LE Magazine October 2002
Page 4 of 4
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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