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: 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-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 [...]... [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... 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- Important for Yku70-related functions Negatively regulates telomere positioning and telomere elongation Plays an important role in telomere. .. 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. .. counting mechanism that measures the telomere length, which probably signals other proteins and protein complexes 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... on telomeres 1) Yeast and Mammals 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. .. 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... important mediator of TRF1 function An essential factor for the regulation of telomere length Telomeric silencing and telomere position effects both of which are essential to telomere replication Important for Rap1 functions Interacts with YKu70 Yeast Interacts with SET domain proteins Set 1 Binds G-rich overhangs Yeast Sir protein complex (Sir2/3/4) Rif1p and Rif2p (Rap 1p- interacting factor 1 and 2) Mlp2... 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 syndrome, and ataxia telangiectasia [78] In the latter case, the mutated gene that is responsible... positively and negatively regulates telomere replication Genes Dev 15, 404–414 113 Grandin, N., Damon, C & Charbonneau, M (2001) Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13 EMBO J 20, 1173–1183 114 Marcand, S., Wotton, D., Gilson, E & Shore, D (1997) Rap1p and telomere length regulation in yeast Ciba Found Symp 211, 76–93; discussion 93–103 Telomere length. .. about 95% of cancers telomerase is up-regulated with most having stabilized telomere lengths Several investigations 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 . 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. 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.