telomerase cancer aging
TRANSCRIPT
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Critical Reviews in Oncology/Hematology 41 (2002) 2940
Telomerase in cancer and aging
Meaghan P. Granger, Woodring E. Wright, Jerry W. Shay *
Department of Cell Biology, The Uni6ersity of Texas Southwestern Medical Center, 5323 Harry Hines Boule6ard, Dallas, TX 75390-9039, USA
Accepted 2 August 2001
Contents
1. T elomere biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.2. End-replication problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301.3. Telomere hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.4. Telomere configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
1.5. Telomerase holoenzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1. Senescence and telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2. Genetic disorders and telomeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3. Hematopoietic system and telomerase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.1. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.2. Peripheral blood leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3. C ancer and telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.1. Survey of telomerase and cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2. Role of telomerase in malignant transformation. . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3. Methods of telomerase acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.4. Prognostic implications of telomerase detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.5. Residual disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.6. Diagnostic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.7. Therapeutic potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.7.1. Telomerase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.7.2. Immunotherapy and telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.7.3. Chemoprevention and telomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
www.elsevier.com/locate/critrevonc
* Corresponding author. Tel.: +1-214-648-3282; fax: +1-214-648-8694.
E-mail addresses: [email protected] (M.P. Granger), [email protected] (W.E. Wright),
[email protected] (J.W. Shay).
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Abstract
The telomeretelomerase hypothesis is the science of cellular aging (senescence) and cancer. The ends of chromosomes,
telomeres, count the number of divisions a cell can undergo before entering permanent growth arrest. As divisions are being
counted, events occur on the cellular and molecular level, which may either delay or hasten this arrest. As humans age, a
particular concern is the accumulation of events that lead to the progression of cancer. Telomerase is a mechanism that most
normal cells do not possess, but almost all cancer cells acquire, to overcome their mortality and extend their lifespan. This review
aims to provide a comprehensive understanding of the role of telomerase in cancer development, progression, diagnosis, and in
the future, treatment. The ultimate goal of telomerase research is to use our understanding to develop anti-telomerase therapies,
an almost universal tumor target. 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Telomere; Telomerase; Senescence; Replicative aging; Cancer; Immunosenescence; Telomerase inhibitors
1. Telomere biology
1.1. Introduction
After fertilization and mixing of 23 paternal and 23
maternal chromosomes, human life begins as a single
cell with 46 chromosomes whose initial function is to
divide. Each new generation of daughter cells succes-
sively divides until it forms and develops into a com-plex, differentiated organism. With each division, the
genetic code is transferred as our chromosomes are
replicated and distributed into the daughter cells. There
are many cellular mechanisms in place to ensure that
the transfer of information is done in a reliable, accu-
rate, and efficient manner throughout the many dupli-
cations required over a human lifetime. Two of the
mechanisms central to the subject of this review are the
semi-conservative replication of DNA and cellular
senescence.
1.2. End-replication problem
Semi-conservative replication of DNA is the process
of duplicating the original DNA such that the finished
products are two double DNA strands, each with one
original and one new strand, to be distributed to the
daughter cells. Replication begins with the separation
of the double-stranded molecule so that the replication
of each strand is done individually. As the two strands
are separated, new bases must be added in the 5% to 3%
direction. That task is straightforward on the leading
strand, whose template is of the opposite polarity, and
the bases are added in serial fashion. On the opposinglagging strand, replication must be done in segments,
called Okazaki fragments, in order to accomplish 5 % to
3% addition of bases. A new RNA primer is synthesized
and used to initiate the synthesis of each fragment and
eventually the fragments are ligated together to create a
continuous strand. A problem occurs when the lagging
strand, or the backwards strand, nears the end of the
chromosome. There is no DNA beyond the end to
serve as a template for the next Okazaki fragment to fill
in the gap between the last Okazaki fragment and the
end of the chromosome. Thus, the extreme end of the
chromosome is not replicated and the telomeres pro-
gressively shorten. This is known as the end-replication
problem.
Fortunately, this problem does not result in the loss
of essential genes in that each of the 46 human chromo-
somes is capped with long repeats of expendable non-
coding DNA bases called telomeres (Fig. 1). Loss of the
telomeric DNA continues with successive divisions untilthe telomeres reach such a critically short length that
replication is halted. Human cells are estimated to have
the potential to undergo on average 60 70 divisions,
and at this point the cells growth arrest and enter
senescence [1].
1.3. Telomere hypothesis
The sequence of human telomeres was identified as
repeats of 6 base pairs (bp), (TTAGGG)n, by Moyzis in
1988 [2], although the name telomere (telos=end;
meros=part) and the observation of the specializedgenetic structures at the ends of chromosomes dates
back to 1938 [2,3]. Human telomeres may vary with age
and cell type and in general range from 6 to 12 kb in
length in somatic cells [1]. Approximately 50 100 bp
are lost with each cell cycle [4].
The shortening of telomeres is responsible for the
counting mechanism that Hayflick observed in normal
cells in tissue culture in 1961. He found that normal
human fibroblasts predictably entered a period where
they ceased replication but continued metabolism (re-
viewed in [5]). The telomere hypothesis is the idea that
progressive telomere shortening is a biologic or mitoticclock of the cell, keeping track of the number of
replications a cell has used and indicating the time for
permanent growth arrest when some of the telomeres
are sufficiently short.
1.4. Telomere configuration
The fact that these bases do not code for any genetic
information does not diminish their importance. We
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Fig. 1. Metaphase spread of human fibroblasts visualized in a fluorescence microscope. Fluorescence in situ hybridization (FISH) using telomeric
probes reveal the red/pink dots at the ends of each chromosome. Each dot identifies a telomere and shows the two telomeres per chromosome
with a total of 96 telomeres per normal human cell.
now know they are a site of dynamic activity beyond
being the biologic timepiece [6]. They have a unique
T-looped configuration where the telomere bends back
on itself [7]. The overhanging guanine-rich single strand
is tucked into the double stranded telomere. This
creates a second smaller d-loop by displacing one of the
telomere strands. This structure appears to protect the
telomeres from end to end fusion with other chromo-somes and from cell cycle checkpoints that would oth-
erwise recognize the telomeres as chromosome breaks
requiring repair (reviewed in [8]).
Proteins that localize specifically to telomeric DNA
are the duplex telomere binding proteins, TRF1 and
TRF2. TRF1 and 2 and their associated proteins have
the primary responsibility of stabilizing the complex
and forming the t-loop. Some degree of stabilization is
intrinsic to the telomere overhang due to the G-rich
nature of the TTAGGG repeats that form quadruplex
structures. TRF1 is important in intratelomeric coiling
[7]. TRF2 also binds along the length of the telomerebut appears to be particularly abundant at the base of
the t-loop and is important for its stabilization and
formation [7]. Their cooperation is similar to two hands
tying a knot, the first hand (TRF1) forms a loop and
the second hand (TRF2) tightens the strand and secures
it.
These duplex telomere DNA binding proteins also
have their own associated proteins [9]. Human rap1p is
integrated into the t-loop complex and interacts with
TRF2, but its specific role in humans is unknown [10].
Tankyrase has the ability to inhibit TRF1, thereby
releasing it from the t-complex and allowing telomerase
and other enzymes to bind. TIN2 promotes TRF1
function and causes it to bind to the telomere [9]. The
DNA damage response complex RAD50/MRE11/
NBS1 also cooperates with TRF2. The MRE11 com-
plex functions conventionally in homologous
recombination to repair DNA double strand breaks[11]. At the telomere, however, it is thought to stabilize
the d-loop where the single stranded tail invades the
duplex telomere. Based on its function in vitro, the role
of NBS1 during the S phase may be to unwind the
t-loop via a helicase [12].
1.5. Telomerase holoenzyme
Telomerase is a reverse transcriptase enzyme that can
add the hexameric repeats, TTAGGG, to chromosome
ends, extending and maintaining the length of thetelomeres and thereby extending the number of divi-
sions the cell may undergo (Fig. 2) [13]. The holoen-
zyme is composed of a RNA subunit, hTR, a protein
subunit, hTERT, and many associated proteins. The
reverse transcriptase complex catalyzes the addition of
DNA bases, TTAGGG, to the telomere ends that are
complementary to the RNA template sequence of hTR
[14,15]. The human holoenzyme requires foldosome
proteins p23 and hsp90 to assemble the telomerase
components in vivo, which is confirmed in vitro since
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Fig. 2. Telomerase holoenzyme. The telomerase holoenzyme adds
telomeric repeats, TTAGGG, in two steps (1) elongation and (2)
translocation in succession. The enzyme is composed of two primary
parts: hTR is the telomerase functional or template RNA portion,
and hTERT is the telomerase reverse transcriptase enzymatic portion.
The telomeric end can binds to the template region of hTR and is
elongated by the addition of the bases complementary to the template
via the catalytic subunit (hTERT). The complex then pauses and
translocates and repeats the elongation of the telomere (e.g. the
human telomerase complex is processive).
Normal cells have a finite number of divisions they
can undergo before entering retirement or replicative
senescence. Cells removed from older individuals, in
general, divide fewer times in culture when compared to
cells obtained from younger patients. Replicative senes-
cence is the process by which cells stop dividing due to
a genetically programmed event. Normal cells reach a
period of growth arrest termed M1, or mortality stage
1, that is controlled by cell cycle regulatory genes
p53/p21 and perhaps p16/Rb. There is speculation that
M1 might be initiated by the presence of at least one
sufficiently short telomere and activation of the DNA
damage response, although at this growth point most of
the 92 telomeres still have several kilobase pairs of
telomeric repeats. Other possibilities include regulation
by subtelomeric genes or by transcription factors asso-
ciated with the telomere [9]. If p53 function is altered or
blocked (as with SV40 T antigen or E6/E7 papillo-
mavirus proteins) cells continue to divide with progres-
sive telomere shortening until they reach a second stage
known as M2, mortality stage 2. It has been establishedthat telomere shortening controls both M1 and M2
[15,24]. The M2 stage is often referred to as crisis at
the point where many telomeres have been critically
shortened and can no longer protect the telomeres so
that chromosome fusion and breakage cycles occur and
the cells eventually undergo apoptosis. In human
fibroblasts in vitro that express viral oncogenes, a small
number of cells (1107), are able to escape M2 crisis
and immortalize by the acquisition of a method for
maintaining stable telomeres. This is accomplished
through a reactivation oftelomerase in most cells, but
alternative lengthening of telomere mechanisms (ALT)exist that use recombination and copy switching to
move DNA from one telomere to another [25].
It is believed that replicative senescence decreases the
number of mutations that can occur bylimiting the
number of times the cell can divide. Properties of
senescence are dependent on the number of cell divi-
sions not time. It entails cells entering an irreversible
state incapable of proliferation and with altered func-
tion. Cells become growth arrested in G1 and are
unable to replicate their DNA [26].
What is the relationship between senescence, aging,
cancer, and telomerase? Telomeres shorten in aging cellpopulations in vitro and in vivo (Fig. 3). Human
fibroblasts from fetal tissues can typically undergo 60
80 population doublings (PDs), whereas young adult
cells achieve only 20 40 doublings, and older adult
cells 1020 doublings before entering senescence. It is
important to understand the molecular mechanism reg-
ulating senescence in oncology because it is the very
cellular outcome we are seeking, for the cancer cells to
stop dividing [26].
combining recombinant foldosome proteins, hTR, and
hTERT is sufficient to reconstitute the holoenzyme [16].
The telomerase gene was recently mapped to 5p15.33
as one of the most distal genes on chromosome 5p. This
has raised questions about whether its proximity to the
telomere might result in it being regulated by telomere
position effect mechanisms [1719].
The introduction of the catalytic protein (hTERT)
component of telomerase into normal fibroblasts and
epithelial cells prevents shortening of the telomeres, andresults in immortalization [20]. The key role of telom-
erase in immortalization is to maintain telomere length,
not to produce a net increase in length [15]. Transient
expression of a cre-excisable telomerase results in a
preferential lengthening of the shortest telomeres and
an increase in lifespan proportional to the length of the
shortest telomere [21]. Likewise, the inhibition of telom-
erase in immortalized human cells leads to progressive
telomere shortening and cell death [22].
2. Aging
2.1. Senescence and telomerase
Humans are living longer than ever before. Life
expectancy at birth was 47.3 years in 1900 compared to
70.8 years in 1970 and 76.5 years in 1997. Centenarians
are one of the most rapidly growing segments of the
population. By the year 2050, persons greater than 85
years of age are expected to comprise nearly 15% of the
entire population [23].
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Fig. 3. Telomere hypothesis. (a) With progressive cell divisions, telomeres shorten until they reach a critical shortened length. At this point, they
undergo growth arrest or apoptosis (depending on whether other cellular pathways have been altered) unless they are able to maintain their
telomeres to allow for subsequent divisions. (b) Stem cells exhibit a slower rate of telomere shortening because of the intrinsic presence of
telomerase in these cells indicating that they may undergo more doubling prior to becoming senescent. (c) Committed peripheral blood
lymphocytes (PBL, dotted line) are derived from the stem cell compartment and have a telomere length correlating with their age at the time of
commitment. PBLs, upon activation, can have a brief period of telomerase upregulation followed by continued telomeric shortening. (d) Stem cell
transplant recipients have accelerated telomeric shortening following transplant and then continued shortening at a rate proportional to the donor
stem cells. (e) Cancer cells (dashed lines) may develop at any point in normal and hematopoietic cells and, in most cases, have utilized telomerase
to maintain their telomeres. (f) All cells have higher rates of telomere loss from birth to 1 year, somewhat less from 1 to 4 years, followed by
consistent loss of 50100 bp/division.
2.3. Hematopoietic system and telomerase
2.3.1. Stem cells
Telomerase activity can be detected in both hemato-
poietic stem cells and in stem cell populations in other
tissues such as skin, hair follicles, small intestine crypt
cells, and lymphoid cells. Though the hematopoietic cells
possess telomerase, they still have telomeres that shorten.
Stem cells that are CD34+/CD38 have shorter telom-
eres in adults than the same cell type in fetal and newborn
tissue [31]. It is believed that the expression of telomerase
in stem cells may help slow down, but does not com-
pletely prevent telomere attrition in cells that have a high
rate of turnover (Fig. 3). Telomerase activity ensures
that the stem cell compartment will be able to handle
potentially large expansion demands, preserving the
ability to maintain and repair the tissues. Though the
telomeres still shorten, the time to critically shortened
length may be delayed by telomerase [32].
Studies in stem cell transplant patients have shown that
stem cells are on average 0.4 kb shorter in the reconsti-
tuted recipient when compared concurrently with the
donor (Fig. 3). It is likely that the proliferation demands
required to reconstitute the entire hematopoietic system
2.2. Genetic disorders and telomeres
Patients with Hutchison Gilford progeria exhibit ac-
celerated aging effects noticeable by age 2 years. They
have short stature and abnormal posture and possess the
typical aging phenotype of alopecia, joint stiffness,
atrophic and wrinkled skin, atherosclerosis, and coro-
nary artery disease including angina pectoris and my-
ocardial infarction [27,28]. Fibroblasts from these
patients show shorter telomere lengths than age matched
controls and entered senescence in vitro much earlier
than the aged matched control cells. When infected with
hTERT they immortalize and telomere shortening is
prevented without affecting checkpoints, functions, andcellular controls [28]. Similar results are achieved with
cultures of skin fibroblasts from patients with Werners
syndrome. These patients have premature aging effects
of vascular disease, diabetes mellitus, cataracts, skin
atrophy, graying hair, testicular atrophy, and cancer with
an average lifespan of 47 years [28,29]. Trisomy 21 is
another disorder with features of accelerated aging.
Telomeres lengths have been found to shorten in
lymphocytes obtained from Downs syndrome patients
three times faster than normal individuals [30].
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results in aging of the cells prematurely by :15
years [33]. Further, this shortening could eventually be
sufficient to contribute to genetic instability and ac-
count for some of the secondary neoplasms seen in
stem cell transplant patients beyond those attributed
to alkylating agents and etoposide [31]. It is unknown
whether the cumulative dose of stem cells given pa-
tients have an effect on this aging [33].
Telomere lengths in aged hematopoietic stem cells
have not been shown to reach a critically shortenedlength leading to complete senescence. However, the
cellularity of the bone marrow compartment is re-
duced by one-third at the age of 70 years [34]. It has
been suggested that the replicative stress of shortening
telomeres, particularly in lymphocytes, seen in early
childhood and in the elderly might be responsible for
the coinciding with the bimodal distribution of some
hematopoietic disorders [34]. Acute lymphoblastic
leukemia peaks in occurrence in children at an average
age of 4 years [35] and again in adults after the age of
45 years [36]. The fact that this disorder is extremely
rare during the critical years of childbearing and rear-ing and more common in early childhood and the
elderly may be evidence of an evolutionary tumor pro-
tective mechanism.
2.3.2. Peripheral blood leukocytes
The aging immune system involves a complex
change in the entire system, both constitutionally and
functionally. It is clinically apparent that aging indi-
viduals are at increased risk for infection, cancer, de-
creased immunity from previous vaccination, and
reactivation of latent disease such as varicella. An
overview of the global nature of these changes hasrecently appeared [37].
T cells, in general, shift from nave to mature mem-
ory types with an increased proportion found in the
bone marrow rather than peripheral blood. There are
proportionally more CD8+ T-cells than CD4+ . B
cells also show increased levels in the bone marrow
with overall qualitative defects in antibody production.
This is presumed to be from increased somatic muta-
tions affecting Ig-gene rearrangements but is also infl-
uenced by the shift in the T-helper cell population
from Th1 to Th2. In contrast to decreased circulation
of T and B cells, NK cells are found in increasednumbers [37].
Granulocytes show decreased phagocytosis and res-
piratory burst in aging individuals. Monocytes are
more activated, dendritic cells are unchanged, and
macrophages increase their production of cytokines.
Erythrocytes exhibit a shift in proportions of young to
old populations [37].
As in the stem cell compartment, both circulating T
and B cells have progressive telomere shortening with
age and express low levels of telomerase activity at
rest, but levels transiently increase with stimulation by
mitogens (Fig. 3, committed PBL). Interestingly,
hTERT (the mRNA component of telomerase) ap-
pears to be constant among all lymphocyte stages in-
dependent of the level of telomerase activity [38,39].
This is consistent with most normal telomerase posi-
tive somatic cell types that still exhibit shortening in
spite of the presence of telomerase and could reflect
alternatively spliced variants of hTERT that are inac-
tive.Several studies have shown that telomere shortening
with aging in peripheral blood leukocytes, both T
lymphocytes and neutrophils, occurs in at least two
phases. First, there is a rapid shortening from birth to
4 years at about 1 kb per year. Next there is a gradual
shortening until :40-years-old of 2050 bp per year
and more slowly thereafter (Fig. 3) [34,40]. These
reflect the complexities due to the presence of telom-
erase, which may make telomere lengthening and
shortening a more dynamic system depending on the
hematopoietic requirements of the body. Certainly, the
demand for clonal proliferation of a committedlymphocyte may increase or decrease telomere length,
but also the telomere length of the originating stem
cell can play a role [34].
Replicative senescence is intact in normal T-cells
just as in fibroblasts and other cells. However, the
implications of senescence in the immune system are
more significant. Mature T cells are required to give
rise to clonal proliferations of cells to respond to for-
eign antigens upon activation. This cannot occur if the
T cells are senescent. Senescence in culture reliably
occurs in T lymphocytes, both CD4+ and CD8+ ,
after 2540 PDs. Thus, each mature T cell is capable
of producing :240 cells, or 11012 cells, before
senescing [41].
There are significant functional changes in senescent
T cells. The most important being the lack of expres-
sion of CD28, which plays a key role in the transduc-
tion of IL-2 transcription and receptor expression,
cooperation with B cells for antibody production, T
cell homing, and signaling the induction of telomerase
activity [41]. CD28 is present on 99% of neonatal T
cell compared to only 45% of centenarian T cells.
Telomeres of CD28 cells are shorter than CD28+
telomeres [41]. The CD28 cells are primarily of the
CD8+ subset, which play a pivotal role in cytotoxic
functions against cells with endogenously expressed
antigens such as virally infected cells and tumor cells.
Senescent T cells also acquire resistant to apoptosis
that results from an increase in bcl-2 [41].
Telomeric changes in B cells are quite different from
T cells. Rather than the steady but slow decline in
telomeric length of aging T cells, activated B cells in
germinal centers of tonsillar tissue show an increase in
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telomere length from their nave state. The length then
begins to decline once the B cells enter the memory
compartment. Telomerase activity is highest in tonsillar
B cell germinal centers, which corresponds to the point
of longest telomeres. This is possibly a mechanism to
protect the telomeres of highly specific B cells from the
replicative stress placed on B memory cells [42].
3. Cancer and telomerase
Telomerase expression is a hallmark of cancer.
Nearly the complete spectrum of human tumors has
been shown to be telomerase positive (Fig. 4). In gen-
eral, malignant tumors are characterized by telomerase
expression, indicating the capacity for unlimited prolif-
eration and thus immortality. Most benign tumors are
characterized by the absence of telomerase, indicating
their limited proliferative capacity, and ultimate
senescence.
3.1. Sur6ey of telomerase and cancer
An extensive summary of telomerase in human tu-
mors has been surveyed as shown in Fig. 4. Telomerase
can be measured by the TRAP assay, which uses PCR
to amplify the extension products of the telomerase
enzyme. The assay is quite sensitive and can detect as
few as 0.01% positive cells. The background tissue in
most cases is of normal somatic derivation and does
not contribute telomerase activity. However, in cases
where the histological environment of the tumor is
naturally telomerase expressing (such as intestinal ep-
ithelium), a positive result is considered only when
telomerase levels are higher than the matched control
tissue [43].
The hematopoietic tumors present a unique and
difficult assessment since activated lymphocytes have
some inherent telomerase activity. Cells from patients
with chronic lymphoid leukemia, in the early-stages,
have low levels of telomerase that progressively increase
Fig. 4. Summary of telomerase activity expression in human cancers from a review of the literature. Tumor samples were assayed by the TRAP
assay. Percentages in parentheses refer to the number of samples that were telomerase positive compared to matched control tissue. Adapted from
Shay and Bacchetti, 1997. Please refer to original article for details and discussion [43]
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over the course of the disease. This increase is accom-
panied by a net loss in telomeric length [32]. A series of
58 patients showed that high telomerase activity and
shorter telomeres had an adverse prognosis [44].
Chronic myeloid leukemia does not show an increase in
telomerase activity over peripheral mononuclear cells,
however, a shorter TRF length correlates with shorter
time to blast crisis phase. Small studies in acute
lymphoblastic leukemia have found telomerase activity
to be variable [32]. Acute myelogenous leukemia, multi-ple myeloma, plasma cells leukemia, and non-
Hodgkins lymphoma all exhibit telomerase positivity
[45]. However, Hodgkins lymphoma does not exhibit
telomerase activity [46].
3.2. Role of telomerase in malignant transformation
Telomerase expression alone is not the inciting event
in the transformation to neoplasia. While introduction
and expression of telomerase has been shown to im-
mortalize cells, it does not by itself induce a trans-
formed phenotype [47,48]. In human fibroblasts, manyfactors are required to experimentally transform telom-
erase positive cells, including overexpression of a mu-
tant version of the H-ras oncogene to constitutively
activate signal transduction pathways, SV40 large T
antigen to block pRb and p53 cell cycle checkpoints,
and SV40 small t antigen to inhibit phosphatase activity
[15,49]. Human fibroblast cells that express only
hTERT exhibit normal cell cycle activities, maintain
contact inhibition, adherence, growth requirements,
and maintain normal karyotype [40].
It has been suggested that there are at least six
essential alterations necessary for malignancy shared byvirtually all types of cancers. They are the generation of
self-stimulatory growth signals, insensitivity to in-
hibitory growth signals, resistance to apoptosis, unlim-
ited potential for proliferation, capacity for
angiogenesis, and tissue invasion and metastasis [50].
Thus, there is a diverse system of cellular mechanisms
in place to suppress the development of neoplastic cells.
It is further postulated that the multiplicity of these
defenses explains the relative rarity of human cancer
[50]. Indeed, the cancer rate is estimated to be 400 cases
per 100 000 individuals for all types, age, sex, and sites.
However, when viewed adjusted for age the rate risessharply. For individuals over age 65 the estimated
incidence is 2151 cases per 100 000 [51].
3.3. Methods of telomerase acquisition
There is debate among investigators over just how
cancer cells acquire telomerase activity. For example,
does the neoplasm originate from a telomerase-com-
petent stem cell or is telomerase turned on at some
phase in neoplasia? Models for the origin of the former
are based on the idea that cancer arises by clonal
expansion of proliferating cells, and it is the stem cells
of epithelial tissues that constitute the pool of prolifer-
ating cells. Alternatively, it might be expected that
cancer would arise in differentiated cells rather than
stem cells since the mass of most tissues is comprised of
differentiated cells. Following a mutation that initiates
clonal expansion, the pre-malignant cell accumulates
other critical mutations such as p53 resulting in ge-
nomic instability and continued cell division and fur-ther shortening of telomeres. This repetitive, clonal
expansion leads to the acquisition of other mutations,
loss of heterozygosity and the ultimate upregulation or
reactivation of telomerase. This upregulation or reacti-
vation of telomerase permits the stabilization of the
telomeres and an immortal state [52].
3.4. Prognostic implications of telomerase detection
Many studies have been conducted to assess the
prognostic implications of telomerase expression.
Telomerase activity increases in direct proportion tograde of malignancy in a series of cutaneous
melanocytic lesions, from low in benign nevi to very
high in melanoma [53]. One of the classic examples of
clinical outcome as predicted by telomerase activity is
in childhood neuroblastoma. High levels of telomerase
are found in advanced, Stage 4 disease that is of very
poor prognosis. However, Stage-4s neuroblastoma is a
disseminated form of the disease (s is for special)
known to present almost exclusively in infancy and
often spontaneously regress. These particular tumors
have low to absent levels of telomerase activity and
very short telomeres suggesting that inability to main-tain telomere length could contribute to their regression
[54]. These studies also show that a cell does not
necessarily need to have telomerase activity to become
malignant, but a mechanism must be engaged to main-
tain telomere stability to confer long-term growth of
the tumor.
Numerous studies have shown that telomerase activ-
ity in breast carcinomas is an adverse prognostic sign as
it is in other malignancies. In a retrospective prognostic
study of 398 patients with breast carcinoma involving
lymph nodes, telomerase activity as analyzed by the
TRAP assay was shown to strongly correlate with anaggressive phenotype in terms of the fraction of cells in
S-phase, progesterone receptor level, DNA ploidy, and
lymph node status. Increased TRAP also indicated
decreased disease-free survival (P=0.041), decreased
overall survival (P=0.009), strong predictor of death
(P=0.027), but was only moderately predictive of re-
lapse (P=0.08) [55]. Another study examined 125 pa-
tients with various stages of breast carcinoma and also
found that telomerase activity correlated with stage in a
statistically significant manner (P=0.02) [56]. Suspi-
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cious cytogenetic abnormalities in breast cancer corre-
late with increased telomerase activity, namely 3q+
(site of hTR), 8q+ (c-myc), and 17p (p53) [57].
One hundred patients with colorectal cancer were
followed for 3 years after surgery. High telomerase
activity was found in 44/100 patients at the time of
biopsy and correlated with a significantly (P=0.01)
decreased survival, 81% vs. 43% [58].
A retrospective study in patients with meningioma
appeared to predict relapse. In 25 patients that wereexamined, five patients had detectable telomerase activ-
ity and subsequently relapsed. Twenty-five patients had
no detectable telomerase activity and did not relapse
[59]. Glioblastoma is one of the few examples where no
correlation has been seen between grade and telomerase
activity. TRAP levels have been found to be highly
variable both within the same patient and within a
series of glioblastomas [60]. These observations of the
prognostic utility of telomerase assays have not yet
reached the clinic in terms of predicting outcome for
patients.
3.5. Residual disease
Telomerase may play an important clinical role in
assessing the extent of tumor margins. Biopsy speci-
mens from a tumor bed show that telomerase activity
was detectable in 10% of tissue areas that were pre-
sumed disease-free based on morphologic review. This
means that margins that were declared free of tumor
may not in actuality be free of tumor. An assay for
telomerase could theoretically provide a molecular way
of determining margins, and thus identifying patients
who are at increased risk for local recurrence [13].
3.6. Diagnostic potential
Telomerase activity has been proposed as an adjunc-
tive diagnostic tool in urinary tract cancers. It is esti-
mated that nearly 50% of bladder cancers are missed on
initial cytological survey. The specificity for telomerase
activity in cancer cells allows for earlier detection and
identification using bladder washings in combination
with cytology [60].
Fine-needle aspiration is widely used as a diagnostic
tool in breast cancer. A recent prospective blindedstudy included 617 patients and examined 220 FNA
samples by both cytology and telomerase activity in
which the diagnosis was later confirmed histologically
after surgery [61]. The cytology method alone correctly
identified 62 out of 93 tumors (67%) that were initially
classified as malignant or probably malignant. The
telomerase assays correctly identified 72 of the 93 tu-
mors (77%). When both tests were used together on the
FNA samples, 84 of 93 (90%) were correctly identified.
Of the cytologically indeterminate FNA samples, 10/17
with telomerase activity were ultimately diagnosed as
carcinoma and 6/7 without telomerase activity were
ultimately diagnosed as benign lesions with a P=
0.0007. The TRAP assay thus has the potential to
augment the FNA screening tool in combination with
cytology in the early diagnosis of breast cancer [61].
Telomerase activity has all the desired characteristics
to be used as a potential cancer-screening tool. It
requires a small amount of tissue, can be done on a
variety of tissue types or body fluids requiring minimalinvasiveness, has a sensitive assay, is specific to the
malignant state in most instances, and can be done at a
low cost [62].
3.7. Therapeutic potential
In most cases, chemotherapy targets dividing cells. It
has limited effectiveness in specifically targeting cancer
cells, even further limitations in eradicating minimal
residual disease, and can often be evaded through drug
resistance mechanisms. Many would claim that the
current miracles of chemotherapy have been exhaustedand future therapeutic advances require a more sophis-
ticated armamentarium. Telomerase inhibitors are an
attractive weapon against this problem, largely because
of the specificity of telomerase activity in tumor cells.
This is currently and area of intense investigation
worldwide and several classes of potential agents have
been developed.
A key to understanding the role for this class of
agents is that the inhibitory effects are only apparent
after the cancer cells shorten their telomeres sufficiently
through continued proliferation to cause them to enter
crisis. Therefore, time to effectiveness in halting tumorgrowth is dependent on the original length of the
telomeres in the cancer cell. Because the cells will
continue to proliferate before inhibition is sensed by
the cell, they are less likely to be used in up-front
therapy and more likely to play a supportive role to
control minimal residual disease after initial control is
accomplished through conventional surgery,
chemotherapy or radiation. Levels of telomerase are
detectable in the same regenerative tissues that are
vulnerable to the toxic effects of chemotherapy, such as
the hematopoietic tissues, germ cells, skin and hair
cells, and gastrointestinal cells. However, the effectshere are predicted to be minor since the stem cells in
these tissues tend to have much longer telomeres com-
pared to cancer cells. As is always the case, there
remains the possibility that drug resistance mechanisms
would develop [63].
3.7.1. Telomerase inhibitors
The RNA template of the telomerase holoenzyme is
a popular target for inhibition research using antisense
oligonucleotides that are complementary to this region
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of hTR. Regardless of the configuration of telomerase,
the template region of hTR must be accessible to bind
to the telomeric repeats, which exposes it to inhibition
by antisense approaches. The major challenge for this
class of drugs is access and stabilityhow to get the
oligonucleotides into the cell and then to the enzyme
without being degraded by nucleases. One strategy has
been to modify the DNA using sugar-modified RNA,
such as 2%-O-methyl RNA and the 2%-methoxyethoxy
RNA [63].In the laboratory, telomerase can be inhibited by
the introduction of a dominant-negative hTERT gene
into the cell. The gene encodes a point-mutated re-
verse transcriptase crippled hTERT that inhibits wild-
type hTERT both by sequestering the available hTR
and by competing with the wild-type hTERT for ac-
cess to the telomeres. In vitro studies have shown that
the introduction of the dominant-negative (DN-
hTERT) into cancer cells inhibits telomerase and leads
to progressive telomere shortening and cell death [64].
Wild-type hTERT, DN-hTERT, and control vectors
were introduced into 36 M ovarian carcinoma celllines in culture. After several PDs, the cells were intro-
duced into nude mice to assess for tumorigenicity. The
wild-type and control vector cells produced tumors but
the DN-hTERT cells did not. The application of this
design may be more feasible as the area of gene ther-
apy progresses.
Attention has also been given to the reverse tran-
scriptase inhibitor class of drugs, such as AZT, that
have been effective in HIV treatment. Unfortunately,
it has not been shown to date that this class of agents
promotes shortening of telomeres and senescence or
apoptosis of the treated cells [63].
3.7.2. Immunotherapy and telomerase
Recently, Vonderheide, et al. identified a tumor-as-
sociated antigen (TAA) that correlates with hTERT
expression in an HLA subset of patients. He generated
cytotoxic T lymphocytes and demonstrated hTERT
specific cytolysis in many tumor lines that spared
telomerase positive peripheral blood CD34+ cells.
However, since CD40+ activated B cells were lysed,
it is possible that the immune system will not function
optimally in a clinical setting if it is forced to rely
solely on the interaction of antigen processing cellswith cytotoxic T cells without activated B cells in the
germinal centers [65]. Other investigations have shown
similar results with other hTERT peptides that are
able to generate a cytotoxic response against tumor
cells but not telomerase-positive CD34+ stem cells
[66].
3.7.3. Chemopre6ention and telomerase
Telomerase antisense inhibitors have been recently
shown to have potential value as a chemopreventative
agent. Human mammary epithelial cells from women
with Li-Fraumeni syndrome are characterized by a
mutation in the p53 tumor suppressor gene that makes
it nonfunctional. These cells spontaneously immortal-
ize in culture at a reliable frequency. Using a variety
of telomerase inhibitors, such as the 2-O-methyl-RNA
antisense oligonuclotide, the dominant negative
hTERT, or nontoxic concentrations of other
chemotherapeutic agents, the rate of in vitro immortal-
ization was significantly reduced [67]. Other patients athigh risk for spontaneous immortalization could
benefit from this strategy of chemoprevention includ-
ing those at high risk for lung cancer from smoking or
chemical exposure, patients treated for a primary ma-
lignancy with a high probability of recurrence, and
those with conditions considered premalignant with a
high probability of progression.
4. Conclusion
A hypothesis gaining support is that the function ofcellular senescence is to restrict the number of muta-
tions that can be accumulated by a pre-malignant cell.
If one accepts this hypothesis, then counting cell divi-
sions becomes the distinguishing feature of replicative
aging. Determining whether replicative aging has rele-
vance to organismal aging remains a fundamental un-
resolved issue. However, there is mounting
experimental support that restoring mortality by in-
hibiting telomerase in tumors may be an effective ther-
apy and is an area where great progress is anticipated
in the near future. Telomere biology is clearly impor-
tant in replicative aging and cancer. Cancer cells needa mechanism to maintain telomeres, if they are going
to divide indefinitely, and telomerase solves this prob-
lem. The key is to understand how the telomerase
holoenzyme and telomere-complex interact to maintain
telomere length. The challenge is to learn how to in-
tervene in these processes and exploit our increasing
knowledge of telomere biology for cell and tissue engi-
neering as well as the diagnosis and treatment of ma-
lignancies.
Reviewers
Joachim Lingner, PhD, Swiss Institute for Experi-
mental Cancer Research (ISREC), 155, ch. des Bover-
esses, CH-1066 Epalinges, Switzerland.
Petra Boukamp, PhD, Deutsches Krebs-
forschungszentrum (DKFZ), Abteilung B0600/FS2, Im
Neuenheimer Feld 280, D-69120 Heidelberg, Ger-
many.
Dr. Goran Roos, Department of Pathology, Umea
University, S-90187 Umea, Sweden.
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Biographies
Meaghan P. Granger, MD is a clinical fellow in
pediatric Hematology and Oncology at UT Southwest-
ern Medical Center. She received her MD from the
University of Arkansas and completed her residency in
pediatrics at Vanderbilt University. She is currently
conducting telomerase research in the laboratory of
Jerry W. Shay, PhD and Woodring E. Wright, MD/
PhD in the Department of Cell Biology.
Woodring E. Wright received his BA from Harvard
College and then completed his MD/PhD at Stanford
University in California, where earned his PhD in the
laboratory of Leonard Hayflick. He pursued postdoc-
toral studies at the Pasteur Institute in Paris with
Francois Gros and then joined the faculty of South-
western Medical Center where he is currently a profes-
sor of Cell Biology.
Jerry W. Shay earned his BA and MA at the Univer-
sity of Texas at Austin and his PhD at the University ofKansas at Lawrence. He did his postdoctoral work at
the University of Colorado in Boulder with Keith
Porter and David Prescott before moving to Dallas
where he is currently a professor of Cell Biology at the
University of Texas Southwestern Medical Center in
Dallas and an Ellison Medical Foundation Senior
Scholar.
In 1985, Shay and Wright began what has become a
very close and productive collaboration. This led to the
development of the two-stage model for cellular senes-
cence for which they shared the Allied Signal Award
for research on aging in 1995 and in 2001 the AmericanAging Association Hayflick Award. They are both
members of Gerons scientific advisory board and have
over 15 patents allowed on their telomere and telom-
erase-based research. Both have served on the Scientific
Research Board of the American Foundation for Aging
Research.