To investigate telomere function in cancer, through analysis of telomere length and telomerase activity, and to evaluate the prognostic implications of these parameters.
Patients & Methods: Telomere length was determined in four gastric tumour samples and their corresponding normal controls by Southern blot analysis of terminal restriction fragments (TRF). Six tumours of the lung and the paired normal samples were tested for telomerase activity using the TRAP-ELISA assay. Four relevant research papers were surveyed to evaluate the prognostic impact of telomere status and telomerase expression.
Results: Telomere status varied between samples, with one sample showing no significant change in length and another exhibiting telomere elongation. The other two tumour specimens displayed telomere shortening. Telomerase activity was detected in 5 (83%) of 6 lung cancer specimens and only one of the paired normal lung tissues. In general, the presence of (high) telomerase activity in cancer cells was an indicator for poor clinical outcome (i.e. tumour recurrence or death). Telomere status, on the other hand, had changeable effects on prognosis in different cancer types.
TABLE OF CONTENTS
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Telomeres: The Guardian of Chromosome Ends
Telomeres are repeated nucleoprotein structures, sited at the chromosome termini, which help to preserve the integrity and stability of the genome (Bhattacharyya & Lustig, 2006). The protective function owes much to their unusual architecture and composition. Human telomeres consist of tandem repeats of the sequence 5'-TTAGGG-3' (Moyzis et al., 1988) and terminate with a single-stranded 3'-end overhang (Makarov et al., 1997). The ends of telomeres are 'capped' by telomere-binding proteins (collectively called shelterin) (de Lange, 2005), which promote invasion of the duplex telomeric DNA repeat arrays by the 3' overhang to form a higher-order structure known as a T-loop (Figure 1) (Griffith et al., 1999; de Lange, 2004). This is thought to prevent the chromosome ends from being mistaken for DNA breaks, thus avoiding unnecessary activation of repair systems and subsequent chromosome fusions or degradation (de Lange, 2002). Telomeres also play important role in chromosome segregation during mitosis (Kirk et al.; 1997).
Figure 1: Electron Micrograph of Human T-loop (Griffith et al., 1999)
Adjacent to telomeres are variable regions called subtelomeres, which have high sequence similarity to telomeres (Riethman, 2004; Riethman, 2005). Consequently, there is no means of separating these two regions, and so telomere length cannot be measured directly. Instead, digestion of DNA liberates terminal restriction fragments (TRFs), which comprise both regions. In humans, telomere length is estimated to be between 0.5 and 15 kilobase pairs (kbp) long (Aubert & Lansdorp, 2008).
The End-Replication Problem
Telomeres provide a buffer of disposable DNA, which solves the 'end-replication problem' (i.e. inability of DNA polymerase to completely replicate the ends of linear chromosomes) (Watson, 1972; Olovnikov, 1973)
DNA polymerase can only replicate DNA in the 5'â†’3' direction. The leading strand, which has the 3' to 5' parent strand, undergoes continuous DNA synthesis whilst the lagging (discontinuous) strand is replicated as a series of short Okazaki fragments, which require a RNA primer to initiate replication. Removal of the terminal RNA primer at the end of the lagging strand leaves a gap that cannot be filled by the enzyme due the absence of a free 3'-OH to prime synthesis (Figure 1). This results in incomplete replication of telomeres.
The end-replication problem has always been regarded as a 'lagging strand problem' (Levy et al., 1992). However, it could also stem from the inability of leading strand synthesis to produce a 3' overhang (Lingner, 1995).
Figure 2: The End Replication Problem. At the end of the chromosome, there is no template DNA to bind to the primer, therefore, a gap is left in the lagging strand.
Regardless, this phenomenon shortens telomeres by approximately 120 base pairs per cell doubling (Vaziri et al., 1993) and serves as a 'mitotic clock' that defines the proliferative potential of the cells (Harley, 1991; Morin, 1997). The loss of telomeric repeats ultimately leads to permanent cellular growth arrest called replicative senescence (or M1 stage) though initiation of DNA damage signals and activation of p53 and/or p16-Rb tumour suppressor pathways (Hara et al., 1991; Shay et al., 1991; Vaziri & Benchimo, 1996; Lin et al., 1998; d'Adda di Fagagna et al., 2003). By restricting the proliferative capacity of cells, including those with deleterious mutations, telomere shortening and replicative senescence form a major barrier to tumourigenesis.
Replicative Senescence & Beyond
Always on Time
Marked to Standard
Normal somatic cells have a finite replicative capacity, known as 'Hayflick's Limit' (Hayflick, 1965; Shay & Wright, 2000). When telomeres reach a critical length proliferation ceases at the replicative senescence stage (Allsopp & Harley, 1995), which is characterised by changes in cell morphology (Hayflick, 1965), low metabolic rate and Î²-galactosidase expression (Dimri et al., 1995).
Although, there is universal agreement that telomere shortening causes the onset of senescence there remains some doubt about the specific molecular trigger. Some studies suggest that a single short telomere or a group of the chromosomes with the shortest telomeres may induce senescence (Hemann et al., 2001; Zou et al., 2004). Other researchers argue that an altered telomere structure (function) rather than an exact length triggers senescence (Blackburn, 2000; Karlseder et al., 2002). Telomeres are able to repress the transcription of neighbouring genes by a length-dependent mechanism known as the telomere position effect (TPE) (Baur et al., 2001), thus it has been proposed that telomere shortening may lead to reactivation of genes implicated in cell cycle arrest. However, this is unlikely since specific genes controlled by telomere length are yet to be identified.
In the absence of senescence pathways (e.g. due to loss of p53 function), cells continue to divide and telomeres shorten further, destabilising chromosome ends (Duncan & Reddel, 1997). The resulting telomeric fusions can initiate repeated breakage-fusion-bridge (BFB) cycles that lead to extensive chromosome rearrangements and aneuploidy (Lundblad, 2001; Shay & Roninson, 2004). This genomic instability invariably leads to a period of crisis (or M2 stage), which is characterised by mitotic catastrophe and apoptotic cell death (Shay & Wright, 2005). Nonetheless, selected rare cells (10-7) may emerge from crisis with a mechanism for telomere maintenance and often exhibit genomic abnormalities (Shay et al., 1993; Artandi & DePinho, 2000; Murnane, 2006). Thus, telomere dysfunction serves as a mutational mechanism that drives cancer cell formation.
Telomere shortening can be counteracted by activation of a specialised enzyme named telomerase, which was first identified in the protozoan Tetrahymena (Greider & Blackburn, 1985). Telomerase is a ribonucleoprotein composed of a catalytic subunit called telomerase reverse transcriptase (TERT) and an RNA moiety known as telomerase RNA (TERC), which serves as the template for de novo synthesis of telomeric repeats (Feng et al., 1995; Shippen-Lentz & Blackburn, 1990; Nakamura et al., 1997; Autexier & Lue, 2006). The enzyme elongates the 3' end of the DNA, thus enabling other polymerases to synthesise the complementary strand (Autexier & Lue, 2006).
Figure 3: Telomere Extension by Telomerase. The telomeric repeats are synthesised through repeated cycles of elongation and translocation
A short segment within the TERC is repeatedly used to extend the telomere (Figure 3). Firstly, the DNA is recognised by telomerase and hybrid structure is formed between the 3' end of the DNA and the integral RNA template. DNA sequences upstream of this region interact with an anchor site. Next, nucleotides are sequentially added onto the 3' end of the DNA until the 5' end of the template is reached. The enzyme then translocates to 3' end of the newly synthesised DNA sequence and another reaction cycle is initiated. It is remains unclear how the process is halted, but telomere structure could be involved since folding of DNA into a special G-quartet configuration has been shown to prevent elongation (Zahler et al., 1991).
Telomerase & Immortalisation
Telomerase is active in about 80% of cancer cells (Kim et al., 1994, Shay & Bacchetti, 1997), suggesting that its acquisition is critical for tumour progression. These cells characteristically possess very short telomeres (Counter et al., 1994; Schmitt et al., 1994).
Most somatic cells do not display telomerase activity (Forsyth et al., 2002). However forced expression (transfection) of human TERT (hTERT) triggers enzyme activity and extends the replicative lifespan of the cells (Bodnar et al., 1998; Counter et al., 1998; Vaziri et al., 1998). The cells do not undergo changes associated with a malignant phenotype (Jiang et al., 1999), suggesting that telomerase expression alone does not cause tumours.
How telomerase is activated during crisis remains unknown, though amplification of the hTERT gene has been observed in some tumours (Janknecht, 2004; Takuma et al., 2004; Nowak et al., 2006). Another possibility is that the telomerase genes may be translocated alongside a strong promoter causing upregulation of the enzyme.
Alternative Lengthening Telomeres
A small minority of immortalised cells maintain telomere length by a telomerase-independent process termed alternative lengthening of telomeres (ALT) (Bryan et al., 1995; Bryan et al., 1997). These cells usually exhibit long, heterogonous telomeres (Bryan et al., 1995). ALT appears to be a recombination-based mechanism, in which one telomere is used as a template to extend another telomere (Dunham et al., 2000; Muntoni & Reddel, 2005). However, the precise process has not been fully elucidated. The prevalence of ALT ranges 5% to 15% in carcinomas, but can be as high as 60% in sarcomas (Henson & Reddel, 2010).
Possible Clinical Applications
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Investigations of telomeres and telomerase contribute to understanding of cell immortalisation in cancer. Given that, telomere length is often shorter in tumours (Hastie et al., 1990; Hackett & Greider, 2002) and telomerase is expressed in approximately 80% of malignant tumours (Kim et al., 1994), these factors could provide an excellent diagnostic and prognostic tool.
Telomerase may also serve as a target for cancer therapy. It has been predicted that inhibition of the enzyme would reinitiate telomere shortening, leading to chromosome instability and cell death (Shay et al., 2001). There would be fewer side effects, since most somatic cells lack the enzyme. The current approaches under investigation are immunotherapy, gene therapy and the use small molecular inhibitors (Shay & Keith, 2008).
BASIS OF TECHNIQUES USED
Telomere Length Measurement
High-quality, undegraded DNA was an essential requirement for this process. Isolation of DNA was derived from a procedure originally described by Blin and Stafford (1976). In the method, tissue samples were pulverised in the presence of liquid nitrogen to allow rapid homogenisation in lysis solution, whilst preserving DNA integrity. This temperature-lowering effect, which arrests nuclease activity and makes tissue brittle, can also be achieved by snap-freezing samples with dry ice (Graham, 1978).
SDS (sodium dodecyl sulfate) lyses cells by solubilising the lipid and protein components of the cell membrane (Sambrook & Russell, 2001). The detergent also stimulates the activity of proteinase K (Hilz et al., 1975), a protease secreted by the fungus, Tritirachium album (Ebeling et al. 1974), which inactivates endogenous nucleases (Wiegers & Hilz, 1971). Proteins are removed from the lysate by phenol. Kirby (1956, 1957) pioneered a two-phase extraction system, containing phenol and aqueous solutions, to purify nucleic acids. Although, this approach still forms the basis of current methods, mixtures of chloroform and phenol are now used, because high density of chloroform ensures phase separation (Sambrook & Russell, 2001). Isoamyl alcohol reduces foaming of the solution and ethanol precipitates the DNA (Sambrook & Russell, 2001).
Genomic DNA was digested using restriction endonucleases that do not cut within the 5'-TTAGGG-3' telomere sequences, namely Rsa I and Hinf I, but cleave other regions repeatedly. Rsa I cleaves between T and A of the 5'-GTAC-3' restriction site and Hinf I cuts between the G and A of the sequence 5'-GANTC-3' (Lynn et al., 1980; Gingeras et al., 1981). The combination of enzymes produces extremely short non-telomeric fragments and longer TRF fragments.
Southern Blot Analysis
Southern blotting involves the transfer of electrophorectically separated DNA fragments from the gel onto a (nylon) membrane by an upward stream of buffer (Figure 1) (Southern, 1975; Sambrook & Russell, 2001). The buffer migrates through the agarose gel towards a stack of paper towels by capillary action (Sambrook & Russell, 2001).
The DNA molecules are firstly partially depurinated with HCl and denatured with NaOH to facilitate rapid transfer from the gel before it becomes dehydrated (Evans, 1994; Sambrook & Russell, 2001). Once the fragments come into contact with the (positively charged) membrane, ionic bonds are formed. Labelled telomere probes that bind to the target sequence on the membrane are detected by enzyme-coupled monoclonal antibodies (Roche, 2007). On addition of the chemiluminescent substrate, the enzymatic activity causes emission of photons of light, which can be captured on X-ray film.
Detection of Telomerase Activity
The TRAP (telomeric repeat amplification protocol) assay was developed as a highly, sensitive PCR-based assay for detection of telomerase activity in clinical specimens (Kim et al., 1994). The rationale behind the technique is that if telomerase is present within the protein extract, it will add telomeric repeats to the substrate (primer). The resultant products are then PCR-amplified and analyzed by electrophoresis.
An extension of the original protocol replaces the electrophoresis step with a colorimetric-based immunoassay, known as ELISA (enzyme-linked immunosorbent assay), which provides semi-quantitation of telomerase activity (Wu et al., 2000). In ELISA, the PCR product is hybridised to labelled telomere-specific probe and immobilised on a coated microplate. Following, detection with an enzyme-conjugated antibody, the probe is visualised by virtue of substrate metabolism to form a coloured reaction product (Roche, 2007).
AIMS OF RESEARCH
Telomeres and telomerase have been strongly implicated in tumourigenesis. This purpose of this thesis is to explore the changes that occur during this process and the impact of telomeres and telomerase on disease progression.
The aims of research were as follows:
To compare the telomere lengths of tumours and their paired normal samples in order to assess the changes that take place during cellular transformation, and to see if tumour samples exhibit a characteristic telomere status (i.e. shortening, elongation or no change in length).
To detect and measure telomerase activity in tumours and their respective controls in order to prove the importance of activation of the enzyme in cancer.
To investigate the prognostic influence of telomerase expression and telomere status in human cancers.
SAFETY AND ETHICS
Personal protective equipment (PPE) (i.e. fastened laboratory coat, suitable footwear, goggles, masks and nitrile gloves) was worn at all times to prevent exposure to potential hazards. Certain procedures were performed under the fume hood to prevent inhalation of potentially harmful substances. The work area was wiped clean with ethanol before use and spillages were dealt with immediately. In addition, precaution was taken with glassware (e.g. pipettes).
Ethidium Bromide (EtBr) was used to visualise nucleic acid fragments in agarose gels. It is a powerful mutagen that is readily absorbed through the skin and is irritating to the eyes, skin, and upper respiratory tract (Handling 1995, p.310). Minimal quantities of ready-made EtBr stock solutions were utilised in order to minimise risk and avoid handling the (inhalable) powdered form. The agarose solution was sufficiently cooled prior to addition of EtBr to prevent generation of fumes and gloves and pipette tips used were disposed immediately after use into a biohazard box to prevent secondary contamination.
Phenol was used to remove proteins from the DNA samples during extraction. It is a corrosive and toxic substance that can easily permeate the skin, causing CNS depression and liver and kidney damage (Handling 1995, p.310). All work with phenol was conducted with extreme care while wearing impermeable gloves and other appropriate PPE.
UV light was used to fix DNA to the nylon membrane following Southern blot. Since UV rays can be harmful to unprotected eyes, face shields were worn during this procedure.
Care was taken when handling the human tissue samples given the potential risk of exposure to infectious diseases. All waste material was treated as a biohazard and disposed of accordingly.
The ethical considerations pertain to the use of human tissue in the investigation. All the research was given prior approval by the Ethic Committee of San Carlos Clinical Hospital (Madrid) and genuine informed consent (for the storage and use of the samples) was obtained from all the patients ahead of surgery. Confidentiality and privacy was upheld throughout the study and the samples were handled sensitively and responsibly.
MATERIALS AND METHODS
The tissue samples used in the study were acquired from the tumour bank at San Carlos Clinical Hospital (Madrid). Only tumours with at least 80% malignancy and no more than 10% necrosis were included for research. Before usage, the tissues were embedded in TissueTekÂ® (Sakura) and sliced in sections with a cryostat (Leica).
Measurements of Telomere Length
Telomere length was measured by Southern blot analysis of terminal restriction fragments (TRFs). The TeloTAGGG Telomere Length Assay (Roche) was used in this procedure.
The tumour sections and corresponding normal mucosa were powdered on dry ice using a cold spatula. 1.25ml of Solution 1 (Tris HCl 10mM, pH 7.5; EDTA 10mM; NaCl 15M) was added to the each reaction tube followed by 50Î¼l of proteinase K (10mg/ml) and 62.5Î¼l of 20% SDS (20%). The tubes were mixed by inversion and incubated for 3 hours at 65Â°C. Afterwards, 1.25ml of Solution 2 (Tris HCl 10mM, pH 7.5; EDTA 10mM; NaCl 0.65M) and 2.5ml of phenol were added to each lysate. The samples were shaken (using a vortex) and centrifuged at 4000 rpm for 15 minutes, at room temperature. Subsequently, the aqueous phases were transferred into sterile reaction tubes and a volume (equal to the amount collected) of chloroform: isoamyl alcohol (24:1) was added. Again, the samples were vortexed and centrifuged (for 5 minutes), and the aqueous phases were collected. The DNA was precipitated using 2 volumes of absolute ethanol and the extracts were stored overnight at -20Â°C. On the following day, the samples were centrifuged at 4000 rpm for 15 minutes, at 4Â°C. The supernatant was carefully discarded and the pellet was washed with 1ml of cold ethanol (70%). After another centrifugation, the supernatant was decanted and the DNA pellet was cautiously suspended in 100Î¼l of ultrapure sterile water. The samples were stored at 20Â°C until the next step. Note: Samples were kept in ice at throughout of the procedure.
Dilutions of 1 in 50 (2Î¼l of DNA: 98Î¼l of nuclease free water) were prepared and spectrophotometric readings at 260nm (DNA absorption) and 280nm (protein absorption) were obtained. The absorbance at 260nm was used to quantify the DNA and the 260/280 ratio was used to gauge purity. Gel electrophoresis was subsequently performed to check the integrity of the isolated DNA. A gel containing 0.8% agarose in 1x TBE (90mM Tris-borate, 2mM EDTA) was prepared and ethidium bromide added to visualise the bands. Each well was loaded with 1Î¼g of DNA, 2Î¼l of blue xylene loading buffer and nuclease free water. The gel was run for 30 minutes at 100 volts.
A volume of each sample corresponding to 2.5Î¼g of the extracted genomic DNA was made up to 17Î¼l with nuclease free water. 2Î¼l of 10x digestion buffer was subsequently added followed by 1Î¼l of the restriction enzyme mixture (20 U/Î¼l) containing Hinf I and Rsa I. A positive control with high-molecular weight telomeres was also produced. The samples were digested at 37Â°C for 2 hours. 4Î¼l of loading buffer was then added to each sample to stop the reaction.
The DNA fragments are separated by gel electrophoresis alongside digoxigenin (DIG) molecular weight markers. A gel containing 0.8% of agarose in 1x TAE buffer (40mM Tris-acetate, 1mM EDTA) was prepared and the gel was run at 83 volts for approximately 4 hours and 30 minutes. Note: Ethidium bromide was not added to the gel.
Following electrophoresis, the gel was soaked for 10 minutes in 0.25M HCl with gentle agitation on an orbital shaker. The HCl was removed and the gel was rinsed with distilled water. The gel was then submerged in denaturing solution (0.5M NaOH, 1.5M NaCl) and agitated for 30 minutes (2 x 15 minutes). The solution was decanted and the gel was again washed with water. Finally, the gel was treated with neutralisation solution (0.5M Tris-HCl, 3M NaCl) for 30 minutes (2 x 15 minutes) with constant shaking.
Prior to the transfer, the positively charged nylon membrane was cut to the exact size of the gel, activated for 7 minutes with autoclaved water and then wetted in 20x saline-sodium citrate (SCC) transfer buffer (3M NaCl, 0.3M sodium citrate). A large plastic tray holding a support was filled with the transfer buffer. SSC provides the high salt concentration required to transfer the DNA. A piece of 3MM paper (saturated with the 20x SSC) was cut and placed over the support so that each end was submerged in the buffer. The gel was then laid on the bridge and the membrane was placed on the surface of the gel. Two additional pieces of Whatman 3MM paper were cut and positioned on top of the membrane, along with a stack of tissue paper and a weight. The assembly (Figure 4) was left overnight.
Figure 4: Southern Blotting. Typical setup for upward capillary transfer of DNA from agarose gels (Sambrook & Russell, 2001)
Once the transfer was completed, the apparatus was dismantled and the DNA was fixed to the membrane by exposure to UV light (254nm) for 7 minutes, followed by heat treatment for 30 minutes at 80Â°C. The membrane was then washed with 2x SSC and introduced into a roller cylinder. 18ml of preheated prehybridisation solution was added and the cylinder was rotated in the hybridisation oven for 1 hour at 42Â°C. After this time, the solution was discarded and immediately replaced with 10ml of hybridisation solution containing the DIG-labelled probe. The roller cylinder was incubated for 3 hours at 42Â°C.
The membrane was transferred to a tray and washed with twice for 5 minutes at room temperature with Stringent Wash Buffer I (2x SSC, 0.1% SDS), which removes non-specific binding of the probe molecules. A further two agitated washes (2 x 15 minutes) were performed at 50Â°C with Stringent Wash Buffer II (0.2x SSC, 0.1% SDS), which allows only highly complementary sequences to remain bound. The membrane then was washed with 1x Washing Buffer at room temperature.
Subsequently, the membrane was incubated in 70 ml of blocking solution for 30 minutes. 7Î¼l of the DIG-antibody conjugated to alkaline phosphatase (anti-DIG-AP) was added directly to the solution (1:10000) and the membrane was incubated for a further 30 minutes. The antibody solution was discarded and the membrane was washed twice for 15 minutes with 1x washing buffer. The membrane was then equilibrated with for 4 minutes with 100ml of 1x detection buffer. Note: these steps were performed at room temperature, with gentle agitation.
Finally, the membrane was immersed in the CDP-Star chemiluminescent substrate solution (for alkaline phospohtase) for 15 seconds and dried with filter paper. The membrane was exposed to autoradiography film for an optimum time and these films were then scanned with the imaging densitometer (Model GS 700, Bio-Rad) and Quantity One (Bio-Rad) software. The TRF lengths were determined using image analysis software (Image Gauge v3.46, Fujifilm) (Figure 5).
Figure 5: Screenshot of Southern blot analysis using Image Gauge v3.46. The profile of each lane was divided into equal segments and the data pertaining to length of the TRF was extracted.
Determination of Telomerase Activity
Detection of telomerase activity was carried out using the TeloTAGGG Telomerase PCR ELISA (Roche) commercial kit.
200Î¼l of protein lysis reagent was added to eppendorf tubes containing the tissue sections. The mix was homogenised and the samples were placed in ice for 30 minutes. After centrifugation at 12,000 rpm for 20 minutes at 4°C and the supernatant from each sample was collected into two sterile tubes. The samples were stored at 80Â°C until use.
The Bradford (1976) method was used to determine protein concentration. This assay is based on the binding of proteins to Coomassie dye and the subsequent absorbance shift of the dye from 465nm to 595nm (Bradford, 1976; Sedmak & Grossberg, 1977). Dilutions of the samples were prepared depending on the amount of haemoglobin present (i.e. reddishness of the sample) and duplicates of each sample were added to cuvettes containing 1ml diluted Bradford reagent (800Î¼l water: 200Î¼l Bradford reagent). Absorbance was measured at 595nm.
TRAP (Telomeric Repeat Amplification Protocol)
A volume of each corresponding 10-15g of protein was put into PCR tubes. 25Î¼l of the reaction mixture (i.e. Tris buffer containing the biotin-labelled telomerase substrate, nucleotides, primers and Taq DNA polymerase) was then added and the final volume of 50Î¼l was amassed with nuclease free water. All samples were subjected to the PCR program (Figure 6) in the thermocycler.
Figure 6: PCR Program for TRAP
One positive control and two negative controls were also prepared. A cell extract of immortalised telomerase-expressing human kidney cells (1x103 cells/Î¼l) was used as the positive control. 3Î¼l of the positive control was added to 25Î¼l of the reaction mixture and 22Î¼l of water. For one of them, 1Î¼l of lysis reagent was added to 24Î¼l of water and made up to 50Î¼ with the reaction mixture. For the other one, 13Î¼l of the positive control was diluted in 7Î¼l of nuclease free water and incubated for 10 minutes at 85°C. After being cooled in ice for 5 minutes, 1Î¼l of RNase was added to degrade the RNA component of telomerase, thus inactivating the enzyme and the mixture was incubated for 30 minutes at 37°C. Finally, 25Î¼l of the reaction mixture and 15Î¼l of nuclease free water was added to the mix.
Detection by ELISA
5Î¼l of the amplified PCR product of each sample was transferred into sterile tubes and 20Î¼l of denaturation reagent was added. After 10 minutes at room temperature, 225Î¼l of hybridisation buffer (containing a DIG-labelled probe complementary to the telomeric sequence) was added and the mixture was shaken vigorously using a vortex. 100Î¼l of each sample was transferred into the streptavidin-coated wells of the ELISA plate. The plate was covered with an adhesive film to prevent evaporation and incubated for 2 hours at 37°C, with agitation (300 rpm). After incubation, the hybridisation solution was aspirated and each well was washed three times with 250Î¼l of 1x washing buffer to eliminate any non-specific binding. 100Î¼l of the polyclonal antibody (anti-DIG-peroxidase) was added to each well and the plate was agitated for 10 minutes at room temperature. The solution was removed and each well was washed five times with 250Î¼l of the washing buffer. Finally, 100Î¼l of the TMB (3, 3', 5, 5' - tetramethyl benzidine) substrate solution introduced and the plate was shaken for 20 minutes at room temperature. 100Î¼l of the stop reagent (containing 5% sulphuric acid) was added to the wells to halt the reaction. A colour change of blue to yellow was observed (Figure 7).
Figure 7: Colometric ELISA reaction. Colour change observed following addition of the TMB substrate.
The samples were analysed using a microplate reader (Bio-Rad). Absorbance was measured at 450mn against a blank (reference wavelength at 650nm). Samples were regarded as telomerase-positive if the difference in absorbance (Î”A) was higher than 0.2.
The measurements of telomere length were conducted in tumours and the related non-cancerous tissue of five patients suffering from gastric cancer. TRF smears representative of all the telomere lengths within each sample were produced (Figure 8) and the average telomere length (in kbp) was determined by measuring the signal intensity of each TRF smear in relation to the molecular weight marker.
Figure 8: Southern Blot Hybridization of Telomere Probe (Control; N - Normal; T - Tumour; MWM - Molecular Weight Marker)
The mean TRF length was estimated using the formula:
Mean TRF = Î£ (ODx x Lx) / Î£ (ODx)
Equation 1: Calculation of Mean TRF length, where: ODx is the signal intensity from the selected interval (x), and Lx is the length of TRF fragment at the selected interval (x), relative to the markers (Harley et al, 1990).
Two of the tumours exhibited telomere shortening with reductions of 29% and 44%. One tumour was 1.5 times greater than the corresponding normal sample and the other carcinoma showed no significant change in length (Table 1). Note: Sample 6T could not be assessed, because the TRF smear was too faint to interpret.
Table 1: Telomere Length (in kbp) and Telomere Length Status
Telomerase activity was investigated in six tumours of the lung and the respective control samples. Five tumours (83%) displayed positive telomerase activity whereas only one (17%) of normal samples was regarded as telomerase-positive (Table 2).
Table 2: Detection of Telomerase Activity in paired tumours (T) and normal tissues. Absorbance values > 0.2 were considered as telomerase-positive. Absorbance values > 0.5 were regarded as high telomerase activity.
Absorbance (450nm - 690nm)
Level of Telomerase Activity
Absorbance (450nm - 690nm)
Level of Telomerase Activity
Table 3: Absorbance Values for Controls. The manufacturer states the absorbance should be > 1.5 for the positive control and < 0.25 for the negative control.
Absorbance (450nm - 690nm)
In order to assess the prognostic implications of telomere status and telomerase activity in cancer, the survival data of selected reports published in this area were reviewed. Summaries of the results from these papers are outlined below.
NSCLC (Frias et al., 2008)
In an investigation of telomere status in 83 non-small cell lung cancers (NSCLC) (Frias et al., 2008), tumour recurrences were observed in 15% patients displaying telomere maintenance (i.e. T/N ratio â‰¥ 1). In contrast, nearly half (45%) of the patients showing telomeres shortening (i.e. T/N ratio < 1) relapsed.
Figure 9: Kaplan-Meier survival curves showing disease free survival time (DFS) in relation to telomere length status (Frias et al., 2008). The DFS period was calculated from the date of curative surgery until the time of relapse.
Figure 10:Kaplan-Meier survival curves showing disease free survival time (DFS) in relation to telomerase activity (Frias et al., 2008).
In the same study, data analysis revealed that tumours reappeared in 37% of telomerase-positive patients. None of the patients without telomerase activity relapsed (P = 0.04) (Figure 11).
Oesophageal Cancer (Hsu et al., 2005)
In a study of 74 specimens of oesophageal squamous cell carcinomas (Hsu et al., 2005), the 4-year cumulative survival rates of patients with lower T/N ratio (â‰¤ 0.85) and higher T/N ratio (> 0.85) were 38.7% and 15.7%, respectively (P = 0.1307). Telomerase-positive and telomerase-negative patients had 4-year survival rates of 35.8%, and 31.2%, respectively (P = 0.8442)
Liver Cancer (Oh et al., 2008)
Oh et al (2008) measured telomere length and telomerase activity in 49 hepatitis B virus (HBV)-related hepatocellular carcinomas (HCCs) and corresponding non-cancerous tissues. In HCCs with shortened telomeres (TRF T/N < 0.8), patients demonstrated a survival rate of 100%, regardless of telomerase activity. For HCCs with longer telomeres (TRF T/N > 0.8) and low (<0.5) or moderate (0.5 -1.5) telomerase activity, the survival rate was 80%. Patients with HCCs displaying long telomeres and high telomerase activity experienced a survival rate of only (55%) over the 3-year follow up period.
Multiple Myeloma (Wu et al., 2003)
In a study of 183 patients with multiple myeloma, cells with telomere activity 25% or greater than the levels of the neuroblastoma control (59/183) had a 2-year survival rate of 53% (31/59), whereas 82% (102/124) of patients exhibiting low activity levels survived (P = 0.0001). At the 1-year stage, the survival rate in patients with low telomerase activity levels and a telomere length > 5.5 kbp was 82%, while patients with higher telomerase activity and shorter telomere length had a survival rate of 63% (P = 0.004).
The data shows that telomere length status varies from one tumour to another. Two of the samples presented with telomere shortening, which suggests that these cells underwent extensive cell proliferation prior to immortalisation. One of the tumours exhibited telomere elongation, which could have resulted from high telomerase expression (overactivity) or an ALT mechanism. ALT cells characteristically have long heterogeneous telomeres (Bryan et al., 1995).
These results may be insufficient to draw any clear conclusions. However, previous results from the laboratory show similar variability.