Telomerase Cancer Aging

7/28/2019 Telomerase Cancer Aging

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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).

1040-8428/02/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved.

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M.P. Granger et al. /Critical Re6iews in Oncology/Hematology 41 (2002) 294030

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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M.P. Granger et al. /Critical Re6iews in Oncology/Hematology 41 (2002) 2940 31

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


10/12


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.

Documents

Telomerase Cancer Aging