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R E V I E W A R T I C L EAssessment of telomere length and factors that contribute to its stability 1 Department of Biology,2Center for Aging and3Comprehensive Cancer Center, University

R E V I E W A R T I C L E

Assessment of telomere length and factors that contribute

to its stability

1

Department of Biology,2Center for Aging and3Comprehensive 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: trygve@uab.edu

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-FISHFCM, 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)

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 fragmeasure-mented genomic sequences [27–29]

Hybrids of these methods such as Q-FISH, flow FISH and

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.

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

resolved by electrophoresis, hybridized to labeled probes

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

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

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

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

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

Table

C cent

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

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

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

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

variability in the amount of DNA per cell and thus, telomere repeats.A significant correlation has been found

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

method for telomere length measurements due to its

(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

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

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

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

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

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

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

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

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

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 generareplica-tion 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]

BJ foreskin fibroblasts 50–350 16 [55]

IMR90 lung fibroblasts 100–300 14 [55]

MEC (mammary epithelial cells) 175–350 15 [55]

Fibroblasts and lymphocytes < 90 56 [56]

HeLa (cervical cancer cell line) and U937 cells

Fibroblasts, lymphocytes HeLa, and U937 cells

Fibroblasts and lymphocytes 400  1 [56]

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]

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]