Báo cáo khoa học: Assessment of telomere length and factors that contribute to its stability potx

REVIEW ARTICLE
Assessment of telomere length and factors that contribute
to its stability
Sabita N. Saldanha
1
, Lucy G. Andrews
1
and Trygve O. Tollefsbol
1,2,3
1
Department of Biology,
2
Center for Aging and
3
Comprehensive Cancer Center, University of Alabama at Birmingham,
University of Alabama at Birmingham, AL, USA
Short strands of tandem hexameric repeats known as
telomeres cap the ends of linear chromosomes. These repeats
protect chromosomes from degradation and prevent chro-
mosomal end-joining, a phenomenon that could occur due
to the end-replication problem. Telomeres are maintained by
the activity of the enzyme telomerase. The total number of
telomeric repeats at the terminal end of a chromosome
determines the telomere length, which in addition to its
importance in chromosomal stabilization is a useful indica-
tor of telomerase activity in normal and malignant tissues.
Telomere length stability is one of the important factors that
contribute to the proliferative capacity of many cancer cell
types; therefore, the detection and estimation of telomere
length is extremely important. Until relatively recently,
telomere lengths were analyzed primarily using the standard

Southern blot technique. However, the complexities of this
technique have led to the search for more simple and rapid
detection methods. Improvements such as the use of fluor-
escent probes and the ability to sort cells have greatly
enhanced the ease and sensitivity of telomere length meas-
urements. Recent advances, and the limitations of these
techniques are evaluated.
Drugs that assist in telomere shortening may contribute to
tumor regression. Therefore, factors that contribute to
telomere stability may influence the efficiency of the drugs
that have potential in cancer therapy. These factors in rela-
tion to telomere length are also examined in this analysis.
Keywords: telomerase; telomeres; telomere length; inhibitors;
detection methods.
Introduction
When damage to DNA occurs in normal cells, the cell cycle
is arrested until DNA repair mechanisms can restore the
damaged DNA [1,2]. In eukaryotes the ends of the linear
chromosomes, when unprotected, resemble DNA with
broken ends, which can lead to chromosomal aberrations
such as translocations and inversions [1,3]. To prevent such
occurrences, replicating cells synthesize stretches of hexa-
meric repeats at the ends of chromosomes referred to as
telomeres, which protect DNA from end-to-end fusions and
maintain the structural integrity of the genome [4–6]. By
capping the ends of linear chromosomes, the loss of coding
sequences that would occur due to the end-replication
problem [7] is minimized. Thus, telomeres influence and
maintain the proliferative potential of cells [8–10] and
therefore the greater the length of the telomeres, the more

stable is the genome.
During normal somatic cell division, the absence of
telomerase results in the erosion of telomeric repeats and
reduction in telomere length. Critically short telomere lengths
correlate with the cessation of cell division, the onset of the
aging process and the genesis of age-related diseases [9,11–
19]. However, in rapidly proliferating cells, such as germline
and tumor cells, telomerase is expressed and stabilizes the
telomere lengths, thereby maintaining the immortal state
[20,21]. Telomeres are important in various cellular processes
and the stability of these structures depends on the activity of
telomerase. Therefore, telomere length is a potential indicator
of telomerase activity and can be used in the prognosis of
disease, including various malignancies [3,22–25].
Any technique employed for disease prognosis must be
accurate, reliable and rapid. Southern blot analysis has been
the standard method of choice in the detection of telo-
mere length. However, the limitations of this method,
which involves a tedious procedure, have stimulated the
Correspondence to T. O. Tollefsbol, Department of Biology, 175A
Campbell Hall, 1300 University Boulevard, University of Alabama
at Birmingham, Birmingham, AL 35294–1170.
Fax: + 1 205 9756097, Tel.: + 1 205 9344573,
E-mail:
Abbreviations: TRF, telomere/terminal restriction fragment; HPA,
hybridization protection assay; FCM, flow cytometery method; FISH,
fluorescent in situ hybridization; Q-FISH, quantitative fluorescent
in situ hybridization; Q-FISH
FCM
, quantitative FISH and flow

cytometry; TBP, telomere binding proteins; T-OLA, telomeric-oligo-
nucleotide ligation assay; TFV, telomere fluorescent values; TRF,
telomere/terminal restriction fragment; TRF2, telomere repeat factor
2; ATM, ataxia telangiectasia mutant; AE, acridinium-ester labeled
probe; PNA, peptide nucleic acid probe; IFI, integrated fluorescent
intensity; PENT, primer extension/nick translation; ALT, alternative
lengthening of telomeres; HUVEC, human umbilical vein endothelial
cells; nt, nucleotides; DSB, double-strand breaks; ds, double strand;
ss, single strand.
(Received 27 August 2002, revised 1 November 2002,
accepted 3 December 2002)
Eur. J. Biochem. 270, 389–403 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03410.x
development of newer methods of analysis. Several assays
that have eliminated most of the problems with Southern
blotting have been developed but the complexity of these
has not been reduced. Fluorescence in situ hybridization
and flow cytometry [20,26] have greatly increased the
accuracy, speed and reliability of telomere length measure-
ment from whole or fragmented genomic sequences [27–29].
Hybrids of these methods such as Q-FISH, flow FISH and
Q-FISH
FCM
have further improved assays of telomere
length. Recent advances in these techniques and the
advantages and limitations of the various assays are highly
relevant to understanding the role of telomeres in biological
processes such as aging and cancer.
Detection of telomere length
The ability of DNA polymerase to synthesize new DNA
only in the 5¢)3¢ direction results in the incomplete

replication of the lagging strand leading to attrition of
telomere length with each cell division (Fig. 1). In senescent
cells, telomere lengths are short and the cells lose the
capacity to divide. This is in contrast to about 90% of
tumorigenic cell lines, which are immortal, have only
slightly shortened telomere lengths and express high levels
of telomerase (Fig. 1). Thus, there appears to be a strong
correlation between telomerase reactivation and stabiliza-
tion of the short telomere lengths, which could serve as a
Fig. 1. Influence of telomerase activity and telomere length on the processes of cellular aging, senescence, immortalization and tumorigenesis. The
effects of telomerase expression on telomere length in various cell types are depicted. The broad solid line represents the 3¢ terminal portion of a
chromosome and the narrow solid line, the telomere length. Basal or low levels of telomerase are indicated by single upward arrows, double arrows
indicate an intermediate level of telomerase expression, and elevated levels of telomerase are represented by three upward arrows. (A) In the absence
of telomerase in most normal somatic cells, cellular division is accompanied by the loss of telomeric repeats due to the end replication problem.
(B) Repeated cell division leads to the attrition of telomere length resulting in cells acquiring a presenescent phenotype approaching senescence.
(C) With further telomeric attrition to a critical telomere length, cells approach the senescent stage, M1. Some cells in this phase can escape
senescence and become immortal [100]. However, these cells eventually undergo apoptosis or cell death in the absence of telomerase. (D) Cells in the
M1 phase that do not escape senescence enter the M2 crisis stage (towards cell death). (E) A few rare cells in this phase (M2) may escape crisis and
become immortal with the reactivation of telomerase [100]. (F) During transformation the telomere lengths are stabilized and vary depending on the
cell type. The telomeres of transformed cells are short and in most cases are nearly equal to or less than the length at the M2 threshold stage [100].
They are also much shorter than those of telomerase-positive normal cells [101]. It is the reactivation and up-regulation of telomerase that maintains
the stability of the short telomere lengths. Finally, the transforming events (inactivation of tumor suppressor genes, up-regulation of certain
oncogenes such as ras) along with the up-regulation of telomerase impart an immortal and tumorigenic (benign/malignant) phenotype to the cells.
390 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
prognostic indicator for age-related diseases, including
cancer. Telomere length maintenance, a function of telo-
merase activity, is crucial for cell immortalization and is also
important in tumorigenesis.
Southern blotting, which was once the method of choice
used in the detection of telomere length, measures telomere

length as the mean length for all chromosomes [referred to
as the telomere/terminal restriction fragment (TRF)].
However, this procedure often does not provide accurate
measurements of the TRF and is time-consuming and
tedious. The Southern blot procedure has undergone
numerous modifications to increase its simplicity and
reliability for TRF analysis. This review highlights the
current modifications of the standard Southern hybridiza-
tion technique, along with the latest advances in telomere
length measurements.
Southern hybridization/Southern blot
In 1975, E.M. Southern developed a method that
allowed the transfer of DNA fragments from a gel
onto membrane [30] and this procedure has been applied
to the analysis of DNA fragments in combination with
other techniques. The measurement of telomere length
by Southern hybridization requires that the extracted
DNA is unfragmented and pure, which is relatively
difficult to achieve. Although not universally specific for
telomeres, the most commonly used enzymes for the
restriction of telomeric DNA are Hinf1 and Rsa1
[20,31,32]. The fragments obtained by digestion of
genomic DNA with these restriction enzymes are
resolved by electrophoresis, hybridized to labeled probes
specific for the telomere repeats (CCCATT)
3
[20] and
the TRF values obtained by densitometric analysis. The
resulting telomere restriction fragment band represents
the mean telomere length of all chromosomes. Thus, the

TRF values are subject to variation based on the site of
restriction of the subtelomeric region. Another drawback
of this method is that the TRF value that is obtained
represents the measurement of the cell population and
not of an individual chromosome, thereby affecting
interpretation of results. In addition to a low yield of
DNA, the isolation of intact genomic DNA from a
large number of cells (> 10
5
cells) can be difficult to
achieve in some cases.
Most problems encountered with Southern hybridiza-
tion have been eliminated or minimized to some degree
by its combination with other methods [20]. The problem
of genomic fragmentation during the extraction proce-
dure can be overcome if telomere lengths are measured
from whole cells [33]. In this case, the estimated length is
a ratio of the telomere to the centromere, referred to as
the TC ratio. These values can be determined accurately
from as few as 800 whole cells or 9 ng of DNA, thereby
enhancing the sensitivity of the procedure. In addition to
the estimation of TRF values based on band size or
TC ratios, lognormal distributions formulated by mathe-
matical and statistical calculations have proved to be
suitable for the analysis of telomere lengths [34]. Incor-
porating these modifications into the Southern hybrid-
ization procedure has improved the sensitivity of the
method but not its simplicity.
Hybridization protection assay
Unlike Southern blotting, the hybridization protection

assay (HPA) quantifies the telomeric repeats and does not
include subtelomeric regions, thereby avoiding a problem
encountered with the use of Southern blotting. Safety issues
associated with the handling of radioactive isotopes are
eliminated in this method as the telomere-specific probe is
labeled with acridinium ester (AE). In the HPA procedure,
DNA from cells or tissue lysates is treated with a telomere-
specific AE probe and unbound probe is washed off. The
entire procedure can be performed in a reaction tube, as
quantification is by chemiluminescence [31]. Thus, DNA
shearing will not affect the results. DNA in the lysate is
normalized to an Alu probe that is also AE-labeled [31] and
the value obtained is therefore a ratio of telomeric to Alu
DNA. It has been found that a telomere to Alu DNA ratio
of 0.01 corresponds to approximately 2 kb of mean TRF
length [31]. The HPA method has many advantages over the
Southern blot (Table 1), but telomere length cannot be
measured from individual cells using this method. Despite
this weakness of the HPA procedure, its linear range
(10–3000 ng of genomic DNA or 10
3
)10
5
cells) allows for
the analyses of telomere attrition over time as well as
differences in telomere content among different samples.
Studies using normal and transformed clones of human
fibroblast cell lines have shown a comparable assessment of
telomere length measurement using the Southern blot and
HPA methods [31]. However, quantification is easier and

faster with the hybridization protection assay (Table 1). In
addition to the ease in quantification by HPA, cell types that
have minute differences in telomere lengths can be distin-
guished easily by the chemiluminescent mode of detection.
Fluorescent
in situ
hybridization
The HPA has reduced most of the limitations encountered
with the standard Southern hybridization technique. How-
ever, the measurement of telomere repeats by HPA includes
all cells and not individual cells or chromosomes [31].
Implementation of techniques such as fluorescence in situ
hybridization (FISH) allows calculation of the telomeric
length based on the number of telomeric repeats [29,35].
Enhanced modifications of FISH such as quantitative FISH
(Q-FISH), quantitative flow cytometry (Q-FISH
FCM
), and
flow cytometry and FISH (flow FISH) have provided a
means for the accurate measurement of telomere length
from individual cells (Table 1) [20,29,36].
The FISH method involves the treatment of cells in a
suitable fixative followed by exposure to a hybridization
mixture containing appropriate amounts of formamide,
blocking reagent and a fluorescent peptide nucleic acid
probe (PNA) that is complementary to the telomeric repeats
[37,38]. Fluorescent labeling of the telomere repeats allows
the direct measurement of the telomere length by a
quantitative method referred to as Q-FISH [26,29]. The
PNA probes have an uncharged glycine backbone that

forms stable PNA–DNA interactions unlike the traditional
probes [20,38,39] and their ability to hybridize at low ionic
strengths prevents reannealing of DNA strands. The
fluorescent signal emitted by a telomere spot corresponds
to its length and the integration of a dedicated image
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 391
Table 1. Comparative assessment of the methods employed in telomere length and G-rich overhang measurements. The advantages of some methods over others are summarized. Compared to Southern blot,
HPA, and T-OLA the FISH-derived methods (Q-FISH, Q-FISH
FCM
,andQ-FISH
FCM
and digital fluorescence microscopy) are far more sophisticated, with the capacity to handle large sample numbers.
The main disadvantage of these methods is the cost due to the expensive equipment and specialized training. Nevertheless, once established in the laboratory these methods would definitely help with time
constraints and with high data turnout, improving the accuracy of the results.
FISH
HPA [31] Southern Blot [36] Q-FISH [29] Q-FISH
FCM
[29]
Q-FISH
FCM
and
digital fluorescence
microscopy [20,29,36] T-OLA [55,58]
Simple, rapid
( 45 min)
and sensitive.
Time consuming. More complex than HPA and
Southern blot. Labor intensive.
Complexity level may be similar to
or even more than Q-FISH.

However, the entire process takes
 30 h.
A high degree of complexity
but the final output is probably
faster due to the additional
refinements of FCM and digital
microscopy with state of the art
computerized softwares.
Better suited for measuring
G-rich overhangs than
telomere lengths per se but
comparatively less complex
than the FISH-derived
improvizations.
Can measure
telomere repeats
from purified,
sheared DNA,
as well as
unpurified DNA
in cell and
tissue lysates
(1000 cells).
Requires intact and pure
DNA. Large numbers of
cells are needed in the
extraction of genomic
DNA.
Requires intact metaphase
spreads.

The addition of FCM allows the
determination of telomere lengths
in an individual cell and even
subset of cells present in small
numbers in suspension. Can
determine telomere lengths in
various cell cycle phases.
Additional features such as
digital fluorescence microscopy
enhance the measurement of
telomere lengths not only of an
individual cell, but also of an
individual chromosome.
Requires purification of
telomeres which involves
the separation of telomeres
from subtelomeric
sequences. About 30 lg
of DNA is required.
The direct
quantitative
measurement
of telomere
repeats is done
in cell and
tissue lysates.
The TRF measured includes
subtelomeric regions.
The direct labeling of telomere
repeats and the utilization of

fluorescent probes provides a
more precise quantitative
estimate of telomere lengths.
The use of two probes, one that
specifically stains DNA and the
other telomeres, aids better visual
assessment. Utilization of a control
cell population provides for an
internal telomere length standard
that allows comparisons of differ-
ent samples with high precision.
Advantages are further
enhanced by the use of digital
microscopy improving the rate
at which the results can be
obtained.
Visual measurement of
telomere length of the
G-rich overhang. Standards
for internal and terminal
telomeric repeats are gener-
ated for quantitation
against which the visualized
lengths are measured.
Wide linear range
and can measure
telomere repeats
in biopsy
specimens as well
as cells in body

fluids or
washings.
Reading of a smear on the
autoradiogram may be
inaccurate.
Telomere lengths of metaphase
spreads are measured and thus
exclude populations that are
senescent which may yield
biased results. The telomeric
lengths measured may include
subtelomeric regions as well.
The use of FCM with Q-FISH
permits the assessment of telomere
lengths of an individual cell, inclu-
ding a subset of cells present in low
numbers by the use of specific
antibodies. Possible to detect telo-
mere length telomere length less
than 3 kb. Subtelomeric
background signal is low or
even negligible when compared to
Southern blot.
The additional refinement of
utilizing digital microscopy
allows for higher sensitivity,
larger dynamic ranges and
relatively fast readout rates.
Allows the measurement of
telomere length of an individual

chromosome with greater
accuracy from limited number
of cells.
Ranges from less than 90 nt
to about 400 nt. This range
depends on the cell type
and population doubling.
392 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Chemilumines-
cent mode of
detection makes
it safe to use over
long periods of
time.
Safety issues regarding the
use of radioactive probes
may be a concern.
The method is limited in appli-
cability due to the high degree
of technical expertise required.
Peptide nucleic acids are used as
probes and their interaction with
the DNA is more stable producing
stronger fluorescence signals. The
use of two probes enables simulta-
neous visualization of DNA and
telomere repeats with increased
sensitivity and accuracy.
The use of a digital fluorescence
microscope has increased the

accuracy and speed of analysis.
Image acquisition and
analysis can be run independ-
ently. Better accquisition
systems can be used. The soft-
ware program that can perform
telomere segmentation,telomere
fluorescent measurement and
chromosome segmentation for
each metaphase spread is most
appropriate.
This method would be an
excellent one to determine
telomere overhangs after a
certain defined number of
population doublings. Has
the ability to provide data
on intact individual cells.
This method efficiently uses
a system that takes into
account subtelomeric,
internal and terminal
repeats.
The equipment
required for this
method is
relatively simple
and can be
obtained in most
laboratories.

Southern blot requires the
use of densitometer and
autoradiogram for reading
the blots that can be
expensive.
The basic equipment required
for FISH is easily obtained in
most laboratories. However,
the type of microscope used
does affect the quality and
accuracy of the results.
In addition to the general equip-
ment utilized in Q-FISH, this
method employs the use of a flow
cytometer. Depending on the
probes used and the type of signal
measured, several types of flow
cytometers are available on the
market.
The type of image accquisition
and data analysis needed would
dictate the type of software to
be used. Image capture may
require dedicated software.
The basic equipment
required for this method is
an electron microscope.
Overall it is a
reasonably good
method

considering cost
and laboratory
equipment.
Several drawbacks. No information can be
determined on the original cell
type.
The only drawback of the method
is the denaturation conditions used
to maximize probe penetration.
It can be suited for a variety of
applications.
Comparative analysis
between different cell types
can be done.
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 393
analysis software system permits the calculation of a
combined fluorescent value from signals produced by
individual telomere spots [26,36]. The telomere length is
expressed in telomere fluorescence units (TFU), with 1 TFU
corresponding to 1 kb of TTAGGG repeats. Telomere
lengths obtained by Q-FISH have been shown to correlate
well with the TRF values obtained by conventional
Southern hybridization analysis [37].
An interesting system developed by Poon et al. [36] allows
the measurement of telomere length by digital fluorescence
microscopy in cells prepared for Q-FISH. In this system the
cells are hybridized with both a peptide nucleic acid–
cytochrome 3- (PNA–Cy3)- labeled probe that specifically
binds to telomeres and the 4¢,6-diamidino-2-phenylindole
(DAPI) dye specific for the chromosomes [40,41]. The length

of each telomere is an integrated value of the intensities of the
two fluorescent dyes, and is measured as integrated fluor-
escent intensity (IFI) [36]. The system allows the detection of
average telomere length within a cell, of chromosome-
specific telomere lengths in a suspension of cells, and of the
length of individual telomeres. For the measurement of IFI
values of each telomere, a process called segmentation is
performed. This involves the identification of exact bound-
aries of each telomere in the segmented telomere region.
Thresholding or edge detection methods are employed to
determine the approximate location of the telomere spots
[42,43]. For chromosome segmentation, the IFIs are deter-
mined using the
TFL
-
TELO
program. Detailed features of the
program are described elsewhere [36]. The
TFL
-
TELO
gener-
ated output value corresponds to the fluorescence intensity
of each telomere, which is proportional to the number of
probe molecules that hybridize to the region. Utilizing digital
microscopy, telomere length was assessed from two different
samples, same metaphase samples and random metaphase
samples measured on five different days [36]. The average
mean telomere values measured from day one to day five
indicated by the telomere fluorescent values (TFV) were

essentially the same (i.e. 11.3 and 11.2, respectively),
suggesting the method is both accurate and reliable. The
system is very efficient in terms of its sensitivity and telomere
length of chromosomes can be measured in as few as 30 cells.
Modifications such as the use of an automated microscope
focusing process over the manual method or even a three
dimensional volume rather than single image plane [44] may
improve IFI values and telomere length estimates.
Q-FISH and methods used in conjunction with it are
performed on metaphase spreads [20,29,36] which can be a
problem because cells approaching senescence are less able
to enter mitosis. This can lead to variable results in telomere
lengths in a mixed population of cells (i.e. senescent and
proliferating). Although the method is tedious and time
consuming, Q-FISH is suitable for determining changes in
telomeric sequences.
Flow cytometry
In the flow cytometry method (FCM), cells are separated
based on fluorescence intensity and by immunophenotype
(antibody staining) [28]. Therefore, segregation of cells into
subgroups from a large population and at different phases
of the growth cycle is possible by this procedure [28,45,46].
FCM is a highly sophisticated technique with many
advantages [28], among which are that it is simple, rapid,
highly reproducible and can be applied to tissue samples,
fluids, and washings. FCM therefore has much to offer in
terms of accuracy and speed in telomere length measure-
ments. With these advantages, FCM, when used in a
combination with Q-FISH, can eliminate most problems
associated with Q-FISH alone.

Many different fluorescent probes have been utilized to
stain DNA [26,28,31,40] and have contributed greatly to
telomeric analysis. The importance of using the appropriate
probe as well as the right method of fixation has been
discussed elsewhere [20]. In the Q-FISH
FCM
procedure,
fluorescence-labeled PNA probes are employed in the
hybridization process [29,47]. Cells can also be treated with
specific antibodies of interest, which are tagged with a
fluorescent dye. Using the fluorescence-activated cell sorter
(FACS), the cells are sorted based on the intensity of the
fluorescence signal produced [29] and the signals generated
by the respective probes can be detected by different
channels. Telomere length values are calculated as the ratio
of the telomere fluorescence signal (TFS) of the sample to
that of the internal control, normalized to the DNA values
at the G
0
/G
1
phase. The use of an internal control (e.g. 1301
cell line) is important to monitor the accuracy of the
procedure and also to serve as a standard for telomere
length [29]. Normalization of the relative telomere length to
the DNA index of G
0
/G
1
phase compensates for the

variability in the amount of DNA per cell and thus,
telomere repeats. A significant correlation has been found
between the telomere length values obtained by
Q-FISH
FCM
and Southern blotting [29], and a Q-FISH
FCM
value of 0 corresponds to 3.2 kb in a Southern blot.
The presence of intrachromosomal telomeric repeats may
affect the telomere length values. However, the relatively
low occurrence of these repeats may reduce the effect to a
minimum. Overall, Q-FISH
FCM
is by far the most suitable
method for telomere length measurements due to its
sensitivity, reproducibility and speed (within 30 h)
(Table 1). Also, the use of various controls has increased
the sensitivity of the method thereby allowing the assess-
ment of subtle comparisons between different samples. The
method was originally primarily suited for the detection of
telomere length from samples of hematopoietic origin.
However, Q-FISH
FCM
canalsobeusedforthedetectionof
telomere length in various other cells types and samples,
although the cells must first be separated.
Analyzers and sorters are the two main types of flow
cytometers [48]. Recent advancements in technology have
enabled the development of cytometers that have both of
these features combined [48]. The importance of detecting

minute changes in telomere length from a population of cells
and from individual cells is critical to various aspects of
scientific research. Therefore, in choosing the method for
the detection of telomere length (Table 1), cost considera-
tions as well as time constraints are essential factors that
need to be considered. Flow cytometers have facilitated
analyzing and sorting a large number of cells ranging from
300 cellsÆs
)1
to > 20 000 cellsÆs
)1
, depending upon the type
of flow cytometer used, with high purity and accuracy [48].
In general, flow cytometry sorting and analysis of cells are
based on the staining and intensity of the fluorescent signal.
Thus, the choice of the flow cytometer depends on factors
394 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
such as type of sample (cell/tissue), type of information
required, the number of samples to be analyzed and the
quality [49]. When considering microscopy, the flow
cytometry system parameters such as the type and number
of fluorochromes/probes used, light source, objective,
eyepiece and filters are essential [49].
Due to space limitations, these aspects are not described in
this review. However, these parameters, including technical
aspects, advantages, specifications of the different types of
flow cytometer analyzers and sorters are well described
elsewhere [48,50,51]. Flow cytometers are commercially
available with companies such as Becton Dickinson and
Coulter (Beckman Coulter). FACSCalibur, FACSVantag-

eSE, MoFlo, FACScan, FACSort are examples of some
of the commercially available flow cytometers [48,50].
Although expensive (ranging from about $50 000–175 000)
the scope of their applications may be varied [48]. Telomere
length analysis and DNA replicatory mechanisms are
aspects directly involved and related to cell structure,
function and integrity, and thus the use of these sophisticated
instruments are worth the investment. Choosing the right
probe is important as the signals emitted by these probes are
used in the quantification and analysis of the data. Several
probes/dyes have been used in staining surface, integral or
cytoplasmic proteins and even DNA [28,52,53]. Most of the
probes are used routinely based on the need of the
application. However, the CFSE [carboxyfluorescein diace-
tate succinimidyl ester (CFDA-SE)] dye appears to offer
much more. Utilizing this dye with flow cytometry one can
visualize the number of times a cell divides both in vitro and
in vivo [54] which in terms of telomere length measurements
is very important. This dye has been found to show about
8–10 discrete cell cycles of cell division [54]. Also, viable cells
that have undergone a defined number of divisions can be
recovered by flow cytometric sorting utilizing this dye [54], a
feature that may be applicable for telomere length measure-
ments. This technique has the ability to monitor prolifer-
ation in a minor subset of cells and follow the acquisition of
different markers in internal proteins linked to cell divisions.
In the future, combination of this dye with Q-FISH
FCM
may
find its application in the detection of telomere binding

proteins (TBP) or follow the pattern of TBP at the time of
telomere elongation and replication.
Several software applications are commercially available
[48].
CELLQUEST
appears to be the more commonly used
software for data acquisition and analysis.
CELLQUEST
is
user-friendly and is quite versatile in terms of its functions
[29]. Some of these features include user-defined calculations
on the data, management of data acquisition, ability to
export graphics and documents from a variety of formats,
format plots and text objects, and the ability to adjust the
specifications and instrument settings for each tube. The use
of these software programs with the sophisticated flow
cytometers has enabled high purity and accuracy with
greater speed.
Telomeric-oligonucleotide ligation assay
G-rich overhangs are located at the 3¢-end of each
DNA strand of the chromosome and serve as a
substrate for telomerase. Telomere shortening is found
to be directly proportional to the length of the overhang
[55]. The information obtained from the G-rich over-
hang lengths can be used for analyzing drug efficacy
and disease progression as well as other processes. Also,
based on the values obtained, suitable inhibitors may be
designed to increase the rate of the telomere length
attrition process [55]. Analysis of the molecular structure
of the G-rich overhangs is useful as they are suitable

targets in cancer therapy. Stabilization of the T and D
loops formed by G-rich overhangs by chemicals could
inhibit access of telomerase to the 3¢-end resulting in a
decreased number of telomeric repeats with each cell
division, thereby initiating progression towards a senes-
cent phenotype.
Primer extension/nick translation (PENT) [56–58], elec-
tron microscopy of purified telomeres [57,58], and telo-
meric-oligonuceotide ligation assay (T-OLA) [56] are all
suitable methods for the determination of lengths of
G-rich overhangs. However, it is apparent that smaller
G-rich lengths are undetectable by the PENT assay and
electron microscopy (Table 2). When these methods were
used in the detection of G-rich overhang lengths in
HUVEC cells (Table 2), only the T-OLA assay could
detect lengths of < 90 nucleotides (nt) in a majority of
cells, whereas the PENT assay and electron microscopy
detected lengths ranging from 130–210 nt and 225–650 nt,
respectively. Thus lengths shorter than about 100 nt are
below the detection range of these methods. The ability to
detect shorter lengths is crucial, as chromosomes in
senescent and certain proliferating cell lines contain short
overhangs. This problem is overcome using the T-OLA
procedure which has the ability to detect-3¢-overhangs
ranging from 24–650 nt [56]. The assay involves hybridi-
zation of a highly specific
32
aP-labeled oligonucleotide to
nondenatured DNA. The oligo binds in the presence of
ligase to single-stranded DNA with high base-pairing

specificity and the products are resolved on a denaturing
polyacrylamide gel. However, the T-OLA assay can be a
time-consuming procedure due to the gel-based length
detection. Also, the safety issue regarding the handling of
radioactive oligonucleotides can be a concern. Though it
has a wide detection range and applicability to many cell
types, its use in large-scale screening of samples is
questionable.
Factors influencing telomere length
Telomeres
Telomeres consist of tandem repeats (of a hexameric
sequence in humans), which are positioned at the
extreme ends of chromosomes. The repeats are mostly
G-rich although some organisms such as certain fungi
and invertebrates have interspersed C-nucleotides. The
G-nucleotides in telomeric repeats vary by species (from
one to eight nucleotides) and are flanked by T/A
nucleotides at the 5¢-end (e.g. 5¢-TTAGGG-3¢ in Homo
sapiens,5¢-TTAGGC-3¢ in Ascaris) [59]. Telomeres
impart stability to the chromosomes by facilitating the
formation of stable structures. The structural unit of
telomeres, termed the G-quartet, resembles a square
where the G residues occupy the four corners and T/A
residues form the variable arms, which can form loops
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 395
enclosing the G residues. Stacks of two, three or four
quartets tethered by cations (primarily potassium) can
form dimeric, trimeric or quadruple structures [60,61].
These structures are thermodynamically and kinetically
very stable; hence their contribution to the stability

essential for chromosomes. Telomeres tend to form loop
structures referred to as D or T loops [62–64] which
may be required to shield the chromosomal ends from
nuclease activities.
The synthesis of telomeres occurs simultaneously with
DNA replication. The unwinding of the DNA strands is
essential for the binding of the DNA replication apparatus,
which exposes the telomeres to telomerase. However, in
some instances these exposed telomeres can undergo
recombination in the absence of telomerase and maintain
the telomere length [65,66].
G-rich overhangs or tails
A G-rich tail or overhang at the extreme 3¢-end of each
DNA strand is a structure of approximately 200 ± 75
nucleotides associated with all telomeres and arises due to
the end-replication problem [58]. The overhang serves as a
substrate for telomerase in telomere replication and
participates in the formation of the T- and D-loop
structure. A G-tail has the ability to fold backwards and
bond with one of the two duplex telomere strands
forming a T-structure and the free 3¢-end inserts between
the two strands, forming a minor D-loop [67]. Free
3¢-ends may be recognized as DNA strand breaks that can
activate the check-points of the DNA repair apparatus
[67,68], which probably could initiate the process of
cellular senescence and apoptosis [67]. Thus, the loop
structures sequester the free 3¢-ends, preventing DNA
damage and activation of repair signals and therefore
provide stability to the chromosome.
Proteins associated with telomeres

and their importance
Many investigations have unraveled the importance of
telomerase in maintaining stable telomere lengths [69].
However, in telomerase-negative cells or even in some
species, an alternate mechanism exists that enables main-
taining an average telomere length relative to the species
[69,70]. Nonhomologous end-joining or recombination is
one of the alternative lengthening of telomeres (ALT)
mechanisms believed to maintain stable telomere lengths
and evidence supporting this mechanism has been seen in
smaller eukaryotes and in some cases, even mammals [71].
The expression and activity of telomerase has been known
to be significant in the development of a majority if not all
malignant tumors [71]. However, telomeres are also
important in cancer biology. In chromosomes telomeres
serve as stabilizing caps. Irregularities in telomere replica-
tion or structure may therefore affect the generation of
stable telomere lengths. Given that the end-replication
problem in part causes telomere attrition, abnormal
telomeric synthesis and architecture would further enhance
the rate at which telomere attrition would occur leading to a
destabilized telomere length. The genomic instability created
within the cell due to telomere fusions and formation of
dicentric chromosomes may therefore potentiate the for-
mation of abnormal cellular phenotypes and possibly
trigger the onset of cellular senescence or even apoptosis
[41]. These plausible occurrences necessitate a balance
between telomere replication and telomere length stability.
The rapid pace at which telomere biology has moved has
provided fascinating insights to several factors that contri-

bute to maintaining this delicate balance. Several proteins
are now known to exist, some that bind to the components
of the telomerase complex and others that bind specifically
to telomeres, called TBP (Table 3).
Table 2. Detection of G-rich overhang lengths by primer extension/nick translation (PENT), electron microscopy and telomeric-oligonucleotide
ligation assay (T-OLA). The PENT assay, electron microscopy and T-OLA are established procedures for the assessment of G-rich overhangs. The
detection range of G-rich overhangs are 130–210, 650–175, and < 90–400 for PENT, electron microscopy and T-OLA, respectively. Of the three
methods, T-OLA has the ability of detecting G-rich lengths of < 90 nt. The ability of T-OLA to detect smaller G-rich lengths makes it a preferable
method over electron microscopy and PENT. ND indicates values not given (i.e. not described).
Method Cell type
G-rich lengths detected
(nt)
Percent of cells containing
the defined length Reference(s)
PENT assay Human umbilical vein
endothelial cells (HUVEC)
130–210 > 80 [57,58]
Electron microscopy BJ foreskin fibroblasts 200 ± 75 ND [57,58]
HUVEC 225–650 14 [55]
BJ foreskin fibroblasts 50–350 16 [55]
IMR90 lung fibroblasts 100–300 14 [55]
MEC (mammary epithelial cells) 175–350 15 [55]
T-OLA HUVEC < 90 Majority [56]
Fibroblasts and lymphocytes < 90 56 [56]
HeLa (cervical cancer cell line)
and U937 cells
< 90 62 [56]
Fibroblasts, lymphocytes HeLa,
and U937 cells
108–270 37 [56]

Fibroblasts and lymphocytes 400  1 [56]
396 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Table 3. Telomere and telomerase complex bound proteins.
Protein Component Bound Organism Function Reference(s)
TRF1 (telomere
repeat factor 1)
Binds as homodimers
to double strand (ds)
telomeric repeats.
Mammals May play a role in telomere
replication. When bound to
telomeres, telomerase access to
telomeres is prevented and
thus appears to have a role in
regulating telomere length
through inhibition of telomerase
by its interaction with tankyrase.
[70,71,79,102,103]
TRF2 Binds to ds telomeric
DNA only.
Mammals Although TRF2 binds ds repeats
only, it may have an indirect
role in protecting the G-rich
overhang by recruiting other
TBPs to the G-tails or by
mediating the formation of the
telomeric T–loop.
Prevents chromosome fusion.
Interacts with TRF1 to regulate
telomere length via its interaction

with hRap1.
[70,79,102–104]
UP1 Binds both to telomere
repeats and telomerase.
Mammals This protein is the aminoterminal
portion of the heterogenous
nuclear ribonucleoprotein A1.
It is thought that telomerase
may be recruited at the time of
the proteteolytic processing of A1
to UP1 to the telomeres.
[70]
Ku Complexes with TRF1. Mammals Protects telomeres from fusions.
Possibly aids in the formation
of T-loop structure by its
interaction with TRF1.
[79,105,106]
YKu70/80 Ku binds as a heterodimer
to telomeric DNA. It is
also a double strand
break (DSB) repair
protein. Complexes
with the telomerase
RNA component.
Budding yeast
S. cerevisiae
Appears to have several functions
in addition to its critical role
in end-joining of double-strand
breaks. In S. cerevisiae it is

important in telomere maintenance.
Plays a role in telomere end
structure either by assisting in
the formation of G-tails through
the recruitment/regulation of an
exonuclease or by protecting the
G-tails from degradation. Ku may
be involved in clustering of
telomeres and may be involved in
the interaction with the nuclear
envelope.
[70,79,107,108]
pKu70 Homolog of budding
yeast S. cervisiae.
Fission yeast
S. pombe
By its interaction with the
stem-loop structure of telomerase
RNA, may be involved in the
direct recruitment of telomerase.
Absence of this protein results
in telomere fusions and increased
recombination of subtelomeric
sequences, and therefore may
be important in telomere tract
protection from nuclease
and recombinatorial activities.
[79,109]
Rad50/Mre11/Xrs2 A protein complex that
may bind telomeric DNA.

Yeast Similar to Ku, is primarily involved in
DSB end-joining. It may have a role
in telomere maintenance.
[70]
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 397
Table 3. (Continued).
Protein Component Bound Organism Function Reference(s)
Rad50/Mre11/Nbs1 Forms a complex with
TRF2.
Mammals Aids the formation of T-loop
structures.
[79]
Cdc13p (cell division
cycle 13)
Binds to single strand
(ss) telomeric protein.
Yeast May have dual functionality
not only in protecting the
terminal end but also
in facilitating the access of
telomerase via Est1p. May be
essential in the synthesis and
maintenance of the C-rich strand
of the telomere. Protects DSB that
are juxtaposed to TG1-3 repeats
which can be acted upon by telomerase.
[70,79,110]
Est1p (ever shorter
telomeres 1)
Binds to ss telomeric DNA

with relaxed specificity and
requires a free 3¢-end
to bind. May either be a
component of telomeric
chromatin or a protein
subunit of telomerase.
Yeast Along with Cdc13p may assist in
the extension of the 3¢-end in vivo
by telomerase.
[79,110,111]
Stn1 Forms a complex
with Cdc13p.
Yeast Negative regulator of telomerase
recruitment.
[79,107,110,112]
Ten 1 Associates with
Stn 1 and Cdc13
Yeast
S. cerevisiae
Protects telomere ends and
regulates telomere length.
[113]
TBP Binds ss 3¢-overhang. Ciliates Protects the chromosome end. [110]
Oxytrichia and
Euplotes
rTP (replication
telomere protein)
Binds telomeric DNA. Ciliates Euplotes Expressed at all times of DNA
replication and may be an
important telomere-bound

replication factor regulating
telomere replication.
[110]
p80 Binds to the RNA
subunit of telomerase.
Ciliate
Tetrahymena
thermophilia
Probably this complex
(p80 and RNA) may induce
telomerase activity by its
interaction with the catalytic
subunit.
[110]
p95 Are found crosslinked
to telomeric
oligonucleotides.
Ciliate
Tetrahymena
thermophilia
May provide an active site
for telomerase.
[110]
TLP/TLP1 Interacts with the RNA
subunit of telomerase
Mammals A mammalian homolog of p80. [110]
EST1, EST3,
EST4/Cdc13
May associate with the
telomerase complex.

Yeast Not absolutely essential
for telomerase activity in vitro.
However, required for telomerase
activity and telomere maintenance
in vivo.
[79]
Rap1p (repressor-
activator protein 1)
Binds duplex ds
telomeric DNA.
Budding yeast Negatively regulates telomerase
elongation via its carboxyl
terminus May be involved in telomere
length homeostasis by a negative
feedback mechanism. May
be a part of the counting mechanism
that measures telomere length.
[69,79,114–116]
Tankyrase Associates with TRF1. Yeast and
Mammals
In vitro tankyrase adds poly
ADP-ribose to TRF1, decreasing
the affinity of TRF1 for telomeric
DNA which may signal telomerase
to elongate the telomeres.
[69,79,117]
398 S. N. Saldanha et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Proteins such as telomere repeat factor 1 and 2 (TRF1,
TRF2), Tankyrase 1 and 2 (TANK1, TANK2), Ku70/86
and DNA dependent protein kinases (DNA-PKcs), poly

(ADP-ribose) polymerase (PARP1), and Pot1 are known to
associate with telomeres [3,67,72–77] and to have effects on
telomere length, G-rich overhangs and the cell phenotype
[68]. For example, TRF2, a mammalian telomere binding
protein, is present in more than 100 copies per chromosome
and is associated with the entire length of the telomere.
Inhibition of TRF2 binding or blocking of the binding sites
has been reported to result in a senescent phenotype in a
subset of cells and apoptosis in other cells [67] which
emphasizes the telomere protective function of TRF2, the
absence of which results in telomere dysfunction [68].
Dysfunctional telomeres may lack G-overhangs and the
lack of G-overhangs prevents the formation of T-loops and
contributes to the formation of dicentric chromosomes [67].
Formation of these altered structures is likely to affect the
normal course of cell division resulting in cellular senescence
or apoptosis, and in some cases contribute to premature
aging syndromes such as Werner syndrome, Bloom syn-
drome, and ataxia telangiectasia [78]. In the latter case, the
mutated gene that is responsible for this autosomal recessive
disease is the ataxia telangiectasia mutant (ATM). ATM
function is triggered in response to DNA damage (double
strand breaks) and controls the various cellular processes
required to repair the damage [78]. In addition, ATM plays
a vital role in telomere metabolism. Absence of the ATM
protein results in end-to-end chromosomal fusions, known
as telomere associations [78]. Therefore, it is important to
understand the relationship between the various telomere
binding proteins and their functions as they pertain to
telomere metabolism and stability.

The network that controls telomere replication, telomere
elongation and telomerase activity remains to be ascer-
tained. The vast numbers of proteins involved intensify the
need for further investigations in telomere research. Cur-
rently there are few proposed plausible mechanisms instru-
mental in telomere length homeostasis. Rap1, a protein that
binds to telomeric DNA, is thought to be a part of the
counting mechanism that measures the telomere length,
which probably signals other proteins and protein com-
plexes to control the regulation of telomere synthesis by
telomerase [79]. Although this has led to the speculation that
a regulatory on/off switch exists that controls telomere
lengthening, there is little experimental evidence to support
this [79]. Telomeres that are critically short are elongated at
a faster rate initially which gradually slows down once
equilibrium is attained [79]. In addition, long telomeres are
extended much more slowly. Therefore, the regulatory
mechanisms governing this lengthening process are more
rheostatic in nature [79]. Proteins involved in DNA damage
check-points, telomeric silencing and telomere positioning
effects are essential to telomere replication but their roles in
telomere and telomere length homeostasis are unclear and
need to be investigated further [79,80].
Importance of telomeres and telomere length
Telomeres extend up to 10–15 kb at each human
chromosome end (Fig. 1) and protect the ends of the
chromosomes. The geometric configuration of telomeres
appears to be of great importance. The quartets are
very resistant to nuclease attack [81] and contribute to
protecting the chromosomal termini. Telomeres are

thought to control the expression of subtelomeric genes
[59,81–89] and therefore may paradoxically control the
expression of the enzyme telomerase itself, which is
located at the extreme end of chromosome 5p in
humans. Several studies have implicated telomerase in
Table 3. (Continued).
Protein Component Bound Organism Function Reference(s)
Taz1 (Telomere
associated in
S. pombe 1)
A TRF1 homolog of
fission yeast that binds
telomeric DNA.
Fission Yeast Negatively regulates telomere
elongation.
[79,118]
TIN2 Interacts with TRF1
and localizes to
telomeres.
Mammals An important mediator of TRF1
function. An essential factor for
the regulation of telomere length.
[71,79]
Sir protein complex
(Sir2/3/4)
Interacts with Rap1. Yeast Telomeric silencing and telomere
position effects both of which are
essential to telomere replication.
[79,119]
Rif1p and Rif2p

(Rap 1p- interacting
factor 1 and 2)
Interact with Rap1. Yeast Important for Rap1 functions. [71]
Mlp2 (myosin like
protein 2)
Interacts with YKu70. Yeast Important for Yku70-related
functions.
[79]
MeC3p Interacts with SET
domain proteins Set 1.
Yeast Negatively regulates telomere
positioning and telomere elongation.
[79,80,120]
pot1 (protein on
telomeres 1)
Binds G-rich overhangs. Yeast and
Mammals
Plays an important role in telomere
capping. May protect the G-rich
overhang when the t-loop unfolds.
May prevent telomere elongation
of the 3¢-end within a t-loop.
[121]
Ó FEBS 2003 Telomere length detection (Eur. J. Biochem. 270) 399
the maintenance of stable telomere length although
telomere length can, in some cases, be maintained in
the absence of telomerase by mechanisms referred to as
alternative lengthening of telomeres [90–98]. Other
proposed mechanisms are homologous recombination,
which can involve intertelomeric recombination, T-loop

formation, rolling circle and extrachromosomal telo-
meric repeats [99], and nonhomologous end-joining.
Therefore, the telomere, its structural motifs and length,
as well as recombination mechanisms and telomerase,
all appear to be integral to maintaining the structural
and functional integrity of chromosomes. Assessment
of these telomeric factors could provide a wealth of
information with potential for facilitating therapy or
cures for various age-associated diseases including
cancer.
Conclusion
Telomere length and telomerase are two important markers
that are rapidly gaining importance as targets for cures of
several age-related diseases including cancer. For example,
in about 95% of cancers telomerase is up-regulated with
most having stabilized telomere lengths. Several investiga-
tions have shown that inactivation of telomerase leads to
decreased telomere length. Nonetheless, this effect is gradual
and therefore tumor size reduction is not immediate. Hence,
inactivating telomerase alone may not be sufficient to
obliterate the tumor. Targeting telomerase and telomere
length simultaneously shows great promise as therapeutic
intervention, and inhibitors having these dual effects
therefore need to be designed. A few such inhibitors are
already being tested in laboratories; however, further
research is needed in this area.
In addition to serving as a potential target for cure, the
assessment of telomeres and telomere length is important, as
they can be useful in identifying several abnormal cellular
phenotypes, determining the efficacy of inhibitors, and

providing prognostic information that may help guide the
direction of treatment. For these reasons, the methods
utilized in measuring telomere length should be efficient and
expedite the process of determining minute and subtle
changes in telomere length. Unquestionably, telomere
length serves as a marker of telomerase activity, and its
measurement by various methods as described in this review
is therefore necessary and important. Summarizing the
advantages and limitations of the methods described, it is
apparent that the quantitative flow FISH in combination
with digital microscopy will enable efficient large-scale
processing of samples and this combined technique
shows great promise in facilitating analysis of telomere
lengths.
Acknowledgements
We thank Mark Casillas, Dr Mitchell Pate, Dr Nadejda Lopatina, and
Nathaniel Hansen for critical reading of the manuscript. This work was
supported by grants from the National Institute on Aging (1 R03
AG20375 01), the American Cancer Society (IRG-60-001-41), the John
A. Hartford Foundation (South-East Center for Excellence in Geriatric
Medicine), the Leukemia Research Foundation and the UAB Center
for Aging, Comprehensive Cancer Center and Department of Biology.
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