Repeat Amplification Protocol. Chromosomal rearrangements (dicentrics, translocations and breaks/fragments) analysed by Giemsa staining and chromosome painting were increased significantly following X- Quantitative fluorescence in situ hybridisation using a peptide nucleic acid telomeric probe to measure telomere length at irradiation chromosomal level revealed that all cell lines have very short telomeres in the range of 0.29 to 2.1 kb. Following X-irradiation, an increase in the chromosome end-to-end (telomere) associations was observed. The present results demonstrate that presence or upregulation of telomerase activity did not prevent the formation of chromosome aberrations and/or telomere associations in tumour cell lines after X-irradiation.

Chromosome Aberrations↗
PubMed1998

Telomerase activity in candidate stem cells from fetal liver and adult bone marrow.

Telomerase is a ribonucleoprotein polymerase that synthesizes telomeric repeats onto the 3' ends of eukaryotic chromosomes. Activation of telomerase may prevent telomeric shortening and correlates with cell immortality in the germline and certain tumor cells. Candidate hematopoietic stem cells (HSC) from adult bone marrow express low levels of telomerase, which is upregulated with proliferation and/or differentiation. To address this issue, we stimulated purified candidate HSC from human adult bone marrow with stem cell factor (SCF), interleukin-3 (IL-3), and Flt3-ligand (FL). After 5 days in culture, activity was detected in total cell extracts from IL-3-, SCF + FL-, SCF + IL-3-, FL + IL-3-, and SCF + IL-3 + FL-stimulated cultures, but not from cells cultured in SCF or FL alone. Within the CD34(+) fraction of the cultured cells, significant activity was found in the CD34(+)CD71(+) fraction. In addition, PKH26 staining confirmed that detectable telomerase activity was present in dividing PKH26(lo) cells, whereas nondividing PKH26(hi) cells were telomerase negative. Because in these experiments no distinction could be made between cycling "candidate" stem cells that had retained or had lost self-renewal properties, fetal liver cells with a CD34(+)CD38(-) phenotype, highly enriched for cycling stem cells, were also examined and found to express readily detectable levels of telomerase activity. Given the replication-dependent loss of telomeric DNA in hematopoietic cells, these observations suggest that the observed telomerase activity in candidate stem cells is either expressed in a minor subset of stem cells or, more likely, is not sufficient to prevent telomere shortening.

ADP-ribosyl Cyclase↗