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Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay
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Chinese Chemical Letters 19 (2008) 699–702 www.elsevier.com/locate/cclet
Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay Xiao Ming Zhou *, Li Jia MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China Received 25 December 2007
Abstract A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol (TRAP) and magnetic beads based electrochemiluminescence (ECL) assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer (B-TS) and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and tris-(20 20 -bipyridyl) ruthenium (TBR) labeled ACX (TBR-ACX) as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples. # 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Telomerase activity; TRAP; Electrochemiluminescence; Cancer diagnosis
Telomerase, a ribonucleoprotein enzyme that adds telomeric repeats to the 30 end of chromosomal DNA for maintaining chromosomal integrity and stability [1]. Most somatic cells undergo a steady rate of telomere loss and do not possess detectable telomerase activity. However, in the great majority of tumors cells telomerase expressed with high levels [2]. The strong association of telomerase activity with tumors establishing it is the most widespread cancer marker [3]. TRAP is a standard method for telomerase activity analysis [3]. This PCR-based method to detect telomerase activity enabled exponential amplification of the primer-telomeric repeats generated in the telomerase reaction, and the resulting improvements dramatically increased the efficiency and sensitivity of telomerase activity detection. However, like in other conventional electrophoresis-based PCR methods, inherent problems exist in the conventional TRAP assay using a densitometry to quantitate telomerase activity. For example, the limited dynamic range, as well as end-point detection of the PCR product, makes the accurate measurement of telomerase activity difficult. Moreover, the post-PCR processing is time-consuming and adds further variables during analysis of the PCR products. Alternatives for post-amplification detection have been reported, such as TRAP-scintillation proximity assay (SPA)
* Corresponding author. E-mail address: [email protected] (X.M. Zhou). 1001-8417/$ – see front matter # 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.04.017
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X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
[4], TRAP-enzyme immunoassay (EIA) [5], TRAP-hybridization protection assay (HPA) [6], TRAP-enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) [7], and TRAP-fluorescent assay [8]. However, methods mentioned above are still relatively time-consuming, and they either need elaborate instruments or required use of expensive florescent substances, and lack the simplicity required for routine medical analysis. Therefore, the development of sensitive, rapid, and reliable methods for telomerase activity analysis is highly desirable in cancer research. Recently, ECL techniques have been developed and applied in nucleic acid analysis due to its sensitivity, simplicity, and safety [9-12]. In this work, an ECL-based TRAP assay has been developed for telomerase activity analysis. The method was applied to detected cultured HeLa cancer cells, which was used as a target to validate the feasibility of the method. 1. Experimental Tripropylamine (TPA), and the chemicals to synthesize the Ru(bpy)32+ N-hydroxysuccinimide ester (TBR-NHS ester) were purchased from Sigma (Louis, MO, USA). RNA Secure was acquired from Ambion. HS Taq DNA polymerase was from TaKaRa Bio. Streptavidin microbeads (2.8 mm diameter) were products of Dynal Biotech (Lake Success, NY, USA). The B-TS primer (50 -biotin-AATCCGTCGAGCAGAGTT-30 ) and 50 -amino modified ACX primer (50 -amino-GCGCGGCTTACCCTTACCCTTACCCTAACC-30 ) were synthesized and HPLC purified by SSBE. The 50 -amino modified ACX primer was label with TBR-NHS ester by our lab according to Terpetschnig’s paper [13]. TRAP experiment was a modification as described previously [14]. Briefly, cell was prepared as described in our recent paper [15], and cell extracts were obtained by the CHAPS detergent method, as described by Kim et al. [3]. For TRAP, 2 mL cell lysate was added to 23 mL of a solution containing 1 PCR buffer, 1 mmol/L EGTA, 0.1 mg/mL bovine serum albumin, 200 mmol/L dNTPs (50 mmol/L each), and 0.2 mg of biotin labeled TS primer. The solution was incubated at 30 8C for 30 min, then at 90 8C for 2 min to terminate the reaction, followed by the addition of 25 mL solutions which contains 1 PCR buffer, 200 mmol/L dNTPs, 3 U of HS Taq DNA polymerase, and 0.1 mg of TBR labeled ACX primer. PCR was carried out in an eppendorf AG thermal cycler with the following program: 94 8C for 4 min; 30 cycles at 94 8C for 30 s, 58 8C for 30 s, and 72 8C for 30 s; 72 8C for 5 min; 4 8C hold. For ECL detection, 10 mL amplified telomere repeats fragments were directly added to 200 mL TE buffer (pH 7.4) containing optimal number of streptavidin-coated beads, and incubated this for 20 min at room temperature with gentle shaking. The reaction mixture was separated by using magnetic racks, following washed twice with TE buffer (pH 7.4), resuspended in 100 mL ECL assay buffer containing 0.2 mol/L NaH2PO4, 0.04% Tween 20, 0.1 mol/L TPA, pH 8, then the amount of amplified products is determined by measuring the ECL signal generated by the TBR with TPA at the surface of work electrode. 2. Results and discussion Fig. 1 shows the basic principle of the novel TRAP-ECL method. B-TS primer is interacted with cancer cell extract in the presence of the nucleotide mixture dNTPs. Telomerase bound to the B-TS primer and synthesized telomeric repeat DNA sequence (TTAGGG), the extension products serve as the templates for PCR amplification which uses BTS as forward primer and TBR-ACX as reversed primer. Amplified products were linked on to the surface of streptavidin-coated magnetic beads through the highly selective biotin-streptavidin linkage. Excess TBR-ACX primer was washed away. The amount of PCR products is determined by measuring the ECL signal generated by the TBR and TPA in the electrochemical reaction cell. TRAP products obtained using low concentrations of telomerase (cell equivalents) were analyzed by ECL. Fig. 2 shows the ECL signal obtained from cell free control (a), RNase treated telomerase positive HeLa cells extract control (b), 10 telomerase positive HeLa cells (c), and 100 telomerase positive HeLa cells (d). It shows the average ECL value from the analysis of an extract from 100 cells is 1030 cps, which is significantly higher than the ECL signals obtained from cell free control (91 cps). To determine whether the signals of ECL were dependent on telomerase activity, the telomerase positive HeLa cells extract (100 cells) was treated by RNase, which destroyed the RNA component of telomerase, thus prevented the telomerization reaction. An obvious decrease in the ECL signals was observed. This clearly shows the ECL signals were dependent on telomerase activity. For the 10 HeLa cells samples, the average ECL
X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
701
Fig. 1. The principle of TRAP-ECL method.
Fig. 2. ECL intensity corresponding to (a) cell free control, (b) RNase inactivated HeLa cell extract (100 cells), (c) HeLa cell extract (10 cells), and (d) HeLa cell extract (100 cells). On: potentiostat on. Off: potentiostat off.
value is 246 cps, which is higher than cell free control, indicated 10 HeLa cells can be easily detectable. The limit of detection was one cell, which was evaluated by comparing signals from the readings of cell free control plus three times the standard deviation. A major issue that needs to be addressed relates to the differentiation of normal and malignant cancer cells by the analysis of telomerase activity in respective extracts. The ECL intensities obtained from HeLa cancer cells (1000 cells)
Fig. 3. ECL intensities obtained by analyzing extracts from: cell free control (0 cell), normal blood cells (1000 cells), and HeLa cancer cells (1000 cells).
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X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
are compared to light signals obtained from normal blood cell extracts (1000 cells) (Fig. 3). It was shown that the ECL signals (telomerase activity) obtained from HeLa cancer cells were significantly higher than the ECL signals obtained from cell free control. Whereas no activity could be detected in normal blood cell extracts from a healthy volunteer used as control. To evaluate the influence of interference, cell lysate was prepared in larger number of cells (>1000 cells). It was found when 5000 or more cells were analyzed, an inhibition was observed, which may be accounted for by an increased concentration of PCR inhibitors present in tumor cells. In the study, we describe novel method for telomerase activity analysis. Compared with traditional electrophoresisbased methods, the coupling of telomerase product amplification with the ECL assay leads to an increased speed of assay. In addition, good sensitivity and accuracy, easy quantification and easy handling of a large number of samples are the main merit of the current method. In conclusion, TRAP-ECL provides a new tool for the rapid and reliable quantification of telomerase activity. Acknowledgments This research is supported by the National Natural Science Foundation of China (No. 30600128; 30700155), the National High Technology Research and Development Program of China (863 Program) (No. 2007AA10Z204), and the Natural Science Foundation of Guangdong Province (No. 7005825). References [1] C.M. Counter, H.W. Hirte, S. Bacchetti, C.B. Harley, Proc. Natl. Acad. Sci. 91 (1994) 2900. [2] J.W. Shay, S. Bacchetti, Eur. J. Cancer 33 (1997) 787. [3] N.W. Kim, M.A. Piatyszck, 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, Science 266 (1994) 2011. [4] E. Savoysky, K. Akamatsu, M. Tsuchiya, T. Yamazaki, Nucleic Acids Res. 24 (1996) 1175. [5] A.J. Cheng, R.P. Tang, J.Y. Wang, J.T. Chang, T.C.V. Wang, Jpn. J. Cancer Res. 90 (1999) 280. [6] M. Hirose, J. Abei-Hashimoto, K. Ogura, H. Tahara, T. Ide, T. Yoshimura, J. Cancer Res. Clin. Oncol. 123 (1997) 337. [7] S. Xu, M. He, H. Yu, X. Cai, X. Tan, B. Lu, B. Shu, Anal. Biochem. 299 (2001) 188. [8] S. Gelmini, A. Caldini, L. Becherini, S. Capaccioli, M. Pazzagli, C. Orlando, Clin. Chem. 44 (1998) 2133. [9] D. Zhu, D. Xing, X. Shen, et al. Biosens. Bioelectron. 20 (2004) 448. [10] D. Zhu, D. Xing, X. Shen, et al. Biochem. Biophys. Res. Commun. 324 (2004) 964. [11] J. Liu, D. Xing, X. Shen, et al. Biosens. Bioelectron. 20 (2004) 436. [12] D. Zhu, D. Xing, X. Li, et al. Chin. Chem. Lett. 17 (2006) 499. [13] E. Terpetschnig, H. Szmacinski, H. Malak, J.R. Lakowicz, Biophys. J. 68 (1995) 342. [14] J.P. Jakupciak, P.E. Barker, W. Wang, S. Srivastava, D.H. Atha, Clin. Chem. 51 (2005) 1443. [15] Y.H. Pei, D. Xing, X.J. Gao, L. Liu, T.S. Chen, Apoptosis 12 (2007) 1681.
Chinese Chemical Letters 19 (2008) 699–702 www.elsevier.com/locate/cclet
Detection of telomerase activity by combination of telomeric repeat amplification protocol and electrochemiluminescence assay Xiao Ming Zhou *, Li Jia MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China Received 25 December 2007
Abstract A highly sensitive telomerase detection method that combines telomeric repeat amplification protocol (TRAP) and magnetic beads based electrochemiluminescence (ECL) assay has been developed. Briefly, telomerase recognizes biotinylated telomerase synthesis primer (B-TS) and synthesizes extension products, which then serve as the templates for PCR amplification using B-TS as the forward primer and tris-(20 20 -bipyridyl) ruthenium (TBR) labeled ACX (TBR-ACX) as the reversed primer. The amplified product is captured on streptavidin-coated paramagnetic beads and detected by ECL. Telomerase positive HeLa cells were used to validate the feasibility of the method. The experimental results showed down to 10 cancer cells can be detected easily. The method is a useful tool for telomerase activity analysis due to its sensitivity, rapidity, safety, high throughput, and low cost. It can be used for screening a large amount of clinical samples. # 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Telomerase activity; TRAP; Electrochemiluminescence; Cancer diagnosis
Telomerase, a ribonucleoprotein enzyme that adds telomeric repeats to the 30 end of chromosomal DNA for maintaining chromosomal integrity and stability [1]. Most somatic cells undergo a steady rate of telomere loss and do not possess detectable telomerase activity. However, in the great majority of tumors cells telomerase expressed with high levels [2]. The strong association of telomerase activity with tumors establishing it is the most widespread cancer marker [3]. TRAP is a standard method for telomerase activity analysis [3]. This PCR-based method to detect telomerase activity enabled exponential amplification of the primer-telomeric repeats generated in the telomerase reaction, and the resulting improvements dramatically increased the efficiency and sensitivity of telomerase activity detection. However, like in other conventional electrophoresis-based PCR methods, inherent problems exist in the conventional TRAP assay using a densitometry to quantitate telomerase activity. For example, the limited dynamic range, as well as end-point detection of the PCR product, makes the accurate measurement of telomerase activity difficult. Moreover, the post-PCR processing is time-consuming and adds further variables during analysis of the PCR products. Alternatives for post-amplification detection have been reported, such as TRAP-scintillation proximity assay (SPA)
* Corresponding author. E-mail address: [email protected] (X.M. Zhou). 1001-8417/$ – see front matter # 2008 Xiao Ming Zhou. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.04.017
700
X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
[4], TRAP-enzyme immunoassay (EIA) [5], TRAP-hybridization protection assay (HPA) [6], TRAP-enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA) [7], and TRAP-fluorescent assay [8]. However, methods mentioned above are still relatively time-consuming, and they either need elaborate instruments or required use of expensive florescent substances, and lack the simplicity required for routine medical analysis. Therefore, the development of sensitive, rapid, and reliable methods for telomerase activity analysis is highly desirable in cancer research. Recently, ECL techniques have been developed and applied in nucleic acid analysis due to its sensitivity, simplicity, and safety [9-12]. In this work, an ECL-based TRAP assay has been developed for telomerase activity analysis. The method was applied to detected cultured HeLa cancer cells, which was used as a target to validate the feasibility of the method. 1. Experimental Tripropylamine (TPA), and the chemicals to synthesize the Ru(bpy)32+ N-hydroxysuccinimide ester (TBR-NHS ester) were purchased from Sigma (Louis, MO, USA). RNA Secure was acquired from Ambion. HS Taq DNA polymerase was from TaKaRa Bio. Streptavidin microbeads (2.8 mm diameter) were products of Dynal Biotech (Lake Success, NY, USA). The B-TS primer (50 -biotin-AATCCGTCGAGCAGAGTT-30 ) and 50 -amino modified ACX primer (50 -amino-GCGCGGCTTACCCTTACCCTTACCCTAACC-30 ) were synthesized and HPLC purified by SSBE. The 50 -amino modified ACX primer was label with TBR-NHS ester by our lab according to Terpetschnig’s paper [13]. TRAP experiment was a modification as described previously [14]. Briefly, cell was prepared as described in our recent paper [15], and cell extracts were obtained by the CHAPS detergent method, as described by Kim et al. [3]. For TRAP, 2 mL cell lysate was added to 23 mL of a solution containing 1 PCR buffer, 1 mmol/L EGTA, 0.1 mg/mL bovine serum albumin, 200 mmol/L dNTPs (50 mmol/L each), and 0.2 mg of biotin labeled TS primer. The solution was incubated at 30 8C for 30 min, then at 90 8C for 2 min to terminate the reaction, followed by the addition of 25 mL solutions which contains 1 PCR buffer, 200 mmol/L dNTPs, 3 U of HS Taq DNA polymerase, and 0.1 mg of TBR labeled ACX primer. PCR was carried out in an eppendorf AG thermal cycler with the following program: 94 8C for 4 min; 30 cycles at 94 8C for 30 s, 58 8C for 30 s, and 72 8C for 30 s; 72 8C for 5 min; 4 8C hold. For ECL detection, 10 mL amplified telomere repeats fragments were directly added to 200 mL TE buffer (pH 7.4) containing optimal number of streptavidin-coated beads, and incubated this for 20 min at room temperature with gentle shaking. The reaction mixture was separated by using magnetic racks, following washed twice with TE buffer (pH 7.4), resuspended in 100 mL ECL assay buffer containing 0.2 mol/L NaH2PO4, 0.04% Tween 20, 0.1 mol/L TPA, pH 8, then the amount of amplified products is determined by measuring the ECL signal generated by the TBR with TPA at the surface of work electrode. 2. Results and discussion Fig. 1 shows the basic principle of the novel TRAP-ECL method. B-TS primer is interacted with cancer cell extract in the presence of the nucleotide mixture dNTPs. Telomerase bound to the B-TS primer and synthesized telomeric repeat DNA sequence (TTAGGG), the extension products serve as the templates for PCR amplification which uses BTS as forward primer and TBR-ACX as reversed primer. Amplified products were linked on to the surface of streptavidin-coated magnetic beads through the highly selective biotin-streptavidin linkage. Excess TBR-ACX primer was washed away. The amount of PCR products is determined by measuring the ECL signal generated by the TBR and TPA in the electrochemical reaction cell. TRAP products obtained using low concentrations of telomerase (cell equivalents) were analyzed by ECL. Fig. 2 shows the ECL signal obtained from cell free control (a), RNase treated telomerase positive HeLa cells extract control (b), 10 telomerase positive HeLa cells (c), and 100 telomerase positive HeLa cells (d). It shows the average ECL value from the analysis of an extract from 100 cells is 1030 cps, which is significantly higher than the ECL signals obtained from cell free control (91 cps). To determine whether the signals of ECL were dependent on telomerase activity, the telomerase positive HeLa cells extract (100 cells) was treated by RNase, which destroyed the RNA component of telomerase, thus prevented the telomerization reaction. An obvious decrease in the ECL signals was observed. This clearly shows the ECL signals were dependent on telomerase activity. For the 10 HeLa cells samples, the average ECL
X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
701
Fig. 1. The principle of TRAP-ECL method.
Fig. 2. ECL intensity corresponding to (a) cell free control, (b) RNase inactivated HeLa cell extract (100 cells), (c) HeLa cell extract (10 cells), and (d) HeLa cell extract (100 cells). On: potentiostat on. Off: potentiostat off.
value is 246 cps, which is higher than cell free control, indicated 10 HeLa cells can be easily detectable. The limit of detection was one cell, which was evaluated by comparing signals from the readings of cell free control plus three times the standard deviation. A major issue that needs to be addressed relates to the differentiation of normal and malignant cancer cells by the analysis of telomerase activity in respective extracts. The ECL intensities obtained from HeLa cancer cells (1000 cells)
Fig. 3. ECL intensities obtained by analyzing extracts from: cell free control (0 cell), normal blood cells (1000 cells), and HeLa cancer cells (1000 cells).
702
X.M. Zhou, L. Jia / Chinese Chemical Letters 19 (2008) 699–702
are compared to light signals obtained from normal blood cell extracts (1000 cells) (Fig. 3). It was shown that the ECL signals (telomerase activity) obtained from HeLa cancer cells were significantly higher than the ECL signals obtained from cell free control. Whereas no activity could be detected in normal blood cell extracts from a healthy volunteer used as control. To evaluate the influence of interference, cell lysate was prepared in larger number of cells (>1000 cells). It was found when 5000 or more cells were analyzed, an inhibition was observed, which may be accounted for by an increased concentration of PCR inhibitors present in tumor cells. In the study, we describe novel method for telomerase activity analysis. Compared with traditional electrophoresisbased methods, the coupling of telomerase product amplification with the ECL assay leads to an increased speed of assay. In addition, good sensitivity and accuracy, easy quantification and easy handling of a large number of samples are the main merit of the current method. In conclusion, TRAP-ECL provides a new tool for the rapid and reliable quantification of telomerase activity. Acknowledgments This research is supported by the National Natural Science Foundation of China (No. 30600128; 30700155), the National High Technology Research and Development Program of China (863 Program) (No. 2007AA10Z204), and the Natural Science Foundation of Guangdong Province (No. 7005825). References [1] C.M. Counter, H.W. Hirte, S. Bacchetti, C.B. Harley, Proc. Natl. Acad. Sci. 91 (1994) 2900. [2] J.W. Shay, S. Bacchetti, Eur. J. Cancer 33 (1997) 787. [3] N.W. Kim, M.A. Piatyszck, 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, Science 266 (1994) 2011. [4] E. Savoysky, K. Akamatsu, M. Tsuchiya, T. Yamazaki, Nucleic Acids Res. 24 (1996) 1175. [5] A.J. Cheng, R.P. Tang, J.Y. Wang, J.T. Chang, T.C.V. Wang, Jpn. J. Cancer Res. 90 (1999) 280. [6] M. Hirose, J. Abei-Hashimoto, K. Ogura, H. Tahara, T. Ide, T. Yoshimura, J. Cancer Res. Clin. Oncol. 123 (1997) 337. [7] S. Xu, M. He, H. Yu, X. Cai, X. Tan, B. Lu, B. Shu, Anal. Biochem. 299 (2001) 188. [8] S. Gelmini, A. Caldini, L. Becherini, S. Capaccioli, M. Pazzagli, C. Orlando, Clin. Chem. 44 (1998) 2133. [9] D. Zhu, D. Xing, X. Shen, et al. Biosens. Bioelectron. 20 (2004) 448. [10] D. Zhu, D. Xing, X. Shen, et al. Biochem. Biophys. Res. Commun. 324 (2004) 964. [11] J. Liu, D. Xing, X. Shen, et al. Biosens. Bioelectron. 20 (2004) 436. [12] D. Zhu, D. Xing, X. Li, et al. Chin. Chem. Lett. 17 (2006) 499. [13] E. Terpetschnig, H. Szmacinski, H. Malak, J.R. Lakowicz, Biophys. J. 68 (1995) 342. [14] J.P. Jakupciak, P.E. Barker, W. Wang, S. Srivastava, D.H. Atha, Clin. Chem. 51 (2005) 1443. [15] Y.H. Pei, D. Xing, X.J. Gao, L. Liu, T.S. Chen, Apoptosis 12 (2007) 1681.