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The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors
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The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors
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Ying-Chieh Chen a, Fong-Chun Huang b, Jing-Jer Lin a,b,⇑
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a b
Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 3 March 2015 Accepted 17 March 2015 Available online xxxx Keywords: Telomerase TRAP PCR Inhibitor Anti-cancer
a b s t r a c t Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity. Ó 2015 Elsevier Inc. All rights reserved.
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Telomerase is a ribonucleoprotein that adds the TTAGGG repeat sequences to telomeres [1,2]. In human somatic cells, no or low telomerase activity is detected. Because of incomplete DNA replication of linear chromosomes, telomeres in these somatic cells become shorter after each cycle of cell division, eventually resulting in senescence. By contrast, telomerase activity is detected in 85 to 90% of cancer and immortal cells [3,4]. Telomerase extends telomeric DNA and maintains the proliferation capacity of these cancer cells. Given its involvement in maintaining telomere length in cancer cells, telomerase is considered a target for anticancer drug developments [5]. Two assays are commonly used to determine telomerase activity: the primer extension assay and the polymerase chain reaction (PCR)-based1 assay. The primer extension assay detects telomerase activity by incorporating radioactive deoxyguanosine triphosphate (dGTP) into the extension products. However, because the primer extension assay has lower sensitivity, it requires high levels of radioactive materials to enable the detection of extension products ⇑ Corresponding author at: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan. E-mail address: [email protected] (J.-J. Lin). 1 Abbreviations used: PCR, polymerase chain reaction; TRAP, telomeric repeat amplification protocol; sTRAP, spin column added TRAP; TS primer, 50 -AATCCGTCGAGCAGAGTT-30 ; CX primer, 50 -CCCTTACCCTTACCCTTACCCTAA-30 ; BRACO-19, N-[9[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino)acridine-3-yl]-3pyrrolidin-1-ylpropanamide; BMVC4, 3,6-bis(4-methyl-2-vinylpyrazinium iodine) carbazole; TMPyP4, 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine; dNTP, deoxynucleoside triphosphate.
[6]. The low sensitivity and the use of hazardous reagents are the main concerns for the primer extension assay. The PCR-based assay uses an additional PCR step to amplify the telomerase extension products [3]. The PCR-based assay, termed telomeric repeat amplification protocol (TRAP), has high sensitivity and does not require radioactive materials. TRAP provides a convenient and user-friendly tool to detect telomerase and is widely used in detecting telomerase activities from a variety of sources, including cultured cell lines and tissue biopsies. TRAP is also used to screen for and analyze compounds that inhibit telomerase activity. However, because TRAP requires two enzymatic reactions, telomerase extension and Taq polymerase amplification, its use in telomerase inhibitor studies must avoid off-target effects on Taq polymerase. Methods to minimize these nonspecific effects have been developed, mainly to evaluate the nonspecific effects of tested compounds toward Taq polymerase and to increase primer specificity [7]. Changing telomerase substrate sequences has also been reported to enhance the specificity of the telomerase reaction [7–9]. Evaluating the inhibitory effects of the PCR in parallel by adding the tested compounds prior to the PCR amplification step can also be performed [10]. However, none of these methods fully resolves the problem of nonspecific inhibition caused by targeting Taq polymerase. Removing inhibitors prior to PCR amplification appears to eliminate the off-target effects toward Taq polymerase. Protocols have been introduced to remove inhibitors after the telomerase extension reactions [11–13]. However, these additional steps do not appear to be satisfactory because they are time-consuming
http://dx.doi.org/10.1016/j.ab.2015.03.020 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020
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and might not be applicable to all types of inhibitors. Thus, a protocol that allows easy and reliable removal of inhibitors in the PCR amplification step is required. Here, we report a method to remove telomerase inhibitors after the telomerase extension step using a size exclusion spin column. The resulting protocol, termed spin column added TRAP (sTRAP), provides a better, reproducible, and accurate method to evaluate telomerase inhibitory effects by small molecular weight compounds.
Fig.1. Steps for spin column-added TRAP (sTRAP). sTRAP can be divided into three steps: telomere extension by telomerase, inhibitor removal by a Sephadex G25 spin column, and signal amplification by PCR. The key reagents for the steps are indicated.
Oligonucleotide primers TS (50 -AATCCGTCGAGCAGAGTT-30 ) and CX (50 -CCCTTACCCTTACCCTTACCCTAA-30 ) were synthesized and purified by MDBIO (Taipei, Taiwan). Compounds N-[9[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino) acridine-3-yl]-3-pyrrolidin-1-ylpropanamide (BRACO-19) and 3,6bis(4-methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) were gifts of from S. Ohnmacht (University College London, London, UK) and T-C. Chang (Academia Sinica, Taipei, Taiwan), respectively. Compound 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) was purchased from Sigma–Aldrich. Spectrophotometric analyses were used to determine the amounts of BRACO-19 (265 nm), BMVC4 (440 nm), and TMPyP4 (420 nm) in the reactions. Standard TS and CX DNA primers were used in our modified protocol [3]. The sTRAP has three steps. In the first step, 2 lg of telomerase-positive cell extracts was added to a 50-ll reaction mixture containing 0.2 lM TS primer and 0.2 lM deoxynucleoside triphosphate (dNTP) in TRAP buffer (20 mM Tris–HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM ethyleneglycoltetraacetic acid [EGTA], and 0.05% Tween 20). Varying amounts of telomerase inhibitors were also added to the mixture. The reaction mixtures were incubated at 30 °C for 30 min to extend telomeric DNA sequences on the TS primer. In the second step, inhibitors were removed from the telomere-extended products by applying the extended products directly on a G-25 spin column (cat. no. CG025, Geneaid, Taiwan), followed by centrifugation at 1.5g for 3 min. The third step was performed by mixing 30 ll of the G-25 flow-through with 0.2 lM CX primer, 0.2 lM dNTP, and 2 U of Taq polymerase in 1 TRAP buffer at a 50-ll final volume. PCRs were conducted by first denaturing the
Fig.2. Optimization of PCR cycles and validation of inhibitor removal by sTRAP. (A) Determining the PCR cycles in sTRAP. Telomerase-positive cell extracts from H1299 cancer cells were incubated with TS primer at 30 °C for 30 min. The reaction products were then directly amplified by Taq polymerase with 25, 30, 35, or 40 PCR cycles (left). Aliquots of the reaction products were passed through a Sephadex G25 (middle) or G50 (right) spin column and then amplified using 25, 30, 35, or 40 PCR cycles. The amplified products were then separated by a 10% polyacrylamide gel and stained with SYBER Green. The positions of telomere ladders are indicated. (B) Removal of TMPyP4 by a Sephadex G25 spin column. TMPyP4 at concentrations of 40, 10, 2.5, 0.62, and 0.15 lM was added to the telomerase extension reaction and incubated at 30 °C for 30 min. The extension products were then passed through a Sephadex G25 column, and the levels of TMPyP4 in the eluates were measured by a spectrophotometer using absorption at 420 nm. (C) Inhibition of telomerase activity by TMPyP4, BRACO-19, and BMVC4.
Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020
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reaction mixtures at 94 °C for 5 min, followed by 33 cycles of 94 °C for 30 s, 57.4 °C for 45 s, and 72 °C for 45 s. The reactions were completed by incubating at 72 °C for another 7 min and then cooled to 4 °C. The reaction products were resolved using 10% polyacrylamide gel electrophoresis and stained by SYBER Green (Invitrogen). Extension products were quantified using Image Lab software (Bio-Rad). In all described experiments, total cell lysates derived from lung cancer cell line H1299 cells were used as a source for telomerase. Protein concentrations of the lysates were determined by a Bio-Rad assay kit using bovine serum albumin as a standard. Given that the sizes of the substrate TS primer ( 6 kDa, 18mer) and the telomerase extended products are generally larger than small molecular weight compounds (generally with sizes < 1 kDa), we considered that the addition of a size exclusion spin column after the telomere extension step should effectively remove inhibitors. Thus, the newly established protocol added a spin column step between the telomere extension and PCR amplification reactions (Fig. 1). Two commercially available gel filtration desalting columns, Sephadex G25 and G50, were applied in the analysis. Standard telomerase extension reactions were conducted, and the telomerase-extended products were then amplified directly or passed through a Sephadex G25 or G50 spin column prior to PCR amplification. As shown in Fig. 2A, standard TRAP-detected telomerase products required approximately 25 to 30 cycles of PCR amplification. With the addition of the spin column in sTRAP, clear telomere ladders were visible after 30 PCR cycles when Sephadex G25 was used and a greater number of PCR cycles were required for Sephadex G50 column. The results suggested that telomerase extension products were not fully recovered from either Sephadex G25 or G50 columns. We estimated that 10% and less than 1% of the extended products were recovered using Sephadex G25 and G50 columns, respectively. Thus, additional cycles were applied in sTRAP. We used 33 PCR cycles in sTRAP because the amplified signal was clearly visible and the resulting products were within the linear amplification range. The results also demonstrated that the Sephadex G25 column performed better in this new protocol. We next evaluated whether Sephadex G25 can effectively remove small molecule weight compounds. TMPyP4 is a wellcharacterized telomerase inhibitor with a porphyrin core and a molecular weight of 1364 [14]. Using the absorbance of TMPyP4 at 420 nm as a criterion, we determined the residual compounds after passing through Sephadex G25. As shown in Fig. 2B, no TMPyP4 was detected after Sephadex G25 spin column filtration, indicating that most of the added TMPyP4 was removed by this step (Fig. 2B). Notably, the protocol effectively removed up to 40 lM TMPyP4 from the reaction, a dose that is higher than that used in most drug screening protocols. Together, these results demonstrated that the addition of a Sephadex G25 spin column effectively removes small molecular weight compounds from reactions without greatly affecting PCR amplification efficiency. A potential source affecting the reproducibility of sTRAP is the low recovery efficiency of telomerase extension products by the Sephadex G25 column. In our protocol, we estimated that 10% of the telomerase extended products were recovered. Despite this low efficiency, the recovery was consistent between experiments. This low recovery of telomerase extension products can be overcome by increasing the number of PCR cycles. The PCR amplification step is increased to 33 cycles in sTRAP compared with 27 to 30 cycles in standard TRAP. We also acquired spin columns from GE
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Healthcare and obtained similar results, suggesting that the sources of the Sephadex G25 spin columns do not appear to affect the assay. To evaluate whether the new protocol could accurately determine the inhibitory effects on telomerase, the inhibition of telomerase activity by TMPyP4, BRACO-19, and BMVC4 was determined using sTRAP (Fig. 2C). For all three compounds, the IC50 values determined by the standard TRAP conditions were similar to those determined when they were added after the telomerase extension step. The results suggested that the inhibitory effects observed were because of the inhibition of Taq polymerase. Using sTRAP, we obtained inhibitory values similar to those observed using the primer extension method. Because inhibition analysis using primer extension assays is considered to be more accurate and reliable, we conclude that sTRAP provides a better estimation of telomerase inhibitory activity than standard TRAP. In summary, we have established a spin column-added TRAP that enables the evaluation of telomerase inhibitors. The protocol avoids the disadvantage of using high levels of radioactivity in standard TRAP, is convenient to set up, and can be run in a routine manner in most laboratory settings.
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Acknowledgment
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This work was supported by the Ministry of Science and Technology under grants 100-2311-B-002-016 and 100-2311-B010-001.
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References
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[1] R.K. Moyzis, J.M. Buckingham, S. Cram, M. Dani, L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, J.-R. Wu, A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 6622–6626. [2] C. Autexier, N.F. Lue, The structure and function of telomerase reverse transcriptase, Annu. Rev. Biochem. 75 (2006) 493–517. [3] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L.C. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Specific association of human telomerase activity with immortal cells and cancer, Science 266 (1994) 2011– 2015. [4] J.W. Shay, S. Bacchetti, A survey of telomerase activity in human cancer, Eur. J. Cancer 33 (1997) 787–791. [5] J.W. Shay, W.E. Wright, Telomerase therapeutics for cancer: challenges and new directions, Nat. Rev. Drug Discov. 5 (2006) 577–584. [6] G.B. Morin, The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats, Cell 59 (1989) 521–529. [7] N.W. Kim, F. Wu, Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP), Nucleic Acids Res. 25 (1997) 2595–2597. [8] W.E. Wright, J.W. Shay, M.A. Piatyszek, Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity, and sensitivity, Nucleic Acids Res. 23 (1995) 3794–3795. [9] K. Piotrowska, E. Kleideiter, T.E. Murdter, C. Taetz, C. Baldes, U. Schaefer, C.M. Lehr, U. Klotz, Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors, Lab. Invest. 85 (2005) 1565–1569. [10] A. De Cian, G. Cristofari, P. Reichenbach, E. De Lemos, D. Monchaud, M.-P. Teulade-Fichou, K. Shin-ya, L. Lacroix, J. Lingner, J.-L. Mergny, Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 17347–17352. [11] L. Rossetti, M. Franceschin, A. Bianco, G. Ortaggi, M. Savino, Perylenediimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase, Bioorg. Med. Chem. Lett. 12 (2002) 2527–2533. [12] S.D. Patel, M. Isalan, G. Gavory, S. Ladame, Y. Choo, S. Balasubramanian, Inhibition of human telomerase activity by an engineered zinc finger protein that binds G-quadruplexes, Biochemistry 43 (2004) 13452–13458. [13] R. Reed, M. Gunaratnam, M. Beltran, A.P. Reszka, V. Ramon, S. Neidle, TRAP– LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules, Anal. Biochem. 380 (2008) 99–105. [14] R.T. Wheelhouse, D. Sun, H. Han, F.X. Han, L.H. Hurley, Cationic porphyrins as telomerase inhibitors: the interaction of tetra-(N-methyl-4-pyridyl)porphine with quadruplex DNA, J. Am. Chem. Soc. 120 (1998) 3261–3262.
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Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020
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31 March 2015 Analytical Biochemistry xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors
8
Ying-Chieh Chen a, Fong-Chun Huang b, Jing-Jer Lin a,b,⇑
9 10
11 1 5 3 2 14 15 16 17 18 19 20 21 22 23 24
a b
Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 3 March 2015 Accepted 17 March 2015 Available online xxxx Keywords: Telomerase TRAP PCR Inhibitor Anti-cancer
a b s t r a c t Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity. Ó 2015 Elsevier Inc. All rights reserved.
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Telomerase is a ribonucleoprotein that adds the TTAGGG repeat sequences to telomeres [1,2]. In human somatic cells, no or low telomerase activity is detected. Because of incomplete DNA replication of linear chromosomes, telomeres in these somatic cells become shorter after each cycle of cell division, eventually resulting in senescence. By contrast, telomerase activity is detected in 85 to 90% of cancer and immortal cells [3,4]. Telomerase extends telomeric DNA and maintains the proliferation capacity of these cancer cells. Given its involvement in maintaining telomere length in cancer cells, telomerase is considered a target for anticancer drug developments [5]. Two assays are commonly used to determine telomerase activity: the primer extension assay and the polymerase chain reaction (PCR)-based1 assay. The primer extension assay detects telomerase activity by incorporating radioactive deoxyguanosine triphosphate (dGTP) into the extension products. However, because the primer extension assay has lower sensitivity, it requires high levels of radioactive materials to enable the detection of extension products ⇑ Corresponding author at: Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan. E-mail address: [email protected] (J.-J. Lin). 1 Abbreviations used: PCR, polymerase chain reaction; TRAP, telomeric repeat amplification protocol; sTRAP, spin column added TRAP; TS primer, 50 -AATCCGTCGAGCAGAGTT-30 ; CX primer, 50 -CCCTTACCCTTACCCTTACCCTAA-30 ; BRACO-19, N-[9[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino)acridine-3-yl]-3pyrrolidin-1-ylpropanamide; BMVC4, 3,6-bis(4-methyl-2-vinylpyrazinium iodine) carbazole; TMPyP4, 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine; dNTP, deoxynucleoside triphosphate.
[6]. The low sensitivity and the use of hazardous reagents are the main concerns for the primer extension assay. The PCR-based assay uses an additional PCR step to amplify the telomerase extension products [3]. The PCR-based assay, termed telomeric repeat amplification protocol (TRAP), has high sensitivity and does not require radioactive materials. TRAP provides a convenient and user-friendly tool to detect telomerase and is widely used in detecting telomerase activities from a variety of sources, including cultured cell lines and tissue biopsies. TRAP is also used to screen for and analyze compounds that inhibit telomerase activity. However, because TRAP requires two enzymatic reactions, telomerase extension and Taq polymerase amplification, its use in telomerase inhibitor studies must avoid off-target effects on Taq polymerase. Methods to minimize these nonspecific effects have been developed, mainly to evaluate the nonspecific effects of tested compounds toward Taq polymerase and to increase primer specificity [7]. Changing telomerase substrate sequences has also been reported to enhance the specificity of the telomerase reaction [7–9]. Evaluating the inhibitory effects of the PCR in parallel by adding the tested compounds prior to the PCR amplification step can also be performed [10]. However, none of these methods fully resolves the problem of nonspecific inhibition caused by targeting Taq polymerase. Removing inhibitors prior to PCR amplification appears to eliminate the off-target effects toward Taq polymerase. Protocols have been introduced to remove inhibitors after the telomerase extension reactions [11–13]. However, these additional steps do not appear to be satisfactory because they are time-consuming
http://dx.doi.org/10.1016/j.ab.2015.03.020 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020
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and might not be applicable to all types of inhibitors. Thus, a protocol that allows easy and reliable removal of inhibitors in the PCR amplification step is required. Here, we report a method to remove telomerase inhibitors after the telomerase extension step using a size exclusion spin column. The resulting protocol, termed spin column added TRAP (sTRAP), provides a better, reproducible, and accurate method to evaluate telomerase inhibitory effects by small molecular weight compounds.
Fig.1. Steps for spin column-added TRAP (sTRAP). sTRAP can be divided into three steps: telomere extension by telomerase, inhibitor removal by a Sephadex G25 spin column, and signal amplification by PCR. The key reagents for the steps are indicated.
Oligonucleotide primers TS (50 -AATCCGTCGAGCAGAGTT-30 ) and CX (50 -CCCTTACCCTTACCCTTACCCTAA-30 ) were synthesized and purified by MDBIO (Taipei, Taiwan). Compounds N-[9[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino) acridine-3-yl]-3-pyrrolidin-1-ylpropanamide (BRACO-19) and 3,6bis(4-methyl-2-vinylpyrazinium iodine) carbazole (BMVC4) were gifts of from S. Ohnmacht (University College London, London, UK) and T-C. Chang (Academia Sinica, Taipei, Taiwan), respectively. Compound 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) was purchased from Sigma–Aldrich. Spectrophotometric analyses were used to determine the amounts of BRACO-19 (265 nm), BMVC4 (440 nm), and TMPyP4 (420 nm) in the reactions. Standard TS and CX DNA primers were used in our modified protocol [3]. The sTRAP has three steps. In the first step, 2 lg of telomerase-positive cell extracts was added to a 50-ll reaction mixture containing 0.2 lM TS primer and 0.2 lM deoxynucleoside triphosphate (dNTP) in TRAP buffer (20 mM Tris–HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl2, 1 mM ethyleneglycoltetraacetic acid [EGTA], and 0.05% Tween 20). Varying amounts of telomerase inhibitors were also added to the mixture. The reaction mixtures were incubated at 30 °C for 30 min to extend telomeric DNA sequences on the TS primer. In the second step, inhibitors were removed from the telomere-extended products by applying the extended products directly on a G-25 spin column (cat. no. CG025, Geneaid, Taiwan), followed by centrifugation at 1.5g for 3 min. The third step was performed by mixing 30 ll of the G-25 flow-through with 0.2 lM CX primer, 0.2 lM dNTP, and 2 U of Taq polymerase in 1 TRAP buffer at a 50-ll final volume. PCRs were conducted by first denaturing the
Fig.2. Optimization of PCR cycles and validation of inhibitor removal by sTRAP. (A) Determining the PCR cycles in sTRAP. Telomerase-positive cell extracts from H1299 cancer cells were incubated with TS primer at 30 °C for 30 min. The reaction products were then directly amplified by Taq polymerase with 25, 30, 35, or 40 PCR cycles (left). Aliquots of the reaction products were passed through a Sephadex G25 (middle) or G50 (right) spin column and then amplified using 25, 30, 35, or 40 PCR cycles. The amplified products were then separated by a 10% polyacrylamide gel and stained with SYBER Green. The positions of telomere ladders are indicated. (B) Removal of TMPyP4 by a Sephadex G25 spin column. TMPyP4 at concentrations of 40, 10, 2.5, 0.62, and 0.15 lM was added to the telomerase extension reaction and incubated at 30 °C for 30 min. The extension products were then passed through a Sephadex G25 column, and the levels of TMPyP4 in the eluates were measured by a spectrophotometer using absorption at 420 nm. (C) Inhibition of telomerase activity by TMPyP4, BRACO-19, and BMVC4.
Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020
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reaction mixtures at 94 °C for 5 min, followed by 33 cycles of 94 °C for 30 s, 57.4 °C for 45 s, and 72 °C for 45 s. The reactions were completed by incubating at 72 °C for another 7 min and then cooled to 4 °C. The reaction products were resolved using 10% polyacrylamide gel electrophoresis and stained by SYBER Green (Invitrogen). Extension products were quantified using Image Lab software (Bio-Rad). In all described experiments, total cell lysates derived from lung cancer cell line H1299 cells were used as a source for telomerase. Protein concentrations of the lysates were determined by a Bio-Rad assay kit using bovine serum albumin as a standard. Given that the sizes of the substrate TS primer ( 6 kDa, 18mer) and the telomerase extended products are generally larger than small molecular weight compounds (generally with sizes < 1 kDa), we considered that the addition of a size exclusion spin column after the telomere extension step should effectively remove inhibitors. Thus, the newly established protocol added a spin column step between the telomere extension and PCR amplification reactions (Fig. 1). Two commercially available gel filtration desalting columns, Sephadex G25 and G50, were applied in the analysis. Standard telomerase extension reactions were conducted, and the telomerase-extended products were then amplified directly or passed through a Sephadex G25 or G50 spin column prior to PCR amplification. As shown in Fig. 2A, standard TRAP-detected telomerase products required approximately 25 to 30 cycles of PCR amplification. With the addition of the spin column in sTRAP, clear telomere ladders were visible after 30 PCR cycles when Sephadex G25 was used and a greater number of PCR cycles were required for Sephadex G50 column. The results suggested that telomerase extension products were not fully recovered from either Sephadex G25 or G50 columns. We estimated that 10% and less than 1% of the extended products were recovered using Sephadex G25 and G50 columns, respectively. Thus, additional cycles were applied in sTRAP. We used 33 PCR cycles in sTRAP because the amplified signal was clearly visible and the resulting products were within the linear amplification range. The results also demonstrated that the Sephadex G25 column performed better in this new protocol. We next evaluated whether Sephadex G25 can effectively remove small molecule weight compounds. TMPyP4 is a wellcharacterized telomerase inhibitor with a porphyrin core and a molecular weight of 1364 [14]. Using the absorbance of TMPyP4 at 420 nm as a criterion, we determined the residual compounds after passing through Sephadex G25. As shown in Fig. 2B, no TMPyP4 was detected after Sephadex G25 spin column filtration, indicating that most of the added TMPyP4 was removed by this step (Fig. 2B). Notably, the protocol effectively removed up to 40 lM TMPyP4 from the reaction, a dose that is higher than that used in most drug screening protocols. Together, these results demonstrated that the addition of a Sephadex G25 spin column effectively removes small molecular weight compounds from reactions without greatly affecting PCR amplification efficiency. A potential source affecting the reproducibility of sTRAP is the low recovery efficiency of telomerase extension products by the Sephadex G25 column. In our protocol, we estimated that 10% of the telomerase extended products were recovered. Despite this low efficiency, the recovery was consistent between experiments. This low recovery of telomerase extension products can be overcome by increasing the number of PCR cycles. The PCR amplification step is increased to 33 cycles in sTRAP compared with 27 to 30 cycles in standard TRAP. We also acquired spin columns from GE
3
Healthcare and obtained similar results, suggesting that the sources of the Sephadex G25 spin columns do not appear to affect the assay. To evaluate whether the new protocol could accurately determine the inhibitory effects on telomerase, the inhibition of telomerase activity by TMPyP4, BRACO-19, and BMVC4 was determined using sTRAP (Fig. 2C). For all three compounds, the IC50 values determined by the standard TRAP conditions were similar to those determined when they were added after the telomerase extension step. The results suggested that the inhibitory effects observed were because of the inhibition of Taq polymerase. Using sTRAP, we obtained inhibitory values similar to those observed using the primer extension method. Because inhibition analysis using primer extension assays is considered to be more accurate and reliable, we conclude that sTRAP provides a better estimation of telomerase inhibitory activity than standard TRAP. In summary, we have established a spin column-added TRAP that enables the evaluation of telomerase inhibitors. The protocol avoids the disadvantage of using high levels of radioactivity in standard TRAP, is convenient to set up, and can be run in a routine manner in most laboratory settings.
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Acknowledgment
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This work was supported by the Ministry of Science and Technology under grants 100-2311-B-002-016 and 100-2311-B010-001.
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References
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[1] R.K. Moyzis, J.M. Buckingham, S. Cram, M. Dani, L. Deaven, M.D. Jones, J. Meyne, R.L. Ratliff, J.-R. Wu, A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 6622–6626. [2] C. Autexier, N.F. Lue, The structure and function of telomerase reverse transcriptase, Annu. Rev. Biochem. 75 (2006) 493–517. [3] N.W. Kim, M.A. Piatyszek, K.R. Prowse, C.B. Harley, M.D. West, P.L.C. Ho, G.M. Coviello, W.E. Wright, S.L. Weinrich, J.W. Shay, Specific association of human telomerase activity with immortal cells and cancer, Science 266 (1994) 2011– 2015. [4] J.W. Shay, S. Bacchetti, A survey of telomerase activity in human cancer, Eur. J. Cancer 33 (1997) 787–791. [5] J.W. Shay, W.E. Wright, Telomerase therapeutics for cancer: challenges and new directions, Nat. Rev. Drug Discov. 5 (2006) 577–584. [6] G.B. Morin, The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats, Cell 59 (1989) 521–529. [7] N.W. Kim, F. Wu, Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP), Nucleic Acids Res. 25 (1997) 2595–2597. [8] W.E. Wright, J.W. Shay, M.A. Piatyszek, Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity, and sensitivity, Nucleic Acids Res. 23 (1995) 3794–3795. [9] K. Piotrowska, E. Kleideiter, T.E. Murdter, C. Taetz, C. Baldes, U. Schaefer, C.M. Lehr, U. Klotz, Optimization of the TRAP assay to evaluate specificity of telomerase inhibitors, Lab. Invest. 85 (2005) 1565–1569. [10] A. De Cian, G. Cristofari, P. Reichenbach, E. De Lemos, D. Monchaud, M.-P. Teulade-Fichou, K. Shin-ya, L. Lacroix, J. Lingner, J.-L. Mergny, Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action, Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 17347–17352. [11] L. Rossetti, M. Franceschin, A. Bianco, G. Ortaggi, M. Savino, Perylenediimides with different side chains are selective in inducing different G-quadruplex DNA structures and in inhibiting telomerase, Bioorg. Med. Chem. Lett. 12 (2002) 2527–2533. [12] S.D. Patel, M. Isalan, G. Gavory, S. Ladame, Y. Choo, S. Balasubramanian, Inhibition of human telomerase activity by an engineered zinc finger protein that binds G-quadruplexes, Biochemistry 43 (2004) 13452–13458. [13] R. Reed, M. Gunaratnam, M. Beltran, A.P. Reszka, V. Ramon, S. Neidle, TRAP– LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules, Anal. Biochem. 380 (2008) 99–105. [14] R.T. Wheelhouse, D. Sun, H. Han, F.X. Han, L.H. Hurley, Cationic porphyrins as telomerase inhibitors: the interaction of tetra-(N-methyl-4-pyridyl)porphine with quadruplex DNA, J. Am. Chem. Soc. 120 (1998) 3261–3262.
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Please cite this article in press as: Y.-C. Chen et al., The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.03.020