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Techniques in gerontology: Cell lines as standards for telomere length and telomerase activity assessment
Experimental Gerontology 41 (2006) 648–651 www.elsevier.com/locate/expgero
Techniques in Aging Research
Techniques in gerontology: Cell lines as standards for telomere length and telomerase activity assessment Christine Fehrer a, Regina Voglauer b, Matthias Wieser b, Gerald Pfister a, Regina Brunauer a, Daniel Cioca a, Beatrix Grubeck-Loebenstein a, Gu¨nter Lepperdinger a,* b
a Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18B, 1190 Vienna, Austria
Received 21 November 2005; received in revised form 24 March 2006; accepted 27 March 2006 Available online 4 May 2006
Abstract The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging. q 2006 Elsevier Inc. All rights reserved. Keywords: Telomere; Telomerase; Aging; Cancer research; Standards; Molecular biology
1. Introduction During cell division, the telomeres are shortening because DNA polymerase is not fully replicating till the very end of the linear molecule (Chakhparonian and Wellinger, 2003). Hence, telomere length appears to be a good indicator of the mitotic history of a cell, reflecting the cumulative effect of the proliferative activity of an individual cell, both in vitro and in vivo (Davis et al., 2005; Effros et al., 2003; Von Zglinicki, 2003; Wallis et al., 2004). The loss of telomere repeats has been implicated in both tumorigenesis and aging (Hastings et al., 2004; Passos and von Zglinicki, 2005; Schmucker, 2005). One of the early methods to assess telomere length is Southern hybridisation (de Lange et al., 1990). Alternative techniques have been established such as slot-blot methods, hybridisation protection assays, a combination of fluorescence in situ hybridisation (FISH) and flow-cytometry (flow-FISH) or Abbreviations: PNA, peptide nucleic acid; FISH, fluorescence in situ hybridization; FBS, fetal bovine serum; PCI, phenol/chloroform/iso-amylalcohol; TRAP, telomeric repeat amplification protocol. * Corresponding author. Tel.: C43 512 5839 1940; fax: C43 512 5839 198. E-mail address: [email protected] (G. Lepperdinger). 0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.03.016
nuclear flow-FISH (Wieser et al., 2006), and, last but not least real-time PCR-based methods (Baird, 2005; Cawthon, 2002). A large number of commercial suppliers retail these analytical methods as easy-to-perform kits. In order to ascertain the exact number of telomere repeat units as well as to work out a relative estimate of the change in cellular telomerase activity, we experienced the lack of commonly available standards. We therefore evaluated cell lines that are either available from cell repositories or used by many laboratories. This short technical note is to propose cell lines, TCL1301, K562 and HEK293 as standards for routine use when monitoring telomere length as well as the corresponding telomerase activity in cells and tissues of interest. 2. Protocol 2.1. Cell culture and DNA preparation Reference curves of human K562 leukaemia cells (ATTC number CCL-243) (Akiyama et al., 2002; Derradji et al., 2005) with short telomeres of 6.5 kbp (Akiyama et al., 2002), TCL1301 (for further information and availability: http:// www.biotech.ist.unige.it/cldb/cl5401.html) (Larsson et al., 1979) with long telomeres of 27 kbp (Derradji et al., 2005) as well as HEK293 (ATTC CRL-1573) (Simmons, 1990) were
C. Fehrer et al. / Experimental Gerontology 41 (2006) 648–651
established by means of quantitative PCR and concomitantly confirmed by flow-FISH. TCL 1301 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine; HEK293 were cultured in MEM, 2 mM L-glutamine, 10% FBS and K562 cells in Iscove’s modified Dulbecco’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10% FBS. The cultures were incubated at 37 8C and 5% CO2 and care was taken to grow the cultures to saturation or confluence. Cells were harvested and re-suspended in 100 mM Tris–HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 mg/mL proteinaseK. The suspension was incubated at 55 8C for 2 h. Before phenol/chloroform/iso-amylalcohol (PCI) extraction, debris was removed by centrifugation at 10,000!g. PCI extraction was repeated at least twice in order to yield a clear aqueous phase and interphase. Before alcohol precipitation, the solution was extracted once with chloroform/iso-amylalcohol. Large amounts of precipitated DNA were removed using a pipet tip, otherwise the precipitate was centrifuged and the pellet washed in 70% ethanol. The moist pellet was dissolved in 10 mM Tris– HCl pH 7.5, 1 mM EDTA by incubation over night at 55 8C and the solution was stored at 4 8C.
649
coding for the single copy gene 36B4, or S number was used for calibration. Standard curves for telomere length and 36B4 amplification reactions were generated by plotting relative DNA concentration against Ct. The Ct of a DNA sample is the fractional number of PCR cycles to which the sample must be subjected in order to accumulate enough product to cross a set threshold of magnitude of fluorescent signal. After amplification of the single copy gene as well as of the telomeric repeat units, Ct values were plotted versus DNA concentration. These reference data were further validated using DNA from HEK293 cells, which revealed a mean telomere length of 5 kbp (Fig. 1A). We recommend to perform the evaluation
2.2. Quantitative PCR The quantitative PCR procedure used here is based on the technique published by Cawthon (2002). Measurement of telomeres by PCR was thought to be impossible, because primers that bind the repetitive telomere sequences TTAGGG and CCCTAA tightly, would form stable dimers and consequently induce self priming during PCR. Yet, this problem has been overcome by specifically designing primers to match this purpose. For detailed information on primer design, please see http://www.pnas.org/cgi/content/ full/0407162101/DC1/1 (Epel et al., 2004). The primers for the single copy gene 36B4 (acidic ribosomal phosphoprotein PO) which yield a 74 bp product, were 5 0 CAG CAA GTG GGA AGG TGT AAT CC 3 0 and 5 0 CCC ATT CTA TCA TCA ACG GGT ACA A 3 0 (final concentration 0.3 mM). The primers for the telomere PCR were 5 0 CGG TTT G (TTTGGG)5 TT 3 0 and 5 0 GGC TTG CC(TTACCC)5 T 3 0 , each used at a final concentration of 0.2 mM. PCR reactions with total DNA amount ranging from 180 ng to 247 pg were carried out using a Roche Light Cycler. Duplicate reactions with 5 mL of each DNA dilution were performed in a 10 mL volume using the Light Cycler Fast Start DNA Master SYBR Green I Kit (Roche), with MgCl2 added to a final concentration of 3 mM. The PCR conditions were as follows: the enzyme was first activated for 10 min at 95 8C. Next, the reaction for the single copy gene was 40 cycles at 95 8C for 5 s, 58 8C for 10 s and 72 8C for 40 s, and for the telomeres 30 cycles at 95 8C for 5 s, 56 8C for 10 s and 72 8C for 60 s, respectively. All transition rates were set to 20 8C/s except the annealing temperature for the determination of telomere repeats was set to 4 8C/s. In order to evaluate the telomeric repeat copies or T number of different DNA samples, the exact amount of genetic material
Fig. 1. (A) Telomere length assessment by quantitative PCR: mean telomere length was revealed by amplifying telomeric repeat regions and a short amplicon corresponding to the single copy gene 36B4 in parallel by means of quantitative PCR and further processing of the data as described in Section 2. When processing sample DNA in parallel to DNA derived from HEK293, K562 or TCL1301, the corresponding mean telomere length could be simply estimated using this graph as a reference curve. (B) Telomere length assessment by flow-FISH: relative telomere length of HEK293 (dashed line), K562 (full line) and TCL1301 (filled curve) were determined after hybridisation with a PNA probe and monitoring by flow cytometry (detector FL-4). (C) Relative telomerase activity of K562 and TCL1301 cells were determined as described in Section 2. Thousand or 500 cells of the standard cell lines K562 and TCL1301 were analysed in triplicates and the corresponding activity was expressed relative to the activity found in HEK293 cells.
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C. Fehrer et al. / Experimental Gerontology 41 (2006) 648–651
procedure on serially diluted DNA samples and fitting of the resulting Ct for S values to the respective reference curve. DNA samples diluted to a concentration, which yield Ct values for S ranging from 20 to 25 were best-suited for further quantitative analyses. Measurements should be carried out in duplicates of three different DNA concentrations. 2.3. Flow-FISH The described cell lines rapidly expand in culture and can therefore easily be used to measure telomere length by flowFISH. A potential drawback of this method is the integration of fluorescence intensity derived from PNA hybridisation leading to deceptive results, because diploid cells that contain the same number of telomeric repeats as their haploid counterpart display double the fluorescence signal after hybridisation. Therefore, without precise standards, this approach hardly yields quantitative measures. Cells were suspended in Ca2C/Mg2C-free PBS containing 0.1% BSA. For further analysis, a modified protocol based on (Hultdin et al., 1998) was used. 1!106 cells were centrifuged for 10 min at 500!g and the pellet was re-suspended in hybridisation buffer consisting of 70% deionised formamide, 0.1% BSA, 20 mM Tris–HCl, pH 7.0 (hybridisation buffer) and centrifuged again at 750!g for 10 min. Subsequently, the pellet was re-suspended in hybridisation buffer containing 0.3 mg/mL of allophycocyanin (APC)-conjugated PNA probe (Applied Biosystems). The mixture was first heated to 85 8C for 12 min, followed by a 2 min incubation on ice. Hybridisation was carried out for 90 min at 25 8C in the dark. Subsequently, the cellular suspension was centrifuged at 750! g for 10 min and washed twice with 70% formamide, 0.1% BSA, 0.1% Tween20, 10 mM Tris–HCl, pH 7.0 at 40 8C for 10 min. Eventually, the cells were re-suspended in PBS containing 0.1% BSA and 10 mg/mL DNase-free RNaseA. The suspension was subjected to flow cytometry using a FACS Calibur (BD Biosciences). Acquisition and analysis was performed using Cell Quest Pro (BD Biosciences). The mean fluorescence intensity (detector FL4) was used to estimate relative telomere length. The histogram representation of Fig. 1B refers relative telomere length to fluorescent intensity. Comparison of results derived from quantitative PCR and flowFISH showed equivalent measures for all three cell lines. 2.4. Telomerase activity The activation of telomerase in tumor cells or stem cells is believed to be responsible to maintain sufficient telomeric length. When telomerase activity is absent, telomeres progressively shorten with each cell division. This may lead to diminished cell viability as cells approach the limits of their replicative capacity. Since these aspects are most relevant in experimental gerontology, we also monitored telomerase activity in these cell lines. The resulting measures were referred to the level of activity detectable in HEK293 cells. Cell lysis for estimation of telomerase activity was performed as described earlier (Kim et al., 1994). Briefly,
cells were rinsed once with PBS followed by a wash in 10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl and 1 mM DTT. After centrifugation for 1 min at 10,000!g and 4 8C, the resulting cell pellet was resuspended in lysis buffer (4000 cells/mL) containing 10 mM Tris–HCl pH 7.5, 1 mM MgCl2 , 1 mM EGTA, 0.1 mM PMSF, 5 mM 2-mercaptoethanol, 10% glycerol, 0.5% CHAPS. The suspension was incubated for 30 min on ice. After centrifugation (16,000!g, 30 min, 4 8C), the supernatants were rapidly frozen at K80 8C. Telomerase activity was determined using a real-time telomeric repeat amplification protocol as described previously (Voglauer et al., 2005). The original TRAP-assay protocol was modified to meet modern real-time PCR technology, and therefore SYBRGreen I was added as a fluorescent dye for online measurement of newly synthesized telomeric repeats. Analysis was performed using various concentrations of cell extract and for standardization, an equivalent of 1, 10, 100 or 1000 HEK293 cells was applied. Lysis buffer and nuclease-free water as well as samples treated with RNAse (2 mL per sample) served as negative controls. A total volume of 20 mL per reaction containing 20 mM Tris–HCl pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 1 mM EGTA, 2.5 mM dNTPs, 5 ng/mL TS modIV primer (5 0 AAT CCG TCG AGA ACA GTT 3 0 ), 5 ng/mL Cxa primer (5 0 GTG TAA CCC TAA CCC TAA CCC 3 0 )(Falchetti et al., 1998), 0.3 mg/mL BSA, 0.005% Tween20, 0.002 units/mL TaqPolymerase and SYBR-Green I (1:20,000) were added and PCR was performed using a Rotor Gene 3000 Real-Time Cycler (Corbett Research) with a 36 Well Carousel High Speed Rotor. First, samples were incubated for 30 min at room temperature, subsequently denatured for 2 min at 95 8C and amplified during 45 cycles applying the following regimen: 10 s at 94 8C, 20 s at 50 8C and 1 min at 72 8C. The fluorescence intensity was monitored both at 72 and 65 8C. A standard curve from serial dilutions of HEK293 was drawn, representing the logarithmic increase in fluorescence versus cycle number. Standards were analyzed in duplicates, samples in triplicates and the resulting telomerase activities of the samples were expressed relative to that determined for HEK293 cells. K562 cells exhibited low levels of telomerase activity, i.e. less than 50% when compared to HEK293 cells. TCL1301 showed an increased level of telomerase activity that amounts to twice as much as found in HEK293 cells (Fig. 1C). In conclusion, HEK293, K562 and TCL1301 cells now represent well-characterized standards in order to gain comparable measures, when conducting assays for the precise determination of mean telomere length or telomerase activity. Acknowledgements G.L. is an APART fellow of the Austrian Academy of Sciences and supported by the Jubilee Fund of the Austrian National Bank (OeNB) and the Austrian Science Foundation (FWF NRN 093).
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References Akiyama, M., Yamada, O., Kanda, N., Akita, S., Kawano, T., Ohno, T., Mizoguchi, H., Eto, Y., Anderson, K.C., Yamada, H., 2002. Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors. Cancer Lett. 178, 187–197. Baird, D.M., 2005. New developments in telomere length analysis. Exp. Gerontol. 40, 363–368. Cawthon, R.M., 2002. Telomere measurement by quantitative PCR. Nucleic Acids. Res. 30, e47. Chakhparonian, M., Wellinger, R.J., 2003. Telomere maintenance and DNA replication: how closely are these two connected? Trends Genet. 19, 439– 446. Davis, T., Skinner, J.W., Faragher, R.G., Jones, C.J., Kipling, D., 2005. Replicative senescence in sheep fibroblasts is a p53 dependent process. Exp. Gerontol. 40, 17–26. de Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M., Varmus, H.E., 1990. Structure and variability of human chromosome ends. Mol. Cell Biol. 10, 518–527. Derradji, H., Bekaert, S., Van Oostveldt, P., Baatout, S., 2005. Comparison of different protocols for telomere length estimation by combination of quantitative fluorescence in situ hybridization (Q-FISH) and flow cytometry in human cancer cell lines. Anticancer Res. 25, 1039–1050. Effros, R.B., Dagarag, M., Valenzuela, H.F., 2003. In vitro senescence of immune cells. Exp. Gerontol. 38, 1243–1249. Epel, E.S., Blackburn, E.H., Lin, J., Dhabhar, F.S., Adler, N.E., Morrow, J.D., Cawthon, R.M., 2004. Accelerated telomere shortening in response to life stress. Proc. Natl Acad. Sci. USA 101, 17312–17315. Falchetti, M.L., Levi, A., Molinari, P., Verna, R., D’Ambrosio, E., 1998. Increased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction. Nucleic Acids Res. 26, 862–863.
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Hastings, R., Li, N.C., Lacy, P.S., Patel, H., Herbert, K.E., Stanley, A.G., Williams, B., 2004. Rapid telomere attrition in cardiac tissue of the ageing Wistar rat. Exp. Gerontol. 39, 855–857. Hultdin, M., Gronlund, E., Norrback, K., Eriksson-Lindstrom, E., Just, T., Roos, G., 1998. Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res. 26, 3651–3656. Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L., Shay, J.W., 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2015. Larsson, I., Lundgren, E., Nilsson, K., Strannegard, O., 1979. A human neoplastic hematopoietic cell line producing a fibroblast type of interferon. Dev. Biol. Stand. 42, 193–197. Passos, J.F., von Zglinicki, T., 2005. Mitochondria, telomeres and cell senescence. Exp. Gerontol. 40, 466–472. Schmucker, D.L., 2005. Age-related changes in liver structure and function: Implications for disease ? Exp. Gerontol. 40, 650–659. Simmons, N.L., 1990. A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. Exp. Physiol. 75, 309–319. Voglauer, R., Grillari, J., Fortschegger, K., Wieser, M., Sterovsky, T., Gunsberg, P., Katinger, H., Pfragner, R., 2005. Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region. Int. J. Oncol. 26, 961–970. Von Zglinicki, T., 2003. Replicative senescence and the art of counting. Exp. Gerontol. 38, 1259–1264. Wallis, C.V., Sheerin, A.N., Green, M.H., Jones, C.J., Kipling, D., Faragher, R.G., 2004. Fibroblast clones from patients with Hutchinson– Gilford progeria can senesce despite the presence of telomerase. Exp. Gerontol. 39, 461–467. Wieser, M., Stadler, G., Bohm, E., Borth, N., Katinger, H., Grillari, J., Voglauer, R., 2006. Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere length. Exp. Gerontol. 41 (2), 230–235.
Techniques in Aging Research
Techniques in gerontology: Cell lines as standards for telomere length and telomerase activity assessment Christine Fehrer a, Regina Voglauer b, Matthias Wieser b, Gerald Pfister a, Regina Brunauer a, Daniel Cioca a, Beatrix Grubeck-Loebenstein a, Gu¨nter Lepperdinger a,* b
a Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18B, 1190 Vienna, Austria
Received 21 November 2005; received in revised form 24 March 2006; accepted 27 March 2006 Available online 4 May 2006
Abstract The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging. q 2006 Elsevier Inc. All rights reserved. Keywords: Telomere; Telomerase; Aging; Cancer research; Standards; Molecular biology
1. Introduction During cell division, the telomeres are shortening because DNA polymerase is not fully replicating till the very end of the linear molecule (Chakhparonian and Wellinger, 2003). Hence, telomere length appears to be a good indicator of the mitotic history of a cell, reflecting the cumulative effect of the proliferative activity of an individual cell, both in vitro and in vivo (Davis et al., 2005; Effros et al., 2003; Von Zglinicki, 2003; Wallis et al., 2004). The loss of telomere repeats has been implicated in both tumorigenesis and aging (Hastings et al., 2004; Passos and von Zglinicki, 2005; Schmucker, 2005). One of the early methods to assess telomere length is Southern hybridisation (de Lange et al., 1990). Alternative techniques have been established such as slot-blot methods, hybridisation protection assays, a combination of fluorescence in situ hybridisation (FISH) and flow-cytometry (flow-FISH) or Abbreviations: PNA, peptide nucleic acid; FISH, fluorescence in situ hybridization; FBS, fetal bovine serum; PCI, phenol/chloroform/iso-amylalcohol; TRAP, telomeric repeat amplification protocol. * Corresponding author. Tel.: C43 512 5839 1940; fax: C43 512 5839 198. E-mail address: [email protected] (G. Lepperdinger). 0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.03.016
nuclear flow-FISH (Wieser et al., 2006), and, last but not least real-time PCR-based methods (Baird, 2005; Cawthon, 2002). A large number of commercial suppliers retail these analytical methods as easy-to-perform kits. In order to ascertain the exact number of telomere repeat units as well as to work out a relative estimate of the change in cellular telomerase activity, we experienced the lack of commonly available standards. We therefore evaluated cell lines that are either available from cell repositories or used by many laboratories. This short technical note is to propose cell lines, TCL1301, K562 and HEK293 as standards for routine use when monitoring telomere length as well as the corresponding telomerase activity in cells and tissues of interest. 2. Protocol 2.1. Cell culture and DNA preparation Reference curves of human K562 leukaemia cells (ATTC number CCL-243) (Akiyama et al., 2002; Derradji et al., 2005) with short telomeres of 6.5 kbp (Akiyama et al., 2002), TCL1301 (for further information and availability: http:// www.biotech.ist.unige.it/cldb/cl5401.html) (Larsson et al., 1979) with long telomeres of 27 kbp (Derradji et al., 2005) as well as HEK293 (ATTC CRL-1573) (Simmons, 1990) were
C. Fehrer et al. / Experimental Gerontology 41 (2006) 648–651
established by means of quantitative PCR and concomitantly confirmed by flow-FISH. TCL 1301 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine; HEK293 were cultured in MEM, 2 mM L-glutamine, 10% FBS and K562 cells in Iscove’s modified Dulbecco’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10% FBS. The cultures were incubated at 37 8C and 5% CO2 and care was taken to grow the cultures to saturation or confluence. Cells were harvested and re-suspended in 100 mM Tris–HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100 mg/mL proteinaseK. The suspension was incubated at 55 8C for 2 h. Before phenol/chloroform/iso-amylalcohol (PCI) extraction, debris was removed by centrifugation at 10,000!g. PCI extraction was repeated at least twice in order to yield a clear aqueous phase and interphase. Before alcohol precipitation, the solution was extracted once with chloroform/iso-amylalcohol. Large amounts of precipitated DNA were removed using a pipet tip, otherwise the precipitate was centrifuged and the pellet washed in 70% ethanol. The moist pellet was dissolved in 10 mM Tris– HCl pH 7.5, 1 mM EDTA by incubation over night at 55 8C and the solution was stored at 4 8C.
649
coding for the single copy gene 36B4, or S number was used for calibration. Standard curves for telomere length and 36B4 amplification reactions were generated by plotting relative DNA concentration against Ct. The Ct of a DNA sample is the fractional number of PCR cycles to which the sample must be subjected in order to accumulate enough product to cross a set threshold of magnitude of fluorescent signal. After amplification of the single copy gene as well as of the telomeric repeat units, Ct values were plotted versus DNA concentration. These reference data were further validated using DNA from HEK293 cells, which revealed a mean telomere length of 5 kbp (Fig. 1A). We recommend to perform the evaluation
2.2. Quantitative PCR The quantitative PCR procedure used here is based on the technique published by Cawthon (2002). Measurement of telomeres by PCR was thought to be impossible, because primers that bind the repetitive telomere sequences TTAGGG and CCCTAA tightly, would form stable dimers and consequently induce self priming during PCR. Yet, this problem has been overcome by specifically designing primers to match this purpose. For detailed information on primer design, please see http://www.pnas.org/cgi/content/ full/0407162101/DC1/1 (Epel et al., 2004). The primers for the single copy gene 36B4 (acidic ribosomal phosphoprotein PO) which yield a 74 bp product, were 5 0 CAG CAA GTG GGA AGG TGT AAT CC 3 0 and 5 0 CCC ATT CTA TCA TCA ACG GGT ACA A 3 0 (final concentration 0.3 mM). The primers for the telomere PCR were 5 0 CGG TTT G (TTTGGG)5 TT 3 0 and 5 0 GGC TTG CC(TTACCC)5 T 3 0 , each used at a final concentration of 0.2 mM. PCR reactions with total DNA amount ranging from 180 ng to 247 pg were carried out using a Roche Light Cycler. Duplicate reactions with 5 mL of each DNA dilution were performed in a 10 mL volume using the Light Cycler Fast Start DNA Master SYBR Green I Kit (Roche), with MgCl2 added to a final concentration of 3 mM. The PCR conditions were as follows: the enzyme was first activated for 10 min at 95 8C. Next, the reaction for the single copy gene was 40 cycles at 95 8C for 5 s, 58 8C for 10 s and 72 8C for 40 s, and for the telomeres 30 cycles at 95 8C for 5 s, 56 8C for 10 s and 72 8C for 60 s, respectively. All transition rates were set to 20 8C/s except the annealing temperature for the determination of telomere repeats was set to 4 8C/s. In order to evaluate the telomeric repeat copies or T number of different DNA samples, the exact amount of genetic material
Fig. 1. (A) Telomere length assessment by quantitative PCR: mean telomere length was revealed by amplifying telomeric repeat regions and a short amplicon corresponding to the single copy gene 36B4 in parallel by means of quantitative PCR and further processing of the data as described in Section 2. When processing sample DNA in parallel to DNA derived from HEK293, K562 or TCL1301, the corresponding mean telomere length could be simply estimated using this graph as a reference curve. (B) Telomere length assessment by flow-FISH: relative telomere length of HEK293 (dashed line), K562 (full line) and TCL1301 (filled curve) were determined after hybridisation with a PNA probe and monitoring by flow cytometry (detector FL-4). (C) Relative telomerase activity of K562 and TCL1301 cells were determined as described in Section 2. Thousand or 500 cells of the standard cell lines K562 and TCL1301 were analysed in triplicates and the corresponding activity was expressed relative to the activity found in HEK293 cells.
650
C. Fehrer et al. / Experimental Gerontology 41 (2006) 648–651
procedure on serially diluted DNA samples and fitting of the resulting Ct for S values to the respective reference curve. DNA samples diluted to a concentration, which yield Ct values for S ranging from 20 to 25 were best-suited for further quantitative analyses. Measurements should be carried out in duplicates of three different DNA concentrations. 2.3. Flow-FISH The described cell lines rapidly expand in culture and can therefore easily be used to measure telomere length by flowFISH. A potential drawback of this method is the integration of fluorescence intensity derived from PNA hybridisation leading to deceptive results, because diploid cells that contain the same number of telomeric repeats as their haploid counterpart display double the fluorescence signal after hybridisation. Therefore, without precise standards, this approach hardly yields quantitative measures. Cells were suspended in Ca2C/Mg2C-free PBS containing 0.1% BSA. For further analysis, a modified protocol based on (Hultdin et al., 1998) was used. 1!106 cells were centrifuged for 10 min at 500!g and the pellet was re-suspended in hybridisation buffer consisting of 70% deionised formamide, 0.1% BSA, 20 mM Tris–HCl, pH 7.0 (hybridisation buffer) and centrifuged again at 750!g for 10 min. Subsequently, the pellet was re-suspended in hybridisation buffer containing 0.3 mg/mL of allophycocyanin (APC)-conjugated PNA probe (Applied Biosystems). The mixture was first heated to 85 8C for 12 min, followed by a 2 min incubation on ice. Hybridisation was carried out for 90 min at 25 8C in the dark. Subsequently, the cellular suspension was centrifuged at 750! g for 10 min and washed twice with 70% formamide, 0.1% BSA, 0.1% Tween20, 10 mM Tris–HCl, pH 7.0 at 40 8C for 10 min. Eventually, the cells were re-suspended in PBS containing 0.1% BSA and 10 mg/mL DNase-free RNaseA. The suspension was subjected to flow cytometry using a FACS Calibur (BD Biosciences). Acquisition and analysis was performed using Cell Quest Pro (BD Biosciences). The mean fluorescence intensity (detector FL4) was used to estimate relative telomere length. The histogram representation of Fig. 1B refers relative telomere length to fluorescent intensity. Comparison of results derived from quantitative PCR and flowFISH showed equivalent measures for all three cell lines. 2.4. Telomerase activity The activation of telomerase in tumor cells or stem cells is believed to be responsible to maintain sufficient telomeric length. When telomerase activity is absent, telomeres progressively shorten with each cell division. This may lead to diminished cell viability as cells approach the limits of their replicative capacity. Since these aspects are most relevant in experimental gerontology, we also monitored telomerase activity in these cell lines. The resulting measures were referred to the level of activity detectable in HEK293 cells. Cell lysis for estimation of telomerase activity was performed as described earlier (Kim et al., 1994). Briefly,
cells were rinsed once with PBS followed by a wash in 10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl and 1 mM DTT. After centrifugation for 1 min at 10,000!g and 4 8C, the resulting cell pellet was resuspended in lysis buffer (4000 cells/mL) containing 10 mM Tris–HCl pH 7.5, 1 mM MgCl2 , 1 mM EGTA, 0.1 mM PMSF, 5 mM 2-mercaptoethanol, 10% glycerol, 0.5% CHAPS. The suspension was incubated for 30 min on ice. After centrifugation (16,000!g, 30 min, 4 8C), the supernatants were rapidly frozen at K80 8C. Telomerase activity was determined using a real-time telomeric repeat amplification protocol as described previously (Voglauer et al., 2005). The original TRAP-assay protocol was modified to meet modern real-time PCR technology, and therefore SYBRGreen I was added as a fluorescent dye for online measurement of newly synthesized telomeric repeats. Analysis was performed using various concentrations of cell extract and for standardization, an equivalent of 1, 10, 100 or 1000 HEK293 cells was applied. Lysis buffer and nuclease-free water as well as samples treated with RNAse (2 mL per sample) served as negative controls. A total volume of 20 mL per reaction containing 20 mM Tris–HCl pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 1 mM EGTA, 2.5 mM dNTPs, 5 ng/mL TS modIV primer (5 0 AAT CCG TCG AGA ACA GTT 3 0 ), 5 ng/mL Cxa primer (5 0 GTG TAA CCC TAA CCC TAA CCC 3 0 )(Falchetti et al., 1998), 0.3 mg/mL BSA, 0.005% Tween20, 0.002 units/mL TaqPolymerase and SYBR-Green I (1:20,000) were added and PCR was performed using a Rotor Gene 3000 Real-Time Cycler (Corbett Research) with a 36 Well Carousel High Speed Rotor. First, samples were incubated for 30 min at room temperature, subsequently denatured for 2 min at 95 8C and amplified during 45 cycles applying the following regimen: 10 s at 94 8C, 20 s at 50 8C and 1 min at 72 8C. The fluorescence intensity was monitored both at 72 and 65 8C. A standard curve from serial dilutions of HEK293 was drawn, representing the logarithmic increase in fluorescence versus cycle number. Standards were analyzed in duplicates, samples in triplicates and the resulting telomerase activities of the samples were expressed relative to that determined for HEK293 cells. K562 cells exhibited low levels of telomerase activity, i.e. less than 50% when compared to HEK293 cells. TCL1301 showed an increased level of telomerase activity that amounts to twice as much as found in HEK293 cells (Fig. 1C). In conclusion, HEK293, K562 and TCL1301 cells now represent well-characterized standards in order to gain comparable measures, when conducting assays for the precise determination of mean telomere length or telomerase activity. Acknowledgements G.L. is an APART fellow of the Austrian Academy of Sciences and supported by the Jubilee Fund of the Austrian National Bank (OeNB) and the Austrian Science Foundation (FWF NRN 093).
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