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Articles |
1
Department of Medicine, Division of Hematology, Karolinska Hospital and Institute, SE-171 76 Stockholm, Sweden.
a Author for correspondence. Hematological Lab, CMM, L8:03, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Fax 468-5177-3054; e-mail
Dawei.Xu{at}cmm.ki.se.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We developed a real-time quantitative TRAP (RTQ-TRAP) system by combining a real-time PCR technique with the conventional TRAP method. Telomerase activity in human tumor cell lines and in 13 lymphoma samples was measured using the RTQ-TRAP assay, and the results obtained from the samples using the RTQ-TRAP method were compared with the conventional TRAP method.
Results: The RTQ-TRAP method was both accurate and reproducible in measuring telomerase activity in a dilution series of protein extracts from HL60 cells. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-TRAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as determined using RTQ-TRAP, was much shorter than the half-life reported previously.
Conclusions: Our results suggest that the conventional TRAP assay frequently overestimates telomerase activity in tumor samples. The RTQ-TRAP method is thus a useful tool to rapidly and precisely quantify telomerase activity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An accurate quantitative measurement of telomerase activity is required for better evaluation of the biological and clinical importance of telomerase in human malignancies. Traditionally, telomerase activity has been assessed based on a biochemical primer extension assay, the inefficiency and low sensitivity of which, together with the low amounts of telomerase activity in mammalian cells, greatly limit the application of the assay in primary human tumors. A landmark method, termed telomeric repeat amplification protocol (TRAP),1 for the determination of telomerase activity was introduced in 1994 (2). 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. At present, the TRAP assay is widely used for telomerase activity assessment. However, like in other conventional PCR methods, inherent problems exist in the current TRAP assay. 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.
Real-time quantitative (RTQ) PCR, designed to avoid the deficiencies of conventional PCR, has been developed (3)(4). The use of the ABI PRISMTM 7700 Sequence Detector System (PE Applied Biosystems) and the fluorescent dye SYBR® Green, which is capable of binding to the double-stranded amplicons and generating fluorescence signals in a PCR reaction, allows the amount of PCR products to be determined based on the fluorescence produced during the extension step of each cycle in a closed tube. In the present study, we used the RTQ-TRAP method to detect telomerase activity. Our results demonstrate that the RTQ-TRAP assay provides a simple and powerful tool to precisely quantify telomerase activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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80%, as determined by trypan blue
exclusion.
human primary t lymphocytes and lymphoma samples
Human T lymphocytes, isolated from the buffy-coat blood of healthy
adults, were cultured in RPMI-1640 medium containing 100 mL/L fetal
calf serum, 20 kilounits/L interleukin-2, and 1 mg/L anti-CD3
antibody to stimulate proliferation. Frozen biopsy samples from 13
patients with high-grade non-Hodgkin lymphomas were selected for
telomerase activity studies. The study was approved by the ethics
committee.
protein extraction and telomerase activity assay
Cells (1–2 x 106) were lysed in 50
µL of CHAPS buffer containing RNase inhibitor (8)
and incubated at 4 °C for 30 min. The lysate was then centrifuged at
12 000g for 30 min at 4 °C, and the supernatant was
collected. The protein concentration was measured using the DC protein
reagent set (Bio-Rad) (9). Telomerase activity was
determined using a commercially available telomerase PCR ELISA method
(Roche Diagnostics Scandinavia) according to the manufacturer’s
protocol (7)(10).
rtq-trap assay
The total volume of the reaction mixture was 25 µL and
contained 1x SYBR Green buffer (PE Applied Biosystems),
2.5 mM each dNTP, 15 mM MgCl2, 10 mM EGTA, 0.2
µg of T4 gene protein, 0.1 µg each of primers TS
(5'-AATCCGTCGAGCAGAGTT-3') and ACX
[5'-GCGCGG(CTTACC)3CTAACC-3']
(2)(11), 1 U of AmpliTaq Gold polymerase, and
0.25 µg of protein extract. The PCR was performed in a 96-well
microtiter plate on an ABI PRISM 7700 Sequence Detector System. The
reaction mixture was first incubated at 25 °C for 20 min to allow
the telomerase in the protein extracts to elongate the TS primer by
adding TTAGGG repeat sequences. The PCR was then started at 95 °C
for 10 min (to activate the AmpliTaq Gold polymerase), followed by a
40-cycle amplification (95 °C for 20 s, 50 °C for 30 s,
and 72 °C for 90 s). SYBR Green, a fluorescence
dye, is known to bind double-stranded DNA. When new amplicons were
produced, SYBR Green bound to them at once and generated fluorescence
signals, which were collected and analyzed with Sequence Detector
software (Ver. 1.6; PE Applied Biosystems) during the late
extension step of each cycle. The fluorescence threshold was calculated
as 10 SD of the baseline fluorescence intensity at the default
setting of 3–15 cycles. Telomerase activity in cell lines or samples
was calculated based on the threshold cycle (Ct).
All samples were run in duplicate or triplicate, and protein extracts
from the telomerase-deficient cell line Saos2 or the lysis buffer were
used as negative controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 µg (Fig. 1A
). However, when >2 µg of protein extract was added,
inconsistently decreased signals were obtained (data not shown),
indicating that too much protein interfered with PCR amplification,
which was in accordance with the results reported in previous studies
using the conventional TRAP assay
(8)(12)(13). Therefore, we chose to
use 0.25 µg of protein extract in the RTQ-TRAP assay. In addition,
combinations of different amounts of TS and ACX primers were tested,
and we found that 0.1 µg of each primer per reaction produced an
optimal result (data not shown).
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To test the reproducibility of the RTQ-TRAP assay, we examined the intra- and interassay CVs. When the telomerase activity in extracts containing 0.25 and 0.05 µg of protein from HL60 cells was measured using the RTQ-TRAP method, the CV for quadruplicate samples containing the same amount of protein in the same reaction was 1% for telomerase activity based on the Ct values. The intraassay CV obtained from the analysis of telomerase activity in the same samples with the same amount of protein on 3 different days was 5%.
Because SYBR Green binds to all double-stranded
DNA, we needed to determine whether the fluorescence signals detected
with an ABI PRISM 7700 Sequence Detector System actually came from the
amplified TS-telomerase products in the reaction containing the HL60
cell protein extracts. After PCR, the amplified products were purified
using the QIAquick gel extraction method (Roche), end-labeled with
[
-32P]dATP, and resolved in polyacrylamide
gels. Fig. 1C
reveals typical ladder pattern bands after a 40-cycle
amplification of TS-telomerase products using the ABI PRISM 7700
Sequence Detector System, confirming the specificity of the RTQ-TRAP
assay. However, fluorescence signals were sometimes detected in the
negative control wells, presumably because of the formation of
primer-dimer artifacts. The signals generated by the primer-dimer
artifacts were different from a real amplification curve and usually
appeared at a late stage of amplification (35–39 cycles; Figs. 1A
and 2A
).
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comparison between rtq-trap and conventional trap assays
It is well known that telomerase activity is suppressed during the
terminal differentiation of HL60 leukemic cells. Using the conventional
TRAP method, several previous studies showed that the
differentiation-related suppression of telomerase activity occurred
48–72 h after exposure of the cells to differentiation-inducing agents
(5)(7)(9)(14)(15)(16). We
compared quantitative results of telomerase activity in DMSO-treated
HL60 cells, using the RTQ-TRAP and conventional TRAP methods. A 75%
reduction in telomerase activity was seen 24 h after the DMSO
treatment, as determined with the RTQ-TRAP assay. In contrast, there
was no detectable decrease in telomerase activity during this period
when the conventional TRAP method with a 28-cycle amplification was
used. Because the results obtained by the RTQ-TRAP method (Fig. 1A
)
showed that a plateau could be approached within 28–30 amplification
cycles, even with a 25-fold difference in protein input, we wanted to
determine whether the discrepancy observed between the RTQ-TRAP assay
and the conventional TRAP assay was attributable to the limited
kinetics of the conventional TRAP method. The same samples were further
analyzed using the conventional TRAP method with 25 and 22 PCR cycles,
respectively. The difference in telomerase activity during
differentiation became clearer with the decreased number of
amplification cycles (Fig. 2B
). This result demonstrated that the plateau-related events
made subtle differences in telomerase activity indistinguishable in the
conventional TRAP assay.
We further used the RTQ-TRAP method to determine telomerase activity in
human lymphoma and healthy human lymphocyte samples. Resting
lymphocytes are known to express low telomerase activity, whereas a
dramatic increase in telomerase activity occurs after the activation of
the cells (17)(18)(19). Healthy human lymphocytes were
stimulated with interleukin-2 and anti-CD3 for 3 days and then analyzed
for telomerase activity using the RTQ-TRAP method. The
Ct values were 30 and 21 in the resting and
activated lymphocytes, respectively. This represents a >1000-fold
difference in telomerase activity, whereas only a 30-fold increase in
telomerase activity in the activated lymphocytes could be detected by
the conventional TRAP method (data not shown). In 13 lymphoma samples,
the Ct values were 23–31. The
Ct value for the positive reference REH cells in
the same reaction as the lymphoma samples was 20. Telomerase activity
in the lymphoma samples, calculated from the
Ct values, was 0.05–14% (mean, 5%) of the
activity recorded in the REH cells. However, based on the results
obtained from the conventional TRAP assay, these same lymphoma samples
exhibited much higher telomerase activity (0.5–84%; mean, 41% of
that in the REH cells; Fig. 3
). The data suggest that telomerase activity in lymphoma
specimens was overestimated by the conventional TRAP assay. However,
despite the huge difference, the results obtained by these two methods
were significantly correlated (r2 =
0.691; P = 0.0004).
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half-life of telomerase activity measured by the rtq-trap assay
The half-life of telomerase activity has been reported as 24
h with the conventional TRAP method
(5)(6)(9). However, when the
RTQ-TRAP assay was used to assess telomerase activity in
differentiated HL60 cells, only 25% of the original concentrations of
the enzyme were left after 24 h. It is therefore unlikely that
telomerase activity displays a half-life as long as the one indicated
by the conventional TRAP assay. We measured the half-life of telomerase
activity again with the RTQ-TRAP assay. K562, HL60, and HeLa cells were
incubated with CHX to block new protein synthesis and harvested at
various time points for telomerase activity analysis. In both K562 and
HL60 cells, a 50% reduction of telomerase activity occurred 5 h
after CHX treatment. In HeLa cells, it took 11 h for telomerase
activity to decrease to 50% of the original values (Fig. 4
). On the basis of these results, the half-life of telomerase
activity is 5–11 h, depending on the different types of tumor cells,
and thus is much shorter than the half-life reported previously.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the major advantages of the RTQ-TRAP is that measurement of the
PCR products is performed at the early phase of exponential
amplification, when reaction components are not limiting and the
accumulation of inhibitory PCR products is unlikely to occur. This
ensures the accuracy of the quantification of telomerase activity and
prevents potential variations associated with the end-point assay in
the conventional TRAP assay. Using the RTQ-TRAP assay, we found that
even with a 25-fold difference in the amount of protein (derived from
HL60 cells) added, a plateau was approached at 28 cycles of PCR
amplification. Therefore, the conventional TRAP assay, which involves
28 cycles and depends on an end-point assay of the PCR products,
probably gives a poor quantitative result. It was indeed found
that a several-fold difference in telomerase activity between
undifferentiated and differentiated HL60 cells could not be easily
discriminated with the conventional TRAP method involving 28-cycle
amplification. Moreover, the data obtained from the conventional TRAP
method revealed much higher telomerase activity in human lymphoma
samples (41% of that in REH cells) than the activities found with the
RTQ-TRAP method (5% of that in REH cells). The cycle number of the
conventional TRAP assay for the detection of telomerase activity is
usually 28–31 in the majority of published studies. This might lead to
overestimation of telomerase activity and contribute to variations or
discrepancies in telomerase expression between studies.
The conventional TRAP method requires post-PCR manipulation, in which
each sample must be separated by polyacrylamide electrophoresis or
detected using ELISA. This is not only laborious, time-consuming, and a
possible source of carryover contamination, but also adds further
uncontrolled variables to the analysis of results. In contrast, in the
RTQ-TRAP assay, the PCR reaction and data analysis are done
simultaneously and can be finished in 3 h. This makes the telomerase
activity assay simpler and faster, allowing a higher throughout and
making it suitable for large-scale sample analyses.
To avoid the formation of primer-dimers, Kim and Wu (11) designed an anchored reverse CX primer known as ACX, instead of the original CX primer used for the TRAP assay (2). ACX reverse primer was used in the present study, but primer-dimer artifacts could not be completely eliminated, and SYBR Green could have bound to them, generating fluorescence signals. To prevent the formation of primer-dimers, we tried to use smaller amounts of the primers in the RTQ-TRAP, but the efficiency of PCR was noticeably affected. A possible concern in the RTQ-TRAP assay could be the false-positive signals caused by the primer-dimer artifacts. However, the signals derived from primer-dimers were too weak to be detected during the first 34 amplification cycles, and the accumulation of fluorescence occurred only at cycles 35–39. In the present study, the Ct value for the lymphoma samples with the lowest telomerase activity was 31 and corresponded to only 0.05% of the telomerase activity found in REH cells. From a practical standpoint, telomerase activity with a Ct of >31 cycles is negligible and can be regarded as negative. Therefore, the signals produced by primer-dimer artifacts should not interfere with analysis of results. In addition, like the conventional TRAP assay, the RTQ-TRAP assay may be inhibited by the PCR inhibitors present in samples, which consequently leads to a false-negative result. To rule out this possibility, telomerase-negative samples need to be tested for the presence of PCR inhibitors.
In conclusion, it has been suggested that telomerase is an attractive target for cancer therapy (1). Conceivably, precise quantification of telomerase activity is required for the development of an antitelomerase strategy in the future. For example, information about the half-life of this enzyme would be critical for the design of antitelomerase treatment protocols. Using the RTQ-TRAP assay, we found that the half-life of telomerase activity was 5–11 h, much shorter than the half-life reported previously. Moreover, evaluation of the efficacy of antitelomerase therapy also depends on the reliable measurement of telomerase activity. We believe that the RTQ-TRAP assay may provide a powerful tool for these various purposes.
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Acknowledgments |
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Footnotes |
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2 Parts of this work were presented during the TaqMan User
Meeting of Nordic Countries, February 1999, Oslo, Norway.
3 These authors contributed equally to this work.
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Rn)
of SYBR Green, shown on the y-axis, was plotted against
the cycle number. Ct represents the threshold
cycle at which fluorescence is first detected above the baseline signal
plus 10 SD within 15 cycles. Curves 1–5
represent a protein input of 1, 0.2, 0.04, 0.008, and 0.0016 µg.
Curve 6 is a negative control.
(B), linear relationship between the protein input and
Ct values. The Ct values (obtained from
A) were plotted vs the amounts of protein extracts. All
points represent the mean of quadruplicate amplifications. The SDs were
too small for the error bars to be visible. (C),
specificity of the RTQ-TRAP assay. The amplicons generated by the
RTQ-TRAP were purified, end-labeled with [
