REVIEW

Review

Telomere maintenance and disease

Judy M Y Wong, Kathleen Collins

The proliferative capacity of human cells is regulated by telomerase, an enzyme uniquely specialised for telomeric DNA
synthesis. The critical role of telomerase activation in tumour progression and tumour maintenance has been well
established in studies of cancer and of oncogenic transformation in cell culture. New evidence suggests that
telomerase activation has an important role in normal somatic cells, and that failure to activate sufficient telomerase
also promotes disease. We review the evidence for premature telomere attrition in proliferative deficiencies of the
human haemopoietic system, and discuss the potential use of telomerase activation in telomere-restorative gene
therapy.

Telomere biology Short telomeres

Telomeres are found at the ends of linear chromosomes.
In most eukaryotes, functional telomeres are constituted
by short, tandem DNA repeats and a multitude of
associated proteins.1,2 The presence of telomeres
distinguishes the natural ends of chromosomes from
random DNA breaks, thereby preventing unwanted end-
to-end fusion or nucleolytic degradation. In addition to
this physical protection of chromosome ends, eukaryotic
telomeres have important roles in cellular processes
including chromatin organisation and control of cell
proliferation.3,4
DNA-dependent DNA polymerases fail to replicate
linear chromosome ends completely. Consequently, up to
a few hundred basepairs of telomeric DNA can be lost
with each mammalian cell division. Telomere erosion is
cumulative. It eventually produces an altered chromatin
structure, which either activates a damage-sensing
checkpoint to halt cell growth, or escapes checkpoint
control and undergoes repair-mediated rearrangement.2,5-7
Cultured normal human fibroblasts with critically short
telomere length are restrained from cell division in a
quiescent state termed replicative senescence. In other cell
types, short telomeres can instead provoke apoptosis. In
all cells with an intact telomere checkpoint, telomere
attrition can serve as a mitotic clock that safeguards
normal somatic cells from deregulated proliferation by
counting down the lineage allotment of cell divisions
(figure 1).
If the cellular pathways monitoring and responding to
short telomeres are inactivated, continued proliferation
will erode telomeres enough to prevent their end-
protective function. Telomeres that become uncovered as
double-stranded breaks are subject to DNA repair,
creating chromosome fusions or translocations. Aberrant
chromosomes are further damaged by rounds of anaphase
bridge, breakage, and fusion. Cell cultures with genomic
instability caused by unstable telomeres are said to be in
crisis phase, which is characterised by a high probability of
Lancet 2003; 362: 983–88. Published online May 13, 2003
http://image.thelancet.com/extras/02art7027web.pdf
Department of Molecular and Cell Biology, University of California
at Berkeley, 401 Barker Hall, Berkeley, CA 94720-3204, USA
(J M Y Wong PhD, K Collins PhD)
Correspondence to: Dr Kathleen Collins
(e-mail: kcollins@socrates.berkeley.edu)
Checkpoint Checkpoint bypassed:
intact additional telomere shortening
Proliferative Chromosome Non-reciprocal
Apoptosis
arrest fusion recombination
Rampant genomic
instability (crisis)
Reactivation of telomerase:
Cell death
genome stabilisation
Cancer
Figure 1: Cellular responses to short telomeres
Telomeres shorten with cell proliferation when not balanced by telomere
synthesis. In healthy somatic cells, critically short telomeres activate a
checkpoint that induces either apoptosis or the proliferative arrest of
replicative senescence. In the absence of checkpoint function, telomeres
erode until they become substrates for aberrant DNA repair. Infrequently,
spontaneous activation of telomerase during the crisis phase of genomic
instability stabilises and allows maintenance of the rearranged genome,
conferring indefinite renewal capacity.
cell death. Rarely, tumorigenic cells that have activated a
mechanism for stable maintenance of telomere length can
survive (figure 1).
Telomere maintenance by telomerase
Several distinct strategies for telomere length maintenance
have evolved in eukaryotes. The physiological pathway for
telomere maintenance in human cells involves the
ribonucleoprotein enzyme telomerase. This enzyme is a
specialised reverse transcriptase that copies a region
Search strategy
We searched PubMed with keywords including telomerase,
telomeres, proliferative senescence, diseases of the
haemopoietic system, gene therapy, and others. We aimed to
cite recent studies directly; helpful review articles were cited
for greater coverage of articles published before 2002.

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 REVIEW
Figure 2: The human telomerase ribonucleoprotein complex
Telomerase RNA (red) must interact with H/ACA proteins (including
dyskerin, green) and with other unknown proteins, to accumulate in cells
as stable, fully processed functional RNA. A stable but inactive
telomerase ribonucleoprotein is present in most human somatic cells. To
become catalytically active, TERT (blue) and probably other proteins must
join the ribonucleoprotein complex. Additional subunits may be required
to mediate interaction of telomerase with the telomeric repeats at a
chromosome 3 end (black).
within its integral RNA component to extend
chromosome 3 ends by synthesis of telomeric simple
sequence repeats.8 An alternative mechanism for
lengthening of telomeres, which is dependent on
homologous recombination, can arise in human cells
under selective pressure. This second pathway is
detectable in some transformed fibroblast cultures and a
small minority of cancers.9
Telomerase ribonucleoproteins are only partly
characterised in subunit composition. Our knowledge of
human telomerase-associated proteins has been gained
from candidate approaches (figure 2). The telomerase
RNA component is ubiquitously transcribed and
assembled into a stable but catalytically inactive
ribonucleo protein.10 Precursor processing and stability of
human telomerase RNA (hTR) require binding of small
nucleolar ribonucleoproteins such as dyskerin, NHP2,
NOP10, and GAR1 to the H/ACA motif of hTR.11-15
Catalytic activity is acquired by binding of the telomerase
reverse transcriptase subunit (TERT)16 and is modulated
by additional factors including chaperones and nucleic
acid binding proteins.17 Finally, unknown additional
conformational changes or interaction partners are
necessary to recruit telomerase to telomeres during the
DNA synthesis phase of the cell cycle.
Telomerase activity is absent from most human somatic
cells beyond the early stages of fetal development,18
although telomerase-positive cells are more generally
present in mouse tissues.19 Telomerase inactivation in
human somatic cells is mainly due to transcriptional
repression of TERT, and can be overcome by constitutive
expression of a human TERT (hTERT) transgene.20
Constitutive expression of hTERT in presenescent
fibroblasts, lymphocytes, epithelial cells, and other cell
types can extend their replicative lifespan.4 Other
mechanisms of telomerase inactivation, including
alternative mRNA splicing to express a catalytically
inactive form of hTERT, have been noted in early
development.21,22 The general somatic inhibition of
telomerase activity may function as a tumour suppression
mechanism, which is perhaps most relevant in organisms
with long lives. Because telomere erosion in telomerase-
negative cells limits proliferative capacity, the likelihood of
accumulation of the multiple spontaneous DNA
mutations essential for carcinogenesis should be
diminished. This hypothesis is consistent with data from
some mouse models, although the short telomeres
resulting from telomerase disruption can also enhance
tumorigenesis by promoting genomic instability.23
984
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Telomerase inactivation in the human somatic tissues
has exceptions, in a highly restricted subset of adult
tissues that are undergoing rapid proliferation.22
Telomerase is known to be active in some germline,
epithelial, and haemopoietic cells. In telomerase-positive
stages of these cell lineages, telomerase activation
correlates with induction of hTERT gene transcription. In
some cases, additional stimulation is provided by an
increase in the amount of ribonucleoprotein, post-
translational modification of hTERT, or subnuclear
shuttling of catalytically active ribonuclearprotein.22,24,25
Telomerase in somatic stem cells does not compensate for
proliferation-dependent telomere loss; it does so only in a
restricted phase of progenitor cell proliferation, and is
subsequently downregulated by terminal differentiation.
Intuitively, telomerase activation could be thought to
replenish every telomeric repeat lost during multiple
preceding rounds of DNA replication. Such telomere
length homoeostasis has been extensively characterised in
single-celled organisms such as yeasts1 and is being
intensively studied in long-term cultures of mammalian
cells.26 In cancer cells, the balance between telomere loss
and telomere addition can maintain stable telomere
lengths over an extended period of proliferation. In
normal human somatic tissues, however, telomerase
activation does not equate with the acquisition or
maintenance of stable telomere lengths. The transient
nature of telomerase activation in normal somatic cell
lineages differs strikingly from the constitutive activation
of telomerase in tumour cells. The amount of telomere
shortening offset by somatic cell telomerase activation
depends on telomere length before activation, rate of
telomere shortening with each round of cell division,
amount of telomerase activity, and cellular regulation of
telomere-telomerase interaction. Much remains unknown
about mechanisms that establish telomere length to
account for findings such as proliferation-linked activation
of telomerase in cells that nonetheless undergo rapid
erosion of telomeres.27
The haemopoietic system: telomerase and
telomere dynamics
The haemopoietic system provides an excellent model for
the study of human telomerase and telomere dynamics in
a physiological context.28,29 Haemopoietic stem cells have
longer telomeres than descendant lineages. The low
telomerase activity in stem cells does not seem to
compensate for proliferation, resulting in progressive
telomere loss with age. In the differentiation of myeloid
cells, including granulocytes and monocytes, telomere
length in progenitor cells sets an upper limit on the
proliferative capacity of lineage descendants. Telomere
lengths in differentiated myeloid populations correlate
with their anticipated replicative history.30 By contrast,
lymphoid cell lineages have a complicated telomerase and
telomere length dynamic. Naive and memory T cells and
B cells from peripheral blood undergo an age-progressive
erosion of telomere length.31 However, antigen challenge
can induce telomerase activation and telomere elongation
in these cells.
Germinal centre B cells provide the most striking
example of telomerase-dependent telomere elongation in
the normal human soma.32 Strong telomerase activation in
centroblasts and centrocytes leads to a net gain in
telomere length of 3–4 Kb for germinal centre B cells
relative to naive B cells. Memory B cells downregulate
telomerase, returning the lineage to proliferation-
dependent telomere loss. Although CD4+ memory T cells
possess shorter telomeres than their naive counterparts,33
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memory and naive B cells can have similar telomere
lengths.32 In culture, mitogenic stimulation of B and
T cells induces telomerase activation in a wide range of
conditions.29 However, telomerase activation in cultured
lymphocytes does not increase telomere length; it can
offset the rate of telomere loss for only a restricted number
of cell cycles after the initial stimulus to proliferation.34
Haemopoietic proliferative failure induced by
telomerase deficiency
Mice without genes for telomerase survive for several
generations despite progressive telomere attrition, since
some inbred strains have especially long telomeres.35 After
several generations of knockout breeding, with successive
telomere shortening transmitted through the germline,
knockout mice develop haemopoietic deficiencies. These
problems include small spleen size, decreased follicle
numbers, reduced lymphocyte counts, and impaired
lymphocyte proliferation.36 These findings could not
predict whether telomerase function would be required
for somatic cell renewal within a person’s lifespan.
Dyskeratosis congenita was the first primary telomere
maintenance disorder to be identified in man. The major
cause of death is progressive bone marrow failure. Data
from studies of this disease suggest that the restricted
amount of telomerase-dependent maintenance of
telomeres in human somatic cells is essential for health
and viability, serving to boost cellular renewal in highly
proliferative epithelial and haemopoietic tissues.22
Dyskeratosis congenita is mainly inherited as an
X-linked disorder, and is characterised by reticulate skin
pigmentation, nail dystrophy, mucosal leucoplakia, and
pancytopenia.37 Genetic linkage analysis in many families
with X-linked disease showed that a mutant gene termed
dyskerin might cause the disorder.38 Dyskerin has
homologues in all eukaryotic organisms. The highly
related protein Cbf5p in budding yeast catalyses the post-
transcriptional conversion of uridine to pseudouridine at
target RNA sites, which are specified by hybridisation
with tightly bound H/ACA small nucleolar RNAs.39 In
vertebrate cells, human dyskerin is associated with
H/ACA small nucleolar RNAs and also with hTR12 via the
hTR H/ACA motif (figure 2).11,13 Most mutations in the
dyskerin gene result in substitutions of a single
aminoacid.40 Since the null allele is lethal for all organisms
investigated (including mice41) alleles of dyskerin in
dyskeratosis congenita must alter but not eliminate the
protein’s function. Dyskerin gene mutations noted in
patients with the disease do not seem to affect amounts of
H/ACA small nucleolar RNA, pseudouridine synthesis in
ribosomal RNA, or any other aspect of ribosome
biogenesis tested to date.12,42 Consistent with a partial loss
of function in disease-associated isoforms of dyskerin,
telomerase RNA is decreased but not absent in cells from
X-linked dyskeratosis congenita patients with dyskerin
gene mutations,12 with a 3–5 fold reduction in steady-state
RNA (unpublished). A reduced concentration of
telomerase ribonucleoprotein restricts maximum catalytic
activity of telomerase, inducing premature telomere
shortening that varies in extent according to the type of
cell and its proliferative history.
Mutations in the telomerase RNA gene are a genetic
basis for autosomal dominant dyskeratosis congenita. In a
large family with autosomal dominant inheritance of this
disorder, a heterozygous deletion of the 3 region of the
telomerase RNA gene cosegregated with disease.43 This
deletion removes important regions of the H/ACA motif
that are essential for RNA accumulation, and such a
mutation should therefore create a null allele. If
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REVIEW
transcription limits expression of hTR, steady state
amounts of RNA would be reduced by 50%. Additional
telomerase RNA gene mutations were identified in
individual patients with autosomal dominant dyskeratosis
congenita.43 These mutations would be predicted to have
a range of different effects on telomerase function,
because some mutations arise within hTR regions that are
important for RNA accumulation, whereas others occur
within hTR regions important for catalytic activity but
dispensable for accumulation.13
Defects in different structural parts of the telomerase
enzyme can lead to different phenotypes. The large degree
of cell type-specific telomerase enzyme regulation means
that a particular mechanism of inhibition could markedly
affect enzyme function in one tissue but not in another.22
Disease onset is generally later in patients with autosomal
dominant dyskeratosis congenita than in those with
X-linked disease; this difference might result from a less
severe telomerase deficiency in autosomal dominant disease
inheritance. Different penetrance of dermatological
phenotypes relative to bone marrow deficiency is also noted
among patients with dyskeratosis congenita,37 suggesting
that different amounts of telomerase ribonucleoprotein loss
can have differential effects on epithelial and haemopoietic
tissue renewal.22 The enhanced risk of carcinoma
development in some patients probably indicates that short
telomeres in human epithelial cells have a role in promotion
of tumorigenic genomic instability.44,45
Dyskerin gene mutations have also been noted in
association with X-linked Hoyeraal-Hreidarsson
syndrome.46-48 Patients with this disorder have prenatal-
onset growth retardation, cerebellar hypoplasia,
microcephaly, and immunodeficiency in the first few years
of life. However, they do not present with the set of
epithelial features that is typically diagnostic of dyskeratosis
congenita. Additionally, telomerase RNA gene mutations
have been described in a subset of patients with aplastic
anaemia.49 Consistent with a primary defect in telomerase
function, peripheral blood leukocytes in these patients can
have substantially shorter telomeres than age-matched
controls.50-52 Possibly, a large range of proliferative
deficiency phenotypes could result from primary
deficiencies of telomerase function, with variations
dependent on genetic background, environmental
challenge, and the mechanism of telomerase inhibition.
Disease with premature telomere loss due to
increased proliferative demand
Defects in genes other than those encoding telomerase
subunits themselves can result in premature telomere loss.
For example, an increased burden of proliferative demand
can drive chronologically premature telomere shortening,
even with an unaltered rate of telomere loss per cell
division. Phenotypes distinct from dyskeratosis congenita
would arise dependent on which tissues are subject to the
highest rates of turnover and which cell types have the least
telomerase-dependent compensation for proliferation.
Some examples relevant to the human haemopoietic system
are described below.
Chronic infection
The chronic phase of HIV infection is associated with
substantial loss of telomere length in the CD8+ subset of T
lymphocytes.53 The reason why telomere loss is specific to
this group of T cells is unknown. Excessive telomere
shortening has also been shown in patients with chronic
hepatitis or liver cirrhosis,54,55 consistent with accelerated
liver cirrhosis in telomerase knockout mice.56 In liver
disease, and probably in other chronic infections, the rate of
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Telomere erosion
with proliferation
Telomere length
Early telomerase activation
Short telomere checkpoint
Cell divisions
Figure 3: Cellular responses to telomerase activation
Most human cells do not express telomerase and lose terminal telomeric repeats with each round of cell division (blue line). Short telomeres trigger the
proliferative arrest of replicative senescence or apoptosis. Telomerase activation before this checkpoint allows indefinite proliferation with normal cellular
physiology (green curve). Activation of telomerase after onset of genomic instability promotes acquisition of transformed and tumorigenic phenotypes (red
curve, dashed to denote the small proportion of cells that evade death).
cellular renewal rises to meet higher demand from the
increase in cell death.
Bone marrow transplant
Bone marrow transplant is currently the only therapeutic
option for some patients with haematological disorders. For
long-term engraftment after transplantation, an expansion
of the transplanted cells must happen to repopulate the
host’s marrow. This unusually high demand for
haemopoietic system renewal, although transient, could
substantially deplete the telomere reserves of stem and
progenitor cells. Shorter telomere lengths have been shown
in transplant recipients than in their donors.57-60 Although
clinical data is not yet available to implicate telomere
shortening as an important factor in the failure of bone
marrow transplant, some relevant evidence has been
reported.61
DNA damage repair syndromes
Damaged DNA and aberrant DNA replication
intermediates must be repaired to protect genome
stability. When gene mutations compromise the function
of DNA repair, an increase in cell death occurs and the
proliferative demands for renewal are increased. Cell
lines derived from patients with DNA-repair gene
mutations undergo premature senescence, which is
causally linked to chronologically accelerated telomere
shortening.
Ataxia telangiectasia is an autosomal recessive
disorder. Patients with this disease have several defects,
including hypersensitivity to ionising radiation. Death is
mostly due to infection, with increased predisposition to
lymphoid malignant diseases.62 The ataxia telangiectasia
gene locus encodes ATM, a protein kinase involved in
986
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Normal genome structure
Normal physiology
Extended proliferative lifespan
Genome rearrangements
Transformed phenotypes
Extended proliferative lifespan
Genomic
Late telomerase instability
activation
Senescence, Crisis
apoptosis Cell death
DNA damage-triggered signal transduction to p53
activation and checkpoint arrest. T cells from patients
with ataxia telangiectasia have shorter telomeres than
age-matched controls, consistent with a greater rate of
cell turnover due to unrepaired genome damage.63
Constitutive expression of hTERT in patients’ cells
increases telomere length and prevents proliferative
senescence, but does not fully rescue telomere
dysfunction; these findings suggest that ATM may have a
direct role at the telomere as well.64 Similar findings have
been reported for cells from patients with Nijmegen
breakage syndrome.65 This disorder derives from
mutations of an ATM target gene, and shares with ataxia
telangiectasia the features of radiosensitivity, cancer
predisposition, and immunodeficiency.
Fanconi anaemia is an autosomal recessive disorder
characterised by progressive bone marrow failure and an
increased risk of cancer, most commonly acute myeloid
leukaemia.62 Patients with this disease have chrono-
logically accelerated telomere shortening.66 Although the
spectrum of disease phenotypes has some similarities to
dyskeratosis congenita, only cells from patients with
Fanconi anaemia have increased sensitivity to DNA
crosslinking agents. Fanconi anaemia can arise from
mutations in different loci, including the breast cancer
susceptibility gene BRCA2.67 These and other findings
firmly establish a direct role for Fanconi anaemia group
proteins in the response to DNA damage.68 In view of the
raised oxidative stress sensitivity of Fanconi anaemia
cells, an increased rate of cell turnover could
chronologically accelerate telomere shortening.
Alternatively, a faster rate of telomere shortening with
each cell division could result from increased telomere
damage.69 Cells of patients with ataxia telangiectasia show
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increased telomere damage,70 but immortal ataxia
telangiectasia cell lines do not differ from controls in any
measured variable of telomere length maintenance.71
Therefore, rates of telomere shortening will have to be
measured directly in different cell types from individuals
with Fanconi anaemia and from healthy people, to
establish a direct effect of disease on rate of telomere loss.
Clinical applications
Expanded proliferative capacity would reverse or prevent
many adverse symptoms of haemopoietic deficiencies,
regardless of their precise molecular cause. Patients with
primary genetic defects in telomere maintenance
pathways or with a higher burden of demand for cellular
renewal would both be served by boosting proliferative
capacity. Patients who might be helped by a telomere-
based treatment could be identified by use of genetic tests
or telomere length measurements, well before the onset
of severe haemopoietic failure. Constitutive activation of
telomerase before activation of the short-telomere
checkpoint (figure 3, green curve), instead of as a
selection in the crisis phase of telomere dysfunction (red
curve), has been shown to leave typical cellular physiology
unperturbed.4
Several features of hTERT transgene expression as a
gene therapy protocol to allow telomerase activation in
haemopoietic cells seem favourable. First, constitutive
hTERT expression with a standard retroviral vector is
sufficient for telomere length maintenance or gain in most
cell types, including T cells72,73 and fibroblasts from
patients with dyskeratosis congenita (unpublished).
Second, hTERT expression can become downregulated
after restoration of telomere reserves and still provide an
adequate expansion of renewal capacity. Third, because
cells that regain telomere length have a proliferative
advantage, they should compete well after engraftment.
Finally, trangene-mediated expression of hTERT protein
should not provoke rejection because this molecule is
expressed widely in development and in some adult
somatic cells as well. Because many cell types in the
human haemopoietic system can activate telomerase
endogenously, constitutive activation of the enzyme might
not increase cancer risk, and could even decrease risk by
reduction of the genomic instability caused by short
telomeres.4,22 These favourable features should provide a
stimulus for clinical trials. However, putative telomere-
independent roles of telomerase in the stimulation of cell
growth and the prevention of apoptosis will also need to be
considered.74 Experimental analysis will be needed to show
whether manipulation of telomere length by telomerase
activation is clinically useful.
Conflict of interest statement
None declared.
Acknowledgments
We thank staff of the Collins Laboratory for comments about the
manuscript, and many colleagues for general discussion. Both authors
contributed to all aspects of manuscript production. JMYW was funded in
part by a postdoctoral fellowship from the National Cancer Institute of
Canada, with additional funding from a grant of the American Cancer
Society to the laboratory of KC. This review does not reflects the interests
of any funding agency.
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