- Email: [email protected]
Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia
Leukemia Research 28 (2004) 969–972
Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia Marcel E. Gil a,∗ , Thérèsa L. Coetzer a,b a
Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa b National Health Laboratory Service, Johannesburg, South Africa Received 23 September 2003; accepted 7 January 2004 Available online 26 February 2004
Abstract Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC). © 2004 Elsevier Ltd. All rights reserved. Keywords: hTERT; Quantitative RT-PCR; LightCycler
1. Introduction Telomere dynamics is the collective study of telomere length, telomerase activity and telomerase regulation. Telomeres are found at the ends of chromosomes, made up of a 5 -sub-telomeric region and a 3 -telomeric repeat region [1]. Originally thought to simply allow complete replication of chromosomes, telomeres have since been shown to be pivotal in the normal functioning of cells by preventing fusions, recombination and activation of DNA repair by chromosome ends [1]. Telomere shortening due to the end replication problem limits cell life span [2]. To counter telomere erosion in rapidly dividing cells, telomerase adds telomeric repeats to telomeres by a unique reverse transcription process catalysed by hTERT [1]. Expression of the gene is regulated by cell-cycle controls, so it is commonly deregulated in cancers resulting in high level constitutive activation of telomerase which is normally at low to undetectable levels in cells. This deregulation in over 90% of Abbreviations: hTERT, human telomere elongation reverse transcriptase; PBGD, porphobilinogen deaminase; ssBP1, mitochondrial single-stranded DNA binding protein 1; UBC, ubiquitin C; CML, chronic myeloid leukemia ∗ Corresponding author. Tel.: +27-11-717-2418; fax: +21-11-717-2395. E-mail address: [email protected] (M.E. Gil). 0145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2004.01.002
malignancies has possible implications in diagnosis, prognosis, progression monitoring and therapeutics of cancer [2]. Chronic myeloid leukemia (CML) is a neoplastic disease with the characteristic Philadelphia (Ph) chromosome arising from the t(9;22)(q34;q11) translocation [3]. Patients remain in a chronic phase for several years, following which, a blast crisis develops and patients either revert to a chronic phase or no longer respond to therapy [3]. Critical shortening of telomere length increases chromosome instability, so the acquisition of additional chromosomal aberrations in the progression of CML may be related to changes in telomere dynamics. Since telomere length is maintained by telomerase activity, which is a function of hTERT expression, a technique to quantify hTERT will be of great value. Quantification of an RNA transcript is best accomplished using a quantitative reverse transcription polymerase chain reaction (RT-PCR) [4]. The key component of quantitative RT-PCR is the internal control, which should fulfil three criteria: it must remain genetically stable, amplify within the same tube as the transcript of interest and have no pseudogenes [4]. The LightCycler TeloTAGGG hTERT Quantification kit (Roche Diagnostics GmbH. Mannheim, Germany) utilises the porphobilinogen deaminase (PBGD) gene as an internal
970
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
control. It is amplified in a separate tube to hTERT and has a genomic localisation of 11q23, which is unstable in a variety of leukemias [5]. The kit therefore only fulfils the internal standard criterion of possessing no pseudogenes. Thus the suitability of two alternative housekeeping genes, mitochondrial single-stranded DNA binding protein 1 (ssBP1) [6] and ubiquitin C (UBC) [7] was compared for use in real-time RT-PCR of hTERT in CML.
Table 1 PCR primer sequences and size of amplification products Primer
Sequence
RNA
DNA
hTERT sense hTERT anti-sense
5 -AggAgCTgACgTggAAgATg-3
5 -CTgACCTCTgATTCCgACAg-3
302
10906
UBC sense UBC anti-sense
5 -ATTTgggTCgCggTTCTTg-3 5 -TgCCTTgACATTCTCgATggT-3
133
1007
5 -gCgATCAggggATAgTgAAg-3 5 -TTgCTTgTCgCCTCACATTA-3
194
2000
2. Materials and methods
ssBP1 sense ssBP1 anti-sense
2.1. Subjects
2.3. Conventional RT-PCR
Five millilitres samples of blood were collected in EDTA-coated tubes from a CML patient under treatment at the Johannesburg General Hospital, South Africa, and a healthy volunteer within the laboratory (informed consent and ethical clearance obtained). Samples were incubated for 5 min in two volumes 0.17 M ammonium chloride, and white blood cells pelleted at 1400 × g for 10 min. Cells were washed twice in 0.9% saline, the pellet resuspended in 2 ml 0.9% saline and cells counted using an improved Neubauer hemocytometer. 106 cells/ml were cultured in RPMI1640 medium using standard techniques. hTERT positive controls were generated from HL-60 cells (kind gift from C. Drummond) and normal white blood cells by stimulating cells for 4 days with phytohemaglutinin-M (Difco, USA) and 100 U/ml IL-2 (Sigma–Aldrich, USA).
A 20 l reverse transcription reaction of 1 g total RNA was performed using the First Strand cDNA Synthesis kit (Roche Diagnostics GmbH. Mannheim, Germany), with 1.6 g of an oligo(dT)15 primer, as per manufacturer’s instructions. A reagent blank reaction with no RNA was processed in parallel. PCR of 10 l cDNA was carried out in a 50 l volume using the PCR Core Kit (Roche Diagnostics GmbH. Mannheim, Germany). Primers for hTERT, ssBP1 and UBC cDNA amplification (IDT, Coralville, IA, USA) were added to a final concentration of 0.25, 0.3 and 0.3 M, respectively. They were designed to span introns to eliminate possible contaminating DNA amplification interfering with mRNA quantification. Primer sequences and the size of amplification products are listed in Table 1. A “hot-start” was performed, followed by 40 cycles of 94 ◦ C for 30 s, 62 ◦ C for 30 s and 72 ◦ C for 1 min, using the Eppendorf Mastercycler Gradient (Eppendorf Netheler-Huiz GmbH, Hamburg, Germany). PCR products were resolved using a 1.5% agarose gel in 40 mM Tris–acetate, 0.04 mM EDTA, pH 8.0 containing ethidium bromide. The samples were analysed using GeneTools v.2.10.02 on the Syngene Gel-Doc System (Synoptics Ltd., Cambridge, UK).
2.2. RNA extraction Isolation of total RNA was performed using TriPure Isolation reagent (Roche Diagnostics GmbH. Germany) or RNeasy Mini Kit (Qiagen, USA) according to manufacturer’s instructions.
Product size (bp)
Fig. 1. Resolution of conventional hTERT/ssBP1 RT-PCR products. M, 100 bp DNA molecular weight marker; B, blank, lane 1, stimulated HL-60 cells; lane 2, stimulated normal cells; lane 3, normal cells; lane 4, telomerase positive CML patient.
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
2.4. Real-time hTERT quantification cDNA was generated as described above. PCR of 2 l cDNA was carried out in a 20 l volume using the LightCycler Fast-Start DNA Master SYBR Green kit (Roche Diagnostics GmbH. Mannheim, Germany), with MgCl2 added to a final concentration of 3 mM. Primers for hTERT, ssBP1 and UBC cDNA amplification were added to final concentrations of 0.2, 0.1 and 0.1 M, respectively. The enzyme was activated at 95 ◦ C for 10 min, followed by 40 cycles of 95 ◦ C for 5 s, 62 ◦ C (UBC) or 64 ◦ C (ssBP1) for 15 s and
971
72 ◦ C for 15 s. All transition rates were set to 20 ◦ C/s with the exception of the annealing transition rate, which was set to 4 ◦ C/s. Peak areas were determined using the LightCycler Data Analysis programme, which enables relative quantification of the amplicons.
3. Results The hTERT/ssBP1 conventional RT-PCR yielded amplification of both transcripts in hTERT positive samples and
Fig. 2. Melting curve analysis of real-time hTERT/UBC RT-PCR products. (a) Melting curves for amplification products of stimulated normal and HL-60 cells, and a telomerase positive CML patient. The UBC products have a melting temperature of 86.5 ◦ C and hTERT products of 91.5 ◦ C. (b) Melting curves for amplification products of normal cells (in duplicate) show the expected UBC products, which melt at 86.5 ◦ C.
972
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
only the internal control was amplified in the hTERT negative sample (Fig. 1). The hTERT/UBC conventional RT-PCR yielded the correct size UBC product of 133 bp, as well as faint additional amplicons. The conventional RT-PCR protocol was modified for use on the LightCycler and both multiplex reactions yielded amplification of the respective internal standard. The HL-60 cell line, as well as normal stimulated cells, and a telomerase positive CML sample showed amplification of hTERT and both internal controls (Fig. 2a). Only the internal control was amplified in normal unstimulated cells (Fig. 2b).
thus amplification of possible contaminating DNA will not invalidate the results.
4. Discussion and conclusions
[1] Cech TR. Life at the end of the chromosome: telomeres and telomerase. Angew Chem Int Ed 2000;39:34–43. [2] McKenzie KE, Umbricht CB, Sukumar S. Applications of telomerase research in the fight against cancer. Mol Med Today 1999;5:114–22. [3] Kantarjian HM, Deisseroth A, Kurzrock R, Estrov Z, Talpaz M. Chronic myelogenous leukemia: a concise update. Blood 1993;82:691– 703. [4] Freeman W, Walker S, Vrana K. Quantitative RT-PCR. Pitfalls and Potential. BioTechniques 1999;26:112–25. [5] Monni O, Knuutila S. 11q deletions in haematological malignancies. Leuk Lymphoma 2001;40(3–4):259–66. [6] Tiranti V, Rossi E, Ruiz-Carrillo A, Rossi G, Rocchi M, DiDonato S, et al. Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involved in mitochondrial biogenesis. Genomics 1995;25(2):559–64. [7] Warrington J, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2000;2:143–7. [8] Ong ST, Le Beau MM. Chromosomal abnormalities and molecular genetics of non-Hodgkin’s lymphoma. Semin Oncol 1998;25(4):447– 60.
Although the amplification of hTERT and ssBP1 was consistent in both conventional and real-time RT-PCR, reliable amplification of UBC could only be achieved on the LightCycler. This discrepancy may be due to the fact the conditions and reagents are quite different between conventional and real-time RT-PCR. The quantitative hTERT RT-PCR described here is a reliable and rapid method for the quantification of hTERT in peripheral blood mononuclear cells of malignant origin. The 7q34 genomic localisation of ssBP1, however, is not stable in non-Hodgkin’s lymphoma [8], which appears to be the only hematopoietic malignancy where amplification of this internal control may not remain constant between samples. The internal controls are amplified within the same tube as the test cDNA to control for tube-to-tube variation in PCR and both sets of primers were designed to span large introns,
Acknowledgements The authors wish to acknowledge funding received from the Cancer Association of South Africa (CANSA), the University of the Witwatersrand and the National Health Laboratory Service. References
Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia Marcel E. Gil a,∗ , Thérèsa L. Coetzer a,b a
Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, 7 York Road, Parktown, 2193 Johannesburg, South Africa b National Health Laboratory Service, Johannesburg, South Africa Received 23 September 2003; accepted 7 January 2004 Available online 26 February 2004
Abstract Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC). © 2004 Elsevier Ltd. All rights reserved. Keywords: hTERT; Quantitative RT-PCR; LightCycler
1. Introduction Telomere dynamics is the collective study of telomere length, telomerase activity and telomerase regulation. Telomeres are found at the ends of chromosomes, made up of a 5 -sub-telomeric region and a 3 -telomeric repeat region [1]. Originally thought to simply allow complete replication of chromosomes, telomeres have since been shown to be pivotal in the normal functioning of cells by preventing fusions, recombination and activation of DNA repair by chromosome ends [1]. Telomere shortening due to the end replication problem limits cell life span [2]. To counter telomere erosion in rapidly dividing cells, telomerase adds telomeric repeats to telomeres by a unique reverse transcription process catalysed by hTERT [1]. Expression of the gene is regulated by cell-cycle controls, so it is commonly deregulated in cancers resulting in high level constitutive activation of telomerase which is normally at low to undetectable levels in cells. This deregulation in over 90% of Abbreviations: hTERT, human telomere elongation reverse transcriptase; PBGD, porphobilinogen deaminase; ssBP1, mitochondrial single-stranded DNA binding protein 1; UBC, ubiquitin C; CML, chronic myeloid leukemia ∗ Corresponding author. Tel.: +27-11-717-2418; fax: +21-11-717-2395. E-mail address: [email protected] (M.E. Gil). 0145-2126/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2004.01.002
malignancies has possible implications in diagnosis, prognosis, progression monitoring and therapeutics of cancer [2]. Chronic myeloid leukemia (CML) is a neoplastic disease with the characteristic Philadelphia (Ph) chromosome arising from the t(9;22)(q34;q11) translocation [3]. Patients remain in a chronic phase for several years, following which, a blast crisis develops and patients either revert to a chronic phase or no longer respond to therapy [3]. Critical shortening of telomere length increases chromosome instability, so the acquisition of additional chromosomal aberrations in the progression of CML may be related to changes in telomere dynamics. Since telomere length is maintained by telomerase activity, which is a function of hTERT expression, a technique to quantify hTERT will be of great value. Quantification of an RNA transcript is best accomplished using a quantitative reverse transcription polymerase chain reaction (RT-PCR) [4]. The key component of quantitative RT-PCR is the internal control, which should fulfil three criteria: it must remain genetically stable, amplify within the same tube as the transcript of interest and have no pseudogenes [4]. The LightCycler TeloTAGGG hTERT Quantification kit (Roche Diagnostics GmbH. Mannheim, Germany) utilises the porphobilinogen deaminase (PBGD) gene as an internal
970
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
control. It is amplified in a separate tube to hTERT and has a genomic localisation of 11q23, which is unstable in a variety of leukemias [5]. The kit therefore only fulfils the internal standard criterion of possessing no pseudogenes. Thus the suitability of two alternative housekeeping genes, mitochondrial single-stranded DNA binding protein 1 (ssBP1) [6] and ubiquitin C (UBC) [7] was compared for use in real-time RT-PCR of hTERT in CML.
Table 1 PCR primer sequences and size of amplification products Primer
Sequence
RNA
DNA
hTERT sense hTERT anti-sense
5 -AggAgCTgACgTggAAgATg-3
5 -CTgACCTCTgATTCCgACAg-3
302
10906
UBC sense UBC anti-sense
5 -ATTTgggTCgCggTTCTTg-3 5 -TgCCTTgACATTCTCgATggT-3
133
1007
5 -gCgATCAggggATAgTgAAg-3 5 -TTgCTTgTCgCCTCACATTA-3
194
2000
2. Materials and methods
ssBP1 sense ssBP1 anti-sense
2.1. Subjects
2.3. Conventional RT-PCR
Five millilitres samples of blood were collected in EDTA-coated tubes from a CML patient under treatment at the Johannesburg General Hospital, South Africa, and a healthy volunteer within the laboratory (informed consent and ethical clearance obtained). Samples were incubated for 5 min in two volumes 0.17 M ammonium chloride, and white blood cells pelleted at 1400 × g for 10 min. Cells were washed twice in 0.9% saline, the pellet resuspended in 2 ml 0.9% saline and cells counted using an improved Neubauer hemocytometer. 106 cells/ml were cultured in RPMI1640 medium using standard techniques. hTERT positive controls were generated from HL-60 cells (kind gift from C. Drummond) and normal white blood cells by stimulating cells for 4 days with phytohemaglutinin-M (Difco, USA) and 100 U/ml IL-2 (Sigma–Aldrich, USA).
A 20 l reverse transcription reaction of 1 g total RNA was performed using the First Strand cDNA Synthesis kit (Roche Diagnostics GmbH. Mannheim, Germany), with 1.6 g of an oligo(dT)15 primer, as per manufacturer’s instructions. A reagent blank reaction with no RNA was processed in parallel. PCR of 10 l cDNA was carried out in a 50 l volume using the PCR Core Kit (Roche Diagnostics GmbH. Mannheim, Germany). Primers for hTERT, ssBP1 and UBC cDNA amplification (IDT, Coralville, IA, USA) were added to a final concentration of 0.25, 0.3 and 0.3 M, respectively. They were designed to span introns to eliminate possible contaminating DNA amplification interfering with mRNA quantification. Primer sequences and the size of amplification products are listed in Table 1. A “hot-start” was performed, followed by 40 cycles of 94 ◦ C for 30 s, 62 ◦ C for 30 s and 72 ◦ C for 1 min, using the Eppendorf Mastercycler Gradient (Eppendorf Netheler-Huiz GmbH, Hamburg, Germany). PCR products were resolved using a 1.5% agarose gel in 40 mM Tris–acetate, 0.04 mM EDTA, pH 8.0 containing ethidium bromide. The samples were analysed using GeneTools v.2.10.02 on the Syngene Gel-Doc System (Synoptics Ltd., Cambridge, UK).
2.2. RNA extraction Isolation of total RNA was performed using TriPure Isolation reagent (Roche Diagnostics GmbH. Germany) or RNeasy Mini Kit (Qiagen, USA) according to manufacturer’s instructions.
Product size (bp)
Fig. 1. Resolution of conventional hTERT/ssBP1 RT-PCR products. M, 100 bp DNA molecular weight marker; B, blank, lane 1, stimulated HL-60 cells; lane 2, stimulated normal cells; lane 3, normal cells; lane 4, telomerase positive CML patient.
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
2.4. Real-time hTERT quantification cDNA was generated as described above. PCR of 2 l cDNA was carried out in a 20 l volume using the LightCycler Fast-Start DNA Master SYBR Green kit (Roche Diagnostics GmbH. Mannheim, Germany), with MgCl2 added to a final concentration of 3 mM. Primers for hTERT, ssBP1 and UBC cDNA amplification were added to final concentrations of 0.2, 0.1 and 0.1 M, respectively. The enzyme was activated at 95 ◦ C for 10 min, followed by 40 cycles of 95 ◦ C for 5 s, 62 ◦ C (UBC) or 64 ◦ C (ssBP1) for 15 s and
971
72 ◦ C for 15 s. All transition rates were set to 20 ◦ C/s with the exception of the annealing transition rate, which was set to 4 ◦ C/s. Peak areas were determined using the LightCycler Data Analysis programme, which enables relative quantification of the amplicons.
3. Results The hTERT/ssBP1 conventional RT-PCR yielded amplification of both transcripts in hTERT positive samples and
Fig. 2. Melting curve analysis of real-time hTERT/UBC RT-PCR products. (a) Melting curves for amplification products of stimulated normal and HL-60 cells, and a telomerase positive CML patient. The UBC products have a melting temperature of 86.5 ◦ C and hTERT products of 91.5 ◦ C. (b) Melting curves for amplification products of normal cells (in duplicate) show the expected UBC products, which melt at 86.5 ◦ C.
972
M.E. Gil, T.L. Coetzer / Leukemia Research 28 (2004) 969–972
only the internal control was amplified in the hTERT negative sample (Fig. 1). The hTERT/UBC conventional RT-PCR yielded the correct size UBC product of 133 bp, as well as faint additional amplicons. The conventional RT-PCR protocol was modified for use on the LightCycler and both multiplex reactions yielded amplification of the respective internal standard. The HL-60 cell line, as well as normal stimulated cells, and a telomerase positive CML sample showed amplification of hTERT and both internal controls (Fig. 2a). Only the internal control was amplified in normal unstimulated cells (Fig. 2b).
thus amplification of possible contaminating DNA will not invalidate the results.
4. Discussion and conclusions
[1] Cech TR. Life at the end of the chromosome: telomeres and telomerase. Angew Chem Int Ed 2000;39:34–43. [2] McKenzie KE, Umbricht CB, Sukumar S. Applications of telomerase research in the fight against cancer. Mol Med Today 1999;5:114–22. [3] Kantarjian HM, Deisseroth A, Kurzrock R, Estrov Z, Talpaz M. Chronic myelogenous leukemia: a concise update. Blood 1993;82:691– 703. [4] Freeman W, Walker S, Vrana K. Quantitative RT-PCR. Pitfalls and Potential. BioTechniques 1999;26:112–25. [5] Monni O, Knuutila S. 11q deletions in haematological malignancies. Leuk Lymphoma 2001;40(3–4):259–66. [6] Tiranti V, Rossi E, Ruiz-Carrillo A, Rossi G, Rocchi M, DiDonato S, et al. Chromosomal localization of mitochondrial transcription factor A (TCF6), single-stranded DNA-binding protein (SSBP), and endonuclease G (ENDOG), three human housekeeping genes involved in mitochondrial biogenesis. Genomics 1995;25(2):559–64. [7] Warrington J, Nair A, Mahadevappa M, Tsyganskaya M. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiol Genomics 2000;2:143–7. [8] Ong ST, Le Beau MM. Chromosomal abnormalities and molecular genetics of non-Hodgkin’s lymphoma. Semin Oncol 1998;25(4):447– 60.
Although the amplification of hTERT and ssBP1 was consistent in both conventional and real-time RT-PCR, reliable amplification of UBC could only be achieved on the LightCycler. This discrepancy may be due to the fact the conditions and reagents are quite different between conventional and real-time RT-PCR. The quantitative hTERT RT-PCR described here is a reliable and rapid method for the quantification of hTERT in peripheral blood mononuclear cells of malignant origin. The 7q34 genomic localisation of ssBP1, however, is not stable in non-Hodgkin’s lymphoma [8], which appears to be the only hematopoietic malignancy where amplification of this internal control may not remain constant between samples. The internal controls are amplified within the same tube as the test cDNA to control for tube-to-tube variation in PCR and both sets of primers were designed to span large introns,
Acknowledgements The authors wish to acknowledge funding received from the Cancer Association of South Africa (CANSA), the University of the Witwatersrand and the National Health Laboratory Service. References