Current Molecular Medicine 2005, 5, 187-196 187

Telomere Dynamics in Response to Chemotherapy

N. Beeharry and D. Broccoli*

Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA
19111, USA
Abstract: The use of chemotherapy provides an essential arm in the treatment of a number of
cancers. The biological feature common to all cancerous cells that sensitizes them to
chemotherapeutic agents is their elevated division rate. Rapidly dividing cells, such as tumor cells,
are more sensitive to chemotherapeutic agents that act to initiate pathways leading to cell death, a
process enhanced in cells with compromised DNA damage responses. The toxicity accompanying
chemotherapy is due to side-effects induced in normal regenerative tissues which also have relatively
high replication rates, such as hair follicles, the hematopoietic system, the gastrointestinal system,
the germline and skin cells. While the side-effects of chemotherapy may be tolerated by the patient,
the long term impact of the cytotoxic effects of chemotherapy on healthy tissues is only now
becoming apparent. Chemotherapy-induced cytotoxicity in regenerative tissues requires multiple cell
divisions in order to reconstitute the affected tissues. At least in part as a consequence of these
extra divisions, telomeres in individuals treated with chemotherapy are shorter than age-matched
control individuals who have never been exposed to these drugs. Given the essential role of telomeres
in regulating cellular aging and chromosomal stability, it is possible that the prematurely shortened
telomeres that arise following chemotherapy may impact the long-term replicative potential of these
tissues. This review is focused on how telomeres may be modulated, directly or indirectly, by
anticancer drugs and the potential long-term consequences of accelerated telomere shortening in
healthy tissue as a result of current cancer treatment protocols.

INTRODUCTION individual. While the current data indicate that

Chemotherapy is successfully used to treat a
wide range of neoplasms, including hematological
malignancies and a variety of carcinomas. Most
anticancer drugs work by inducing apoptosis, either
through perturbation of cellular signaling pathways
or via induction of an excessive amount of DNA
damage. Chemotherapy and radiation therapy
target tumor cells primarily on the basis that
cancerous cells have a higher mitotic index than
normal cells. Since chemotherapy is damaging to
cells with a high replicative rate, it is reasonable to
expect regenerative tissues to be inadvertently
affected by anticancer drugs. While the bone
marrow and gastrointestinal tract are the two tissues
that generally exhibit the greatest sensitivity to most
chemotherapeutic drugs [1], there is also data
reporting molecular mechanisms underlying toxic
side effects of chemotherapy on hair follicles
(reviewed by [2]) and germline cells (reviewed by
[3]). The cell death induced in healthy tissues as a
result of drug toxicity is countered by regenerative
cell divisions. A growing body of evidence supports
the hypothesis that the tissue reconstitution
following each round of chemotherapy is
accompanied by telomere attrition, thereby
producing cells with prematurely shortened
telomeres relative to the actual age of the
*Address correspondence to this author at the Department of Medical
Oncology, Fox Chase Cancer Center, 333 Cottman Avenue,
Philadelphia, PA 19111, USA; Tel: (215) 728-7134; Fax: (215) 728-4333;
E-mail: K_Broccoli@fccc.edu
telomere attrition is at least in part a side-effect of
chemotherapy-induced replicative stress, an
alternative possibility is that erosion of telomeric
sequences is a result of mechanisms acting directly
on telomere structure or telomere maintenance
pathways. For example, there is evidence
suggesting that under certain conditions telomeric
DNA may be a more favorable target of DNA
damaging chemotherapy than bulk genomic DNA
[4]. In this review, we summarize putative
mechanisms by which chemotherapy may
influence telomere dynamics in normal tissues and
suggest potential long-term consequences for
patients requiring chemotherapeutic treatment.
TELOMERE ATTRITION IN RESPONSE TO
CHEMOTHERAPY
It is now evident that an unanticipated side
effect of cytotoxic chemotherapeutic protocols is
telomere attrition in healthy replicative cells. To
date, there is a paucity of studies that have
investigated the effect of chemotherapy on
telomere dynamics in regenerative tissues such as
hair follicles, germline, and gastrointestinal cells. In
contrast, hematopoietic cells provide a tractable
model system whereby the impact of chemotherapy
on telomere length can be investigated because of
the relative ease and minimally invasive procedure
through which samples may be collected. Thus,
telomere length in peripheral blood mononuclear
cells (PBMCs) of patients who have been exposed
to myelotoxic chemotherapeutics is shorter than

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188 Current Molecular Medicine, 2005, Vol. 5, No. 2
that observed in age-matched control individuals
who have never been exposed to such treatments
(see below). Complicating issues that one must be
sensitive to when analyzing telomeres of
hematopoietic cells are lineage-specific
differences in telomere dynamics and telomerase
activation during differentiation and clonal
expansion (reviewed in [5]). These differences in
telomere length between lineages and through
differentiation are paralleled by cell-type specific
differences in telomerase regulation [6, 7].
Furthermore, the rate of telomere attrition varies
among the different hematopoietic lineages, with
greater attrition per year in T cells than in B cells
[7, 8]. This makes it difficult to directly extrapolate
telomere attrition in a specific lineage of
hematopoietic cells to other cell types, much less
other tissues. However, while tissue-specific
differences in both the sensitivity to chemotherapy
and telomere metabolism accompanying cellular
divisions are probable, it is anticipated that the data
obtained from the hematopoietic system will be
generally relevant to other regenerative tissues.
The effect of chemotherapy on telomere
dynamics has been measured by determining the
mean telomeric restriction fragment length in
hematopoietic cells of patients before, during and
after treatment. This methodology has the benefit of
being able to reliably correlate the induction of
chemotherapy with any changes in telomeric
restriction fragment values within individual patients
over a period of time. It is important to stress that
those studies performed using bulk PBMCs will
contain cells of a number of lineages [7, 8] and
thus the resulting telomere length analysis will be
complicated by inter-lineage variability in average
telomere length. Additionally, inter-lineage
variability in sensitivity to drug and/or regenerative
divisions will be masked when analyzing bulk
PBMCs. A final caveat for using such approaches is
that while treatment with chemotherapy may be
correlated with an alteration in telomere length,
little if any, information is gained regarding
underlying mechanisms.
The studies investigating telomere dynamics in
the hematopoietic system in response to
chemotherapy can be broadly divided into those
investigating effects in the context of treatment for
hematological malignancies and those in the
context of treatment for solid tumors. Analysis of
telomere length changes before and after exposure
to chemotherapy in hematological malignancies is
complicated by the mixture of tumor and normal
cells in the samples being tested. The mixed
population of cells results in a biphasic distribution
of telomere lengths, with the shorter population of
telomeres representing those present in the tumor
cells [9]. Following successful treatment, the
population with shorter telomeres is reduced or
absent resulting in an apparent increase in average
telomere length. By comparing length changes
within the longer population of telomeres, one can
Beeharry and Broccoli
establish the effects of myelotoxic chemotherapy
and forced regeneration upon the healthy
hematopoietic cells within the population.
Accelerated telomere shortening of normal cells
as a result of exposure to chemotherapy was first
noted by Engelhardt and co-workers [10]. Telomere
lengths of mononuclear cells (MNCs) and
granulocytes from pediatric patients with acute
lymphocytic leukemia (ALL), acute myeloid
leukemia (AML) or solid tumors were determined
prior to and following a course of chemotherapy.
Telomeres were shortened in normal hematopoietic
cells in all patients following chemotherapy,
regardless of tumor type. However, the extent of
telomere shortening after repeated cycles of
chemotherapy was different between the cell types.
Telomere loss in granulocytes exceeded that
observed in MNCs for both leukemias and solid
tumors, with a mean sequence loss of
approximately 1 kb for MNCs and 1.8 kb for
granulocytes. The type of chemotherapy that these
patients underwent for the treatment of their cancer
was myelosuppressive, with repeated exposure
being followed each time by proliferation. Since
the effect of chemotherapeutic treatment on
telomere loss was more pronounced in granulocytes
compared with MNCs, the authors concluded that
the chemotherapy treatment had a greater effect on
the myeloid than the lymphoid lineage. The
variation of telomere attrition between different
hematopoietic lineages is not surprising given the
variation in telomere dynamics among
hematopoietic lineages [5]. Alternatively, cells may
vary in their sensitivity to other mechanisms of
chemotherapy-induced telomere loss, such as that
induced by oxidative stress (reviewed in [11]),
discussed in more detail below.
In a follow-up study by the same group, the
medium-term outcome of chemotherapy on
telomere length in pediatric patients was
investigated [12]. In MNCs from patients with ALL
telomere length was 9.8±0.9 kb at the time of
diagnosis and had decreased, on average, to
9.0±1.4 kb at a latest follow up period of 20 months,
similar to the average 1 kb loss observed by
Engelhardt et al. [10]. In granulocytes from the
same individuals, average telomere length at
diagnosis was 9.3±1.6 kb and decreased to 8.7±1.8
kb at follow up [12], less than that observed
previously [10]. The erosion of telomeric sequences
in these cells corresponds to a loss rate of 30-40
bp/month (360-480 bp/year), significantly higher
than the range of 20-60 bp/year of erosion that
accompanies natural human aging in the
hematopoietic system [7, 10, 13]. An even greater
rate of telomere loss, approximately 100 bp/month
(1200 bp/year), was observed in the MNCs of
children being treated for solid tumors Standard risk
ALL treatment is generally a low intensity non-
myleoablative regimen compared to the high
intensity chemotherapy that is used to treat solid
tumors. The authors reasoned that since a more
Telomere Dynamics in Response to Chemotherapy
intense treatment protocol was used for patients
with solid tumors as compared to treating patients
with ALL, the greater toxicity associated with the
high dose treatment underpinned the differences in
the effect of chemotherapy between ALL patients
and solid tumor patients.
The relationship between chemotherapeutic
dosage and effect on telomere loss has been
directly studied by Schroder et al.[14]. In this
analysis, individual patients were followed through
a course of standard dose or high dose
combinatorial chemotherapy for treatment of breast
cancer. Leukocyte telomere length was measured
by obtaining blood samples from patients directly
prior to the start of chemotherapy, and 5 months
after completion of therapy [14]. Similar to the two
studies discussed above [10, 12], although there is a
trend towards telomere shortening in individuals
exposed to chemotherapy, the actual changes
measured were highly variable between individuals.
In the standard dose group of 17 patients, 9 patients
exhibited a decrease in telomere length, while 4
patients had an increased telomere length. The
range of telomere length changes following
standard dose chemotherapy was +0.4 to -2.2 kb.
Similarly, in the high dose group of 16 patients, 9
patients had decreased telomere length, while 5
patients exhibited an increase in telomere length.
The range of telomere length changes in patients
who received high dose chemotherapy was +0.8 to
-1.1 kb. By following individual patients, the raw
data demonstrated that some patients are more
affected than others by chemotherapy treatment.
The information from this 'susceptible' group of
patients is 'masked' when the total data are
amalgamated, since telomere length had only
decreased by on average 0.2 kb in this study. It may
be argued that because 52% of patients who
received standard dose treatment and 56% of
patients who received high dose therapy showed a
decrease in telomere length of leukocytes,
resources to alleviate the potential deleterious
outcomes associated with 'artificially accelerated'
telomere loss in normal tissues should be
concentrated on this subset of individuals. However,
since the mechanisms contributing to the
susceptibility to telomere loss in healthy tissues
remain unknown, targeted therapy aimed at
subpopulations is currently not feasible. Future
studies comparing susceptible versus resistant
patients may provide information regarding
underlying pathways and allow identification of
those individuals most likely to benefit from
approaches to modulate chemotherapy associated
telomere attrition.
Surprisingly, less telomere loss accompanied
myeloablative treatment than standard dose
treatment [14]. For example, the maximum
telomere loss among patients receiving standard
dose chemotherapy was 2.2 kb, while in the high
dose group the maximum telomere loss measured
was only 1.1 kb. However, since the patients
Current Molecular Medicine, 2005, Vol. 5, No. 2 189
receiving high dose therapy also received stem cell
support, it is possible that fewer divisions were
needed to achieve hematopoietic reconstitution.
These data indicate that multiple cycles of high
dose chemotherapy with peripheral blood stem cell
re-infusion is likely to be different than repeated
doses of standard dose therapy in terms of
hematopoietic regeneration. Standard doses are
myelosuppressive rather than myeloablative, such
that regenerative requirement following each
exposure is less than that in patients receiving high
dose regimens. Under such conditions, the extent of
telomere attrition for individuals receiving standard
dose chemotherapy would be additive over the
cycles. Myeloablative treatment requires greater
proliferation in order to repopulate the tissue
following each cycle. Since re-infusion of
peripheral blood stem cells containing intact
telomeres occurs after each cycle, although
potentially more severe, telomere loss may not be
additive with additional cycles. A final possibility is
that it may be less harmful to completely ablate the
hematopoietic system than to induce DNA damage
within telomeres, which may result in rapid large
losses of DNA due to repair reactions (see below).
MECHANISMS OF CHEMOTHERAPY
INDUCED TELOMERE LOSS
1. Proliferation Associated Telomere Loss
Although there are limited studies providing
mechanistic insight as to how chemotherapeutic
drugs might induce loss of telomeric DNA, there is
evidence that telomere attrition may be a
consequence of a number of distinct mechanisms.
The simplest explanation is based on the fact that
many anticancer drugs are toxic to the
hematopoietic system of the patient. Stem and
progenitor hematopoietic cells will under-go a
number of cell divisions during reconstitution of the
hematopoietic system. These additional cell
divisions may lead to an apparent enhancement in
telomere attrition during the time period that
patients are undergoing treatment and recovery,
relative to similarly aged individuals whose
hematopoietic systems have never been
challenged in this way (Fig. 1). According to this
model telomere erosion occurs at the usual rate per
cell division, but this rate appears elevated per unit
time due to the significantly greater number of cell
divisions that are required to repopulate the tissue
during recovery. This hypothesis is supported by
numerous studies determining telomere dynamics
following bone marrow transplantation (BMT) [15-
21]). Under the conditions of BMT, proliferation is
increased in order to achieve reconstitution of the
hematopoietic system and there is a concomitant
reduction of telomere length in peripheral blood
mononuclear cells [16, 21], T cells [17, 19],
neutrophils [17, 20] and granulocytes [15, 18] in
recipients following recovery as compared to the
telomere length in the respective donors. Consistent
190 Current Molecular Medicine, 2005, Vol. 5, No. 2 Beeharry and Broccoli

with the model that the telomere loss is due to the
increased proliferation necessary to regenerate the
hematopoietic system, the accelerated loss of
telomeric DNA was present only for the first year
following transplant [18-20]. Furthermore, the
amount of telomere reduction correlated negatively
with the number of cells infused [15, 16], again
suggesting that the amount of sequence loss was a
result of the total number of cellular divisions
required to achieve reconstitution of the
hematopoietic system. Based on these data from
BMT studies, the accelerated rate of telomere
attrition that accompanies chemotherapy would be
predicted to be limited to the time period of during
which cells are exposed to drug, with the rate of
attrition after removal of replicative stress returning
to one that is similar to that observed during normal
aging (Fig. 1).
2. Telomerase Inhibition
There are a number of possibilities by which
chemotherapeutic drugs may stimulate the loss of
telomeric DNA beyond that which accompanies
cell division. One such possibility is by inhibition of
the telomere maintenance enzyme, telomerase. If
telomerase acts to attenuate sequence loss as stem
cells differentiate to reconstitute tissues, then direct
inhibition of telomerase would lead to an increased
amount of sequence loss per cell division, thereby
contributing to the shorter telomeres induced in
healthy regenerative tissues exposed to
chemotherapy. While there is evidence from in vitro
data suggesting that certain anticancer drugs in
clinical use may be associated with telomerase
inhibition [22, 23], this does not always hold true
[24, 25]. In the breast adenocarcinoma cell line
(MCF-7), telomerase activity was inhibited by
adriamycin and 5-fluorouracil [22]. In addition,
using the human hepatoblastoma cell line HepG2,
it was shown that cell survival was deceased when
treated with tamoxifen as compared to untreated
controls. At higher doses of tamoxifen, inhibition of
telomerase preceded apoptotic cell death [23]. In
contrast, the anticancer drugs 5-fluorouracil, methyl
methanesulfonate, etoposide, cisplatin and

Fig. (1). Chemotherapy-associated telomere loss as a result of regenerative cell divisions. Individuals exhibit telomere loss
in many somatic cells each time a cell divides such that telomeres shorten with age (blue line). In circumstances of
extreme proliferative stress, such as that induced by the cytotoxic effects of chemotherapy, rapid telomere shortening is
observed in healthy regenerative cells of patients (red line). While the amount of telomere loss per cell division remains
similar to that accompanying normal aging, the increase in proliferation per unit time causes an apparent increase in the
rate of telomere attrition. Once chemotherapeutic treatment has ended and regeneration is compete, the increased
proliferative burden ceases, paralleled by the telomere loss rate per unit time returning to a similar level to that observed in
untreated individuals, albeit with the cells having less telomeric DNA.
Telomere Dynamics in Response to Chemotherapy
daunorubicin all failed to inhibit telomerase activity
in nasopharyngeal carcinoma cells [24]. Further,
while cisplatin caused a dose-dependent reduction
of telomerase activity in human testicular cancer
cells, other anticancer agents such as doxorubicin,
bleomycin, methotreate and melphalan were
without effect [25]. These studies are complicated
by the use of different combinations of drugs in
different cell lines harboring a complex and distinct
series of genetic abnormalities. Further, it is unclear
how much of the observed decrease in telomerase
activity is an indirect consequence of cell death.
It is possible that even partial inhibition of
telomerase activity may contribute to telomere
shortening induced by chemotherapy. For example,
during B cell differentiation, activation of
telomerase results in lengthening of telomeres in
early stages of maturation, which helps to offset
overall loss incurred during the cell divisions
accompanying the differentiation program [26, 27].
In addition, mice that are deleted for one copy of
the catalytic subunit of telomerase, hTERT, are
unable to maintain telomere arrays at wildtype
lengths indicating that the level of telomerase is
limiting [28]. Thus, reduction of telomerase activity
induced as an unanticipated effect of chemothera-
peutic drugs might contribute to the rate of
telomere attrition observed in healthy regenerative
tissues during recovery from cytotoxic treatment.
Targeting telomerase as part of a chemothera-
peutic protocol would also be predicted to
exacerbate the telomere attrition that accompanies
regeneration in healthy tissues, although this has
yet to be demonstrated in the clinic. If deleterious
long-term consequences accompany the chemo-
therapy-induced, prematurely aged telomeres in
regenerative tissues (see below), it may affect the
use of telomerase inhibition as a therapeutic
strategy (Harley this issue).
3. Modification of Telomere Structure
Certain drugs, like cisplatin, may directly modify
telomeric DNA thereby leading to the rapid loss of
terminal sequences, through a variety of
mechanisms. The effect of cisplatin on telomeres,
which is known to cause DNA adducts [29], was
examined using HeLa cells [30]. Cells cultured for
one cycle in the presence of low doses of cisplatin
led to a loss of telomeric DNA without affecting the
replication of bulk DNA. Loss of telomeric DNA was
followed 24 hours later by cell death, leading to the
suggestion that the shortened telomeres might act
as the apoptosis activating signal. The model
proposed to account for the rapid loss of terminal
DNA was based upon incomplete replication of
telomeres. Due to the absence of transcribed
regions directly adjacent within telomeres,
transcription-coupled nucelotide excision repair
was presumed not to be operational in repairing
cisplatin-induced damage to telomeric DNA. The
presence of unrepaired platinum adducts within the
Current Molecular Medicine, 2005, Vol. 5, No. 2 191
telomere would block replication through this
region, leading to the attendant loss of terminal
DNA sequences [30].
Another possibility, and one that would be
consistent with this study, is that platinum
containing anti-cancer drugs, like cisplatin, may
cause DNA base modification [29], which could
perturb telomere structure or function. Since a
number of proteins localize to the telomere and are
required to maintain telomere homeostasis, it is
possible to envisage that chemotherapeutic agents
may cause DNA adducts in telomeric DNA,
preventing essential telomere/protein interactions
and thereby resulting in perturbed telomere
maintenance. This could lead to an increased rate
of telomere degradation per cell division or
alternatively block the ability of telomerase to gain
access to chromosome termini. Either mechanism
might contribute to shortened telomeres through
increased loss of telomeric sequences per cell
division. In addition, cisplatin-induced DNA
adducts, which may be enriched at telomeres due
to the G-rich content [31] are preferentially
recognized by DNA mismatch repair (MMR) proteins
[32]. Repair of tracts of platinum adducts via the
MMR pathway may result in net loss of telomeric
DNA. It remains to be determined whether any of
these mechanisms contribute to the telomere
attrition observed in patients in vivo following
chemotherapeutic treatment.
4. Generation of Reactive Oxygen Species (ROS)
Many classes of anticancer drugs, such as
anthracyclines, alkylating agents, platinum
containing complexes, epipodophyllotoxins and
camptothecin, generate high levels of ROS
(reviewed in [33]). These may be produced through
direct perturbation of the electron transport system
or as a consequence of activation of the apoptotic
program. Intriguingly, a number of studies support
the hypothesis that oxidative stress plays an active
role in stimulating telomere loss (reviewed in [11],
see von Zglinicki and Martin-Ruiz in this issue).
Thus, culturing cells under hyperoxic conditions is
associated with accelerated telomere shortening
and a concomitant reduction in proliferative
potential [34, 35]. Furthermore, exposure of
endothelial cells to homocysteine, a pro-oxidant,
significantly increased the rate of telomere
sequence loss per cell division as compared to
untreated cells, a process blocked by inclusion of
catalase, an antioxidant that abolishes ROS [36].
Oxidative damage can lead to a variety of base
modifications, one of which is from guanine to 7,8-
dihydro-8-oxoguanine (8-OG). A DNA fragment
containing telomeric repeats was five times more
likely to be modified than one not containing the
GGG motif present in human telomeres, suggesting
that 8-OG damage may be enriched within
telomeric repeats [4]. The kinetics of telomere loss
when cells are cultured under mild oxidative stress
192 Current Molecular Medicine, 2005, Vol. 5, No. 2
is consistent with unrepaired ROS-induced damage
within telomeric DNA inhibiting complete
replication of this region, analogous to the
mechanism proposed above for cisplatin-induced
telomere shortening. Telomeres containing 8-OG
are poor substrates for telomerase in vitro [37],
suggesting that oxidative damage induced at
telomeres may impede the ability of telomerase-
dependent maintenance, thereby contributing to
the telomere shortening seen in patients who have
undergone antineoplastic treatment. In contrast,
more dramatic and rapid loss of telomeric DNA may
be associated with ROS generated early during
DNA-damage-induced apoptosis [38]. This telomere
shortening may play a role in amplifying the
apoptotic signal but is unlikely to contribute directly
to the production of the shorter average telomere
length seen in cancer patients who have received
chemotherapy.
In vivo studies suggest that chemotherapeutic
treatment can cause oxidative stress in
hematopoietic cells. Free radicals, such as
superoxide and hydrogen peroxide were generated
in human polymorphonuclear leukocytes from
patients having undergone chemotherapy [39].
Using 8-OG as a measure of DNA damage induced
by oxidative stress, an increase in oxidative related
damage was found in mononuclear cells of patients
with ALL following aggressive chemotherapy [40].
Likewise, in patients treated with doxorubicin, there
was an increase in 9 different oxidation-dependent
DNA base modifications [41]. Interestingly, telomere
loss following chemotherapy is greater in patients
who have a polymorphism of the gene encoding
NADPH-quinone oxidoreductase [42]. The NQO1-
187Ser polymorphism decreases the ability of the
protein to protect against oxidative and free-radical
induced damage [43]. This finding is consistent
with oxidative stress contributing to the telomere
shortening observed in hematopoietic cells after
chemotherapy. Furthermore, these data are the first
that directly demonstrate genetic factors that may
affect the susceptibility of a patient to suffer
detectable telomere attrition in normal regenerative
tissues as a consequence of chemotherapy.
WHAT IS A SAFE AMOUNT OF TELOMERE
LOSS?
The amount of telomere attrition caused in
patients due to anticancer drugs is dependent on a
number of variables. Chemotherapeutic dose [14],
cell type [10] and the presence of other contributory
genetic factors [42] may each play a role in
determining overall telomere attrition induced by
chemotherapy. Having established a link between
chemotherapy treatment and telomere shortening
in hematopoietic cells, the functional relevance of
telomere shortening is of particular importance
raising the question: “what is a safe amount of
telomere shortening?”
Beeharry and Broccoli
The studies investigating the effect of
chemotherapy on telomere dynamics, as discussed
above, did not distinguish between 'artificially
accelerated' telomere loss as a result of
hematopoietic reconstitution versus that induced
via modification of telomeric structure or
maintenance pathways by anticancer drugs. It is
likely that both processes play, in part, a causal role
in the loss of telomeric DNA in normal cells after
treatment with chemotherapy. However, depending
on which pathway is favored, there may be different
outcomes. If we consider that chemotherapy-
induced telomere shortening occurs as a result of
increased replication of regenerative cells to
repopulate the tissue, then it is reasonable to
expect telomere shortening at chromosome ends
within a given cell to occur at a similar rate in all
chromosomes. Alternatively, if we consider that
chemotherapy-induced damage causes loss of
telomeric DNA through modification of telomere
metabolism or structure, this process would
presumably act at random telomeres, potentially
resulting in sequence loss at only one or a few
chromosome ends. Although more limited in scope,
the second pathway might be immediately
detrimental to cell survival, depending on the
extent of sequence loss. In addition, any telomere-
free chromosomes might be expected to
compromise genome stability and would be
available to interact with other chromosomes to
instigate inappropriate fusions, possibly giving rise
to oncogenic mutations. In contrast, the gradual
loss associated with proliferation may be tolerated
for a number of years before any detrimental effects
are manifested. The distinction as to which
mechanism acts is pertinent to the potential of
modulating telomere attrition (see below).
FUNCTIONAL CONSEQUENCES OF 'ARTIFI-
CIALLY ACCELERATED'TELOMEREATTRI-
TION
Having thus far limited ourselves to evaluating
data regarding the effect of chemotherapy on
human telomeres, there are observations in murine
models that may shed light on potential functional
consequences of chemotherapy-induced telomere
attrition in humans (reviewed in [44]; Fig. 2). During
serial transplantation, murine hematopoietic stem
cells progressively lose telomeric DNA despite
having robust telomerase activity in the stem cell
compartment (see Zimmermann and Martens in this
issue). Telomere attrition was correlated with a
reduction of proliferative potential in secondary
recipients [45]. Similarly, hematopoietic progenitor
cells from late generation mice deficient for the
RNA template component of telomerase (mTERC -/-
), had a reduced capacity to differentiate into all
lineages following cytokine stimulation, although
immune function was not compromised in these
animals as measured by intravenous challenge with
Listeria [46]. While bone marrow-derived stem cells
from late generation mTERC-/- mice could still
Telomere Dynamics in Response to Chemotherapy Current Molecular Medicine, 2005, Vol. 5, No. 2 193

reconstitute the hematopoietic system of shortening in hematopoietic cells [10, 12, 14, 42,
myeloablated animals, the contribution of the 49, 50], the long-term functional outcome of
infused stem cells to the regenerated tissue was accelerated telomere loss induced by exposure to
about half that seen in cells with wildtype telomere chemotherapy remains unclear. We propose that
lengths [47]. These findings provide evidence that the telomere attrition observed in hematopoietic
although the ability of the hematopoietic system to cells of BMT recipients is analogous to that
regenerate vastly exceeds the requirements accompanying chemotherapy, allowing extrapola-
associated with normal lifespan, excessive telomere tion from one system to the other. Lewis et al. [21]
attrition may compromise the degree of concurrently measured both telomere lengths and
regeneration and the ability of some mature readouts of immune function in BMT recipients and
lymphocytes to proliferate. Similarly, liver compared these to the functional measurements
regeneration in the mTERC-/- mouse was obtained from the respective donors. For all donor-
compromised following partial hepatectomy [48], recipient pairs, there was complete engraftment
suggesting that shortened telomeres affect the and reconstitution of the hematopoietic system as
regenerative potential of tissues in addition to the measured using a short tandem repeat locus. It was
hematopoietic system. shown that T-cell proliferation following phytohem-
agglutinin stimulation was severely impaired in
While in vivo studies provide evidence that
transplant recipients as compared to their
chemotherapeutic treatment may lead to telomere

Fig. (2). Chemotherapy-induced telomere attrition in regenerative tissues may have long-term consequences. Following
chemotherapy treatment, regenerative tissues undergo multiple cell divisions in order to repopulate the system, which
causes appreciable telomere loss. Excessively short telomeres may induce cellular senescence or genome instability.
Genome instability can precipitate a number of different outcomes depending on cell type and the status of genome
monitoring and DNA damage response pathways. Induction of cellular senescence and/or activation of apoptosis may lead
to compromised immune function, while telomere dysfunction induced mutations may contribute to the initiation of
secondary malignancies.
194 Current Molecular Medicine, 2005, Vol. 5, No. 2
respective donors. Similarly, the ability of myeloid
progenitor cells to differentiate in response to
cytokine stimulation was attenuated [21]. These
results are similar to observations made in murine
models whereby no obvious dysfunction was
apparent in the mature hematopoietic cell
compartment but where impaired proliferative
ability could be observed when the cells were
challenged in functional assays [46].
Aplastic anaemia (AA) is a condition that results
from a compromised number of hematopoietic stem
cells (reviewed by [51]). This places extra
replicative demand on the remaining viable stem
cells that, in turn, results in distinctly shorter
telomeres in PBMCs from individuals with AA as
compared to age-matched controls [52]. Patients
with AA are at greater risk to develop
myleodysplastic syndrome (MDS), paroxysmal
nocturnal haemoglobinuria [53] or acute leukemia
(reviewed in [54]). It has been suggested that the
replicative stress and resulting shorter telomeres
present in AA directly contribute to the
development of these clonal disorders [55],
although this has yet to be proven.
We suggest that the replicative stress induced by
chemotherapeutic treatment in patients as a result
of tissue reconstitution, may place some patients at
risk of developing secondary complications, akin to
those observed in patients with AA. This idea is
supported by observations demonstrating that in
some patients following chemotherapy there is an
increased risk in developing secondary
malignancies (reviewed by [56]). The most reported
secondary cancer following chemotherapy is
leukemia and the associated MDS. What remains to
be clarified is whether telomere integrity is related
to the occurrence of secondary malignancies. It is
likely that telomere dysfunction may be a risk factor
to developing a secondary cancer, given the
essential role of telomeres in maintaining
chromosomal stability (reviewed in [57] and see
Greenberg in this issue). Although forced telomere-
based crisis in murine models can contribute to the
development of cancer in a permissive genetic
background, studies are still needed to explore
whether telomeres are abrogated to such an extent
during chemotherapy treatment to treat an initial
cancer that the patient is then predisposed to
developing secondary malignancies.
The potential long-term outcome of patients
having received chemotherapy depends on a
number of theoretical predictions regarding rates of
telomere loss. It is likely that the accelerated loss of
telomeric sequences is only evident during
reconstitution and once the tissue has repopulated,
the loss rates are normalized as has been
documented in BMT studies [18-20]. Quantitative
analysis of the amount of telomere loss in
hematopoietic cells ranges from 20-60bp/year [7,
10, 13]. The measured loss of telomeric DNA in
patients after chemotherapy is around 1-2 kb [10,
Beeharry and Broccoli
12, 42, 49]. Depending on the cell type, this
amount of telomere loss corresponds to an
additional aging of 20-40 years. Shortened
telomeres in elderly individuals may contribute to
attenuation of the ability to mount an immune
response (reviewed in [58], see von Zglinicki and
Martin-Ruiz in this issue) which may manifest
significantly earlier in individuals whose immune
systems are prematurely aged following
chemotherapy. This scenario may not affect adults
having undergone chemotherapy since the
reduction in replicative potential of hematopoietic
cells may not be manifested during the lifespan of
the individual. However, rare cases of shortened
telomeres associated with graft failure suggest that
telomere length in human hematopoietic stem cells
may limit regenerative potential. In addition,
genetic influences put some patients at greater risk
to chemotherapy-induced telomere loss [42, 59],
making it important to establish the parameters that
define sensitivity to chemotherapy-induced
telomere attrition.
STRATEGIES TO ATTENUATE CHEMO-
THERAPY-INDUCED TELOMERE LOSS
The studies discussed above provide in vivo
evidence that myelosuppressive or myeloablative
cancer treatment protocols impact on the telomere
length of cells constituting the hematopoietic
system. This concept predicts that the bystander
effect of chemotherapy on hematopoietic cells
would be diminished if telomere attrition could be
attenuated. In support of this model is a study that
evaluated the effects of granulocyte colony-
stimulating factor (G-CSF) on lymphoma patients
receiving chemotherapy [50]. G-CSF was shown to
increase telomerase activity in hematopoietic
progenitor cells expressing the CD34+ antigen that
were isolated from bone marrow and peripheral
blood. The key findings were made in patients
having undergone chemotherapy. MNC telomere
length was decreased significantly in patients after
two cycles of treatment. The range of telomere loss
measured among 5 patients was 200bp-1500bp in
accordance with other studies [10, 12, 42, 49].
However, in the 5 patients that received G-CSF after
chemotherapy treatment, MNC telomere length was
extended in all patients, increasing by 300-3100bp.
The simplest explanation of these data is that the
G-CSF-induced increase in telomerase activity
resulted in telomere length preservation, an
unanticipated beneficial effect of G-CSF. However,
it is also possible that G-CSF specifically stimulates
proliferation of a subpopulation of stem or
progenitor cells with longer telomeres leading to an
apparent increase in telomere length in MNCs.
While the use of G-CSF abolishes telomere loss in
hematopoietic cells by chemotherapy, the long-
term consequences of G-CSF use has not been
evaluated [50]. Nevertheless, this study provides
proof of principle that modulating telomerase or
pathways governing telomere length maintenance
Telomere Dynamics in Response to Chemotherapy Current Molecular Medicine, 2005, Vol. 5, No. 2 195

may circumvent chemotherapy induced telomere LIST OF ABBREVIATIONS

loss in healthy tissues. AA = Aplastic anemia
Based on the discussion above, it seems likely ALL = Acute lymphocytic leukemia
that the generation of free radicals contributes at
least in part to chemotherapy-induced telomere loss AML = Acute myeloid leukemia
(reviewed in [60]). Since several studies show a BMT = Bone marrow transplantation
decrease in the levels of plasma antioxidants
following chemotherapy treatment [61, 62], it is DAHK = Asp-Ala-His-Lys (the four N terminal
plausible that free radical-induced telomere loss amino acids of albumin)
may be further exacerbated due to a decreased G-CSF = Granulocyte-colony stimulating factor
ability to detoxify the free radicals. This proposition
has prompted a number of investigators to examine MDS = Myleodysplastic syndrome
the potential beneficial effect of co-administering MMR = Mismatch repair
antioxidants along with chemotherapy (reviewed in
[33]) despite the potential complication that MNCs = Mononuclear cells
antioxidants may interfere with anticancer drugs mTERC-/-= Mice deficient for the RNA template
and reduce their effectiveness (reviewed in [63]). component of telomerase
Whether or not the beneficial effect of antioxidants
PBMCs = Peripheral blood mononuclear cells
will also protect cells of regenerative tissues against
chemotherapy-induced telomere loss remains to be ROS = Reactive oxygen species
addressed. TRF = Terminal restriction fragment
8-OG = 7,8-Dihydro-8-oxoguanine
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