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LE Magazine October 2002

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Cellular aging

Telomere position effect in human cells.

In yeast, telomere position effect (TPE) results in the reversible silencing of genes near telomeres. Here we demonstrate the presence of TPE in human cells. HeLa clones containing a luciferase reporter adjacent to a newly formed telomere express 10 times less luciferase than do control clones generated by random integration. Luciferase expression is restored by trichostatin A, a histone deacetylase inhibitor. Over expression of a human telomerase reverse transcriptase complementary DNA results in telomere elongation and an additional 2- to 10-fold decrease in expression in telomeric clones but not control clones. The dependence of TPE on telomere length provides a mechanism for the modification of gene expression throughout the replicative life span of human cells.

Science 2001 Jun 15;292(5524):2075-7

Extension of life span by introduction of telomerase into normal human cells.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Science 1998 Jan 16;279(5349):349-52

Telomerase, checkpoints and cancer.

Telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state. In particular, the highly specific correlations and early causal relationships between telomere loss in the absence of telomerase activity and replicative senescence or crisis, on the one hand, and telomerase reactivation and cell immortality, on the other, point to a new and important paradigm in the complementary fields of ageing and cancer. Although the signaling pathways between telomeres and transcriptional and cell cycle machinery remain undefined, recently described homologies between telomeric proteins and lipid/protein kinase activities important in chromosome stability provide evidence for the existence of pathways transducing signals originating in chromosome structure to cell cycle regulatory processes. Similarities between cell cycle arrest at senescence and the response of mortal cells to DNA/oxidative damage suggest overlap in the signal transduction mechanisms culminating in irreversible and stable cell cycle arrest. The feasibility of targeting telomeres/telomerase as a strategy for antiproliferative therapeutics has been shown in studies in yeast, in which mutations in specific telomere associated genes result in delayed cell death. Similarly, antisense oligonucleotide inhibition of telomerase activity in human tumor cells (HeLa) results in delayed cell death. The mechanism of cell death and possible escape from this fate require further study. In human cells, however, it would seem reasonable to predict that in these circumstances, apoptosis is induced in the vast majority of cells either directly in response to a DNA damage signal arising from critically shortened telomeres or as a secondary consequence of genetic instability.

Cancer Surv 1997;29:263-84

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death.

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2’-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Proc Natl Acad Sci U S A 1999 Dec 7;96(25):14276-81

Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative life span. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumor formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

Nat Genet 1999 Jan;21(1):111-4

Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.

S. cerevisiae chromosomes end with the telomeric repeat (TG1-3)n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2- cells) containing white sectors (characteristic of ADE2+ cells). Repression was due to proximity to the telomere itself since an 81 bp tract of (TG1-3)n positioned downstream of URA3 when URA3 was approximately 20 kb from the end of chromosome VII did not alter expression of the gene. However, this internal tract of (TG1-3)n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed. These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control.

Cell 1990 Nov 16;63(4):751-62

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumors. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Nat Genet 1997 Dec;17(4):498-502

Telomerase in brain tumors.

INTRODUCTION: In recent years, many scientists involved in cancer research have directed their attention to telomerase, an enzymatic complex which is specifically involved in duplicating telomeres, the very ends of linear chromosomes. The discovery that most immortal cell lines in vitro and human tumor cells in vivo have telomerase activity, in contrast to telomerase-negative normal somatic cells, has made telomerase a candidate for use as a molecular marker of malignancy and even as a target for anticancer therapies. Thus, the assessment of the role of telomerase activity in neoplastic transformation has become a key issue in oncology, as stated by the exponential increase of papers on telomerase in the last five years. OBJECT: In this paper, we review some recent data from the literature, including our own studies, on the regulation of telomerase activity in brain tumors.

Childs Nerv Syst 2002 Apr;18(3-4):112-7

Replicative aging, telomeres, and oxidative stress.

Aging is a very complex phenomenon, both in vivo and in vitro. Free radicals and oxidative stress have been suggested for a long time to be involved in or even to be causal for the aging process. Telomeres are special structures at the end of chromosomes. They shorten during each round of replication and this has been characterized as a mitotic counting mechanism. Our experiments show that the rate of telomere shortening in vitro is modulated by oxidative stress as well as by differences in antioxidative defence capacity between cell strains. In vivo we found a strong correlation between short telomeres in blood lymphocytes and the incidence of vascular dementia. These data suggest that parameters that characterise replicative senescence in vitro offer potential for understanding of, and intervention into, the aging process in vivo.

Ann N Y Acad Sci 2002 Apr;959:24-9

Regulation of telomerase expression in human lymphocytes.

The function of lymphocytes is highly dependent on the ability of cell to divide. Among the various factors that regulate this cellular process, telomerase-dependent maintenance of telomere length has recently drawn considerable attention. Unlike most normal human somatic cells that express telomerase only during development but not after differentiation, lymphocytes express telomerase during development and retain the ability to express telomerase after maturation in response to antigenic challenge. How telomerase is regulated and its precise role in lymphocytes is not fully understood. The recent progress in characterizing regulation of telomerase expression in human lymphocytes is discussed.

Springer Semin Immunopathol 2002;24(1):23-33

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2.

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.

Mol Cell Biol 2002 May;22(10):3474-87

Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients.

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.

Clin Exp Immunol 2002 Mar;127(3):499-506

Effects of cisplatin on telomerase activity and telomere length in BEL-7404 human hepatoma cells.

Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48 or 72 h in a concentration ranged from 0.8 to 50 microM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process.

Cell Res 2002 Mar;12(1):55-62

Increased life span of human osteoarthritic chondrocytes by exogenous expression of telomerase.

OBJECTIVE: To extend the life span of human osteoarthritic (OA) articular chondrocytes by introduction of the catalytic component of human telomerase while preserving the chondrocyte-specific phenotype. METHODS: Human articular chondrocytes were isolated from the femoral head and tibial plateau of patients undergoing knee joint replacement for OA. The chondrocytes were cultured as monolayers and infected with a retroviral telomerase expression construct followed by selection with G418 for 10 to 14 days. Telomeric-repeat amplification protocol assays and telomere terminal restriction fragment length assays were performed on pools of transduced cells in order to measure telomerase activity and telomere length. Growth kinetics and population doubling capacity were assessed by passaging the cells in monolayer culture. Redifferentiation of the monolayer chondrocyte cultures was induced by transfer to suspension culture on poly-(2-hydroxyethyl-methacrylate) (polyHEMA)-coated dishes. Induction of the chondrocyte-specific phenotype was monitored by analysis of gene expression utilizing reverse transcription-polymerase chain reaction. RESULTS: OA chondrocytes isolated from three different donors (ages 41, 69 and 75 years) were transduced with a retroviral construct expressing telomerase. After selection, pooled populations of cells from all donors and a clonal cell line from one donor expressed telomerase activity and exhibited lengthening of telomeres. Chondrocytes expressing telomerase showed an increase of five to nine population doublings over 234 days of culture in monolayer. The telomerase-transduced cells recovered a chondrocyte-specific gene expression pattern following culture on polyHEMA-coated dishes. CONCLUSION: The exogenous expression of telomerase may represent a way to expand human OA chondrocytes while allowing maintenance of the chondrocyte-specific phenotype. These cells have the potential to be used for restoration of the articular cartilage defects occurring in this disease.

Arthritis Rheum 2002 Mar;46(3):683-93

Telomeres, telomerase and stability of the plant genome.

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.

Plant Mol Biol 2002 Mar;48(4):331-7


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