ã 2003 Oxfrord University Press
2 e4
A rapid biosensor chip assay for measuring of
telomerase activity using surface plasmon resonance
Chihaya Maesawa*, Toru Inaba1, Hidetoshi Sato, Sin Iijima1, Kaoru Ishida1,
Masanori Terashima2, Ryo Sato, Michihiro Suzuki, Akiko Yashima, Satoshi Ogasawara1,
Hiroki Oikawa, Nobuhiro Sato1, Kazuyoshi Saito1 and Tomoyuki Masuda
Department of Pathology and 1Department of Surgery, Iwate Medical University School of Medicine,
Uchimaru 19-1, Morioka 202-8505, Japan and 2Department of Surgery 1, Fukushima Medical University,
Fukushima 960-1295, Japan
Received July 15, 2002; Revised September 24, 2002; Accepted November 2, 2002
ABSTRACT
Considerable interest has been focused on telomer-
ase because of its potential use in assays for cancer
diagnosis, and for anti-telomerase drugs as a
strategy for cancer chemotherapy. A number of
assays based on the polymerase chain reaction
(PCR) have been developed for evaluation of telo-
merase activity. To overcome the disadvantages of
the conventional telomerase assay [telomeric repeat
ampli®cation protocol (TRAP)] related to PCR arti-
facts and troublesome post-PCR procedures, we
have developed a telomeric repeat elongation (TRE)
assay which directly measures telomerase activity
as the telomeric elongation rate by biosensor tech-
nology using surface plasmon resonance (SPR).
5¢-Biotinylated oligomers containing telomeric
repeats were immobilized on streptavidin-pretreated
dextran sensor surfaces in situ using the BIACORE
apparatus. Subsequently, the oligomers associated
with the telomerase extracts were elongated in the
BIACORE apparatus. The rate of TRE was calculated
by measuring the SPR signals. We examined
elongation rates by the TRE assay in 18 cancer and
three normal human ®broblast cell lines, and
12 human primary carcinomas and matching normal
tissues. The elongation rates increased in a concen-
tration- and time-dependent manner. Those of
cancer cells were two to 10 times higher than ®bro-
blast cell lines and normal tissues. Telomerase
activities and its inhibitory effects of anti-telomerase
agents as measured by both the TRE and TRAP
assays showed a good correlation. Our assay allows
precise quantitative comparison of a wide range of
human cells from somatic cells to carcinoma cells.
TRE assay is suitable for practical use in the assess-
ment of telomerase activity in preclinical and clinical
trials of telomerase-based therapies, because of its
reproducibility, rapidity and simplicity.
*To whom correspondence should be addressed. Tel: +81 19 651 5111; Fax: +81 19 629 9340; Email: chihaya@iwate-med.ac.jp
Nucleic Acids Research, 2003, Vol. 31, No.
DOI: 10.1093/nar/gng004
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INTRODUCTION
The telomere is a unique chromosomal structure consisting of
tandem GT-rich repeats (TTAGGG)n, which protects the
termini of linear chromosomes from fusion and degradation
(1). In normal somatic cells, chromosomes lose ~50±200 nt of
telomeric sequence per cell division because of the so-called
DNA end-replication problem (2). This progressive telomere
shortening with each cell division leads to cellular senescence
(1). In contrast to somatic cells, immortal and germ cells have
a mechanism to maintain their telomeric length. Telomerase is
a ribonucleoprotein enzyme complex, including a unique
reverse transcriptase that elongates telomeric DNA (1). Its
activity is thought to be required for the development of
cellular immortality and oncogenesis (1). Thus, considerable
interest is focused on telomerase because of its potential uses
in assays for cancer diagnosis, research into cell biology
and for anti-telomerase drugs as a strategy for cancer
chemotherapy (1).
A number of telomerase assays have been developed over
the last decade (3±7). They are categorized into two major
subgroups: polymerase chain reaction (PCR)-based and
non-PCR-based methods. Telomerase activity was initially
measured in vitro by a primer extension assay, in which
telomerase synthesizes telomeric repeats onto oligonucleotide
(TTAGGG)n primers (3,4). However, this telomerase assay is
not sensitive enough to detect telomerase activity in tissue
samples. To increase the sensitivity of telomerase detection,
Kim et al. (5) developed a telomeric repeat ampli®cation
protocol (TRAP) assay, based on PCR. The TRAP assay still
had several limitations for quantifying telomerase activity, as
described by the authors (5). The major limitations of the
TRAP assay are related to PCR-derived artifacts. There have
been several attempts to improve the methods for quanti®ca-
tion of telomerase activity, such as including internal controls,
a standard for telomerase-positive cell lines or a quanti®cation
control, as supplied in the commercially available TRAP
assays. Despite these efforts, the disadvantages of the TRAP
assay have remained for the necessary post-PCR procedures
that involve the separation of the PCR products by gel
electrophoresis and their evaluation by phosphorimager or
densitometry.
e4 Nucleic Acids Research, 2003, Vol. 31, No. 2 PAGE 2 OF 6

Biosensor technology using surface plasmon resonance

(SPR) is broadly applicable for measurement of biological
activity (8). SPR techniques can measure small local changes
in refractive index, linked directly to alterations in concentra-
tion on a surface (8). Buckle et al. (9) showed that SPR allows
quanti®cation of the relatively small changes in mass associ-
ated with polymerization. Recent reports have also demon-
strated that real-time elongation of oligonucleotides by DNA
polymerase or reverse transcriptase could be measured by SPR
(10±11).
Here, we describe our development of the telomeric repeat
elongation (TRE) assay, a rapid biosensor chip assay using
SPR for the quantitative assessment of telomerase activity.

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The TRE assay eliminates PCR-related artifacts and gives
many advantages (no labeling is required; excellent reprodu-
cibility and rapidity; elimination of PCR-related artifacts; easy
handling using automated one-step analysis).

MATERIALS AND METHODS

Strategy of the TRE assay
The constitution of, and reactions on, the sensor chip surface
that are used in the TRE assay are illustrated in Figure 1.
Initially, 5¢-biotinylated oligomers (TS-5¢B) containing telo-
meric repeats were immobilized on streptavidin-pretreated
dextran sensor surfaces in situ using the BIACORE apparatus
(BIACORE 3000; BIACORE AB, Uppsala, Sweden). After
removing excessive oligomers by washing with a 100-ml pulse
of 1% SDS in 10 mM HEPES, telomerase extracts were
injected across the sensor surface, and we carried out an
association/elongation phase of oligomers and telomerase at
¯ow rates of 5 ml/min. Subsequently, regeneration was
performed by washing with a 100-ml pulse of 1% SDS in
10 mM HEPES at ¯ow rates of 100 ml/min to remove all bound
proteins. SPR measurements were then conducted using a
BIACORE 3000 machine (BIACORE AB). SPR signals were
expressed in resonance units (RU; 1 RU = 1 pg/mm2) and real-
time results were represented as a sensorgram. We de®ned the
elongation value (E-value) as the difference of signals, in RU,
between the baseline value before telomerase extract injection
(baseline 1) and after the regeneration phase (baseline 2). The
rate of TRE was calculated using the E-value.
Samples and preparation of telomerase extracts
Eighteen cancer cell lines and three normal ®broblast cell lines
were maintained under recommended conditions. These cell
lines included three breast cancers (MCF-7, T47-D, ZR75-1;
ATCC, Rockville, MD), ®ve esophageal cancers (TE-2, -3, -4,
-5, -6; Cell Resource Center for Biomedical Research, Tohoku
University, Sendai, Japan), ®ve gastric cancers (MKN7,
MKN28, MKN45, GCIY, GT3TKB; RCB, Tsukuba, Japan),
®ve colonic cancers (SW480, SW837, SW948, SW1417,
LS513; ATCC) and three normal ®broblast cell lines (CSC
2F0-C25 and ACBRI #469; CSC, Kirkland, WA; and CCD
1076Sk; ATCC). Tissues of human carcinomas and matching
normal mucosa were obtained from 12 patients (®ve
esophageal, ®ve gastric and two colonic carcinomas).
Telomerase extracts were prepared by the conventional
method (12). All extracts were diluted to 0.5 mg/ml by 13
CHAPS buffer.
Figure 1. Strategy for the TRE assay. (A) The TS-5¢B oligomers containing
the telomeric repeat sequence (TTAGGG) were immobilized on the strepta-
vidin-pretreated dextran sensor chip surface. This gave the initial baseline
on the sensorgram [baseline 1 in (D)]. (B) Telomerase extracts were injected
through the ¯ow cell and bound to the TS-5¢B oligomers. Elongation of the
immobilized oligomers occurred immediately. This gave the association/
elongation phase on the sensorgram. (C) After ®nishing the injection of the
telomerase extracts, dissociation of binding proteins was observed.
Regeneration was performed to remove all bound proteins. (D) A sensor-
gram of the TRE assay. Real-time monitoring was carried out throughout
the association/elongation, dissociation and regeneration phases. The differ-
ence between baselines 1 and 2 was de®ned as the E-value, which was
equivalent to the weight of the elongated nucleotides.
Immobilization of oligomers containing telomeric
repeats
The TS-5¢B oligomers were as follows: biotin-5¢-AATCCG-
TCGAGCAGAGTTAGGGTTAGGGTTAGGGTTAGGGT-
TAG-3¢. Identical oligomers that were biotinylated at the 3¢
end (TS-3¢B) were also prepared as negative control. The
oligomers (0.125 mg/ml) in 75 ml were injected across a
streptavidin-pretreated dextran sensor surface (SA chip;
BIACORE AB), in situ in the BIACORE apparatus
(BIACORE 3000) at 5 ml/min in 10 mM HEPES, pH 7.4/
150 mM NaCl/10 mM MgCl2 at 37°C. After 1500 RU of
oligomers had been immobilized, the chip surface was washed
three times to remove excess oligomers using a 100-ml pulse of
1% SDS in 10 mM HEPES.
Telomerase extract binding to immobilized DNA
Telomerase extracts were diluted in a TRE buffer (10 mM
HEPES, pH 7.4/150 mM NaCl/10 mM MgCl2/2.5 mM dNTP/
10 mM EGTA) to various concentrations. They were applied
at 5 ml/min across the chip surface for various injection times,
at 37°C. The chip surface was regenerated by washing with a
100-ml pulse of 1% SDS, in 10 mM HEPES, which removed
all protein, at a ¯ow rate of 100 ml/min. The regeneration
PAGE 3 OF 6 Nucleic Acids Research, 2003, Vol. 31, No. 2 e4

conditions were determined by preliminary studies using

NaCl, Gly-HCl and NaOH at several concentrations. After
regeneration, the ¯ow rate was returned to 5 ml/min. We
de®ned the E-value as the difference of RU between the
baseline levels before extract injection and after the regener-
ation phase. All values were determined from three to six
independent assays, and the coef®cients of variation of all data
were con®rmed to be below 5%.
Microrecovery of elongated oligomers and ampli®cation
of 6-bp ladders
First, the mouse monoclonal anti-biotin antibody (DAKO,
Glostrup, Denmark) was immobilized on the CM5 sensor chip

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surface (BIACORE AB) using an amine coupling kit
(BIACORE AB). The TS-5¢B oligomers were injected across
the sensor chip and bound the anti-biotin antibody, with up to
3000 RU. Subsequently, the TRE assay was performed as
described above. The `microrecover' and `bypasswash' pro-
grams of the BIACORE 3000 were used for the automatic
recovery of elongated oligomers. Eluted samples of the sensor
surface were treated with phenol/chloroform and then amp-
li®ed by PCR using the TRAP assay primers. The 5¢ end of the
forward primer was labeled with FAM. The PCR products
were analyzed using an ABI PRISM 310 Genetic Analyzer
with GeneScan Software (ABI, Foster City, CA).
TRAP assay
The TRAP assay was performed using a TRAPeze telomerase
detection kit (Intergen, Purchase, NY). In brief, 2 mg of protein
extract was applied to the TRAP reaction mixture. For each
assay, a negative control and 0.1 amol of the quanti®cation
standard oligonucleotide R8 were used. Telomerase activity
was represented as total product generated (TPG) as described
previously (13). One unit of TPG was de®ned as 0.001 amol of
telomerase substrate primers extended by telomerase present Figure 2. Sensorgrams of the TRE assay, and an electropherogram of 6-bp
in the extract, with at least three telomeric repeats (13). ladders ampli®ed from recovered TS-5¢B oligomers from the sensor chip
surface. (A) The TRE assays on SW480 (red line, E-value 118 RU). Bound
Treatment of telomerase inhibitor for cancer cell lines protein was seen in the sample diluted in the TRE buffer without dNTP, in
which a slight elongation of the oligomers was observed (blue line, E-value
Two breast cancer cell lines (MCF-7 and T47-D) were 12 RU). The heat-inactivated telomerase extract control did not bind to the
subjected to an evaluation of the inhibitory effect of anti- TS-5¢B oligomers, and exhibited no signi®cant increase of E-value above
the baseline level (yellow line, ±1.2 RU). No difference was observed
telomerase agents. Two types of telomerase inhibitors were between the initial baseline and that after the regeneration phase in the TRE
examined. Azidothymidine (AZT) is a reverse transcriptase assay without telomerase extracts (green line, E-value ±4 RU). (B) The
inhibitor that has been studied in great detail, as a possible TRE assay using oligomers biotinylated at the 5¢-biotinylated (TS-5¢B; red
model compound for a telomerase inhibitor (14). Tamoxifen line) and 3¢-biotinylated (TS-3¢B; blue line) in SW480. Extracts bound to
(TAM) is a hormonal agent (anti-estrogen) touted as the TS-3¢B but did not elongate them because of the immobilized 3¢ end on the
sensor chip. (C) Six-base pair ladders were detected as blue peaks from
endocrine treatment of breast cancer. Estrogen activates 44 to 80 bp. Red peaks represent size markers (TAMURA 500, ABI).
telomerase via direct and indirect effects on the hTERT
promotor (15). TAM down-regulates hTERT mRNA (15). Cell
culture and drug exposure have been described (14,15).
Telomerase activity was evaluated by TRE and TRAP assays. When extracts of the telomerase-positive cell line SW480
were injected, the sensorgram immediately showed a steep
slope (Fig. 2A), which gradually became a gentle slope but did
RESULTS not reach a plateau. Equilibrium was not observed during
sample injection on any sensorgrams, because this phase
Preliminary studies involved both the protein binding to the TS-5¢B oligomers and
Preliminary experiments to optimize reaction conditions used elongation of the oligomers. The heat-inactivated telomerase
a cancer cell line (SW480; ATCC) and a human dermal control did not bind to the TS-5¢B oligomers, and showed no
®broblast cell line (CSC 2F0-C25). The optimal buffering and signi®cant increase of the E-value above the background level
temperature conditions were: 10 mM HEPES, pH 7.4/150 mM (<5 RU) (Fig. 2A). Bound protein was seen in the samples
NaCl/10 mM MgCl2/2.5 mM dNTP each/10 mM EGTA at diluted in TRE buffer that did not contain dNTP, and only a
37°C, with a ¯ow rate of 5 ml/min. slight elongation of the oligomers was observed (Fig. 2A). No
e4 Nucleic Acids Research, 2003, Vol. 31, No. 2 PAGE 4 OF 6

Table 1. Telomerase elongation rate in various human biological samples

Cell line Elongation rate Tissue samplea Elongation rate
(mer/min) (mer/min)

Breast cancer Esophageal cancer

T47-D 0.872 EC-T1 0.368
MCF7 0.721 EC-N1 0.056
ZR75-1 0.684 EC-T2 0.624
Esophageal cancer EC-N2 0.089
TE5 0.691 EC-T3 0.452
TE2 0.395 EC-N3 0.012
TE3 0.388 EC-T4 0.553
TE4 0.302 EC-N4 0.026
TE6 0.255 EC-T5 0.321
Gastric cancer EC-N5 0.023

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MKN7 0.739 Gastric cancer
MKN45 0.393 GC-T1 0.669
GCIY 0.370 GC-N1 0.091
GT3TKB 0.331 GC-T2 0.882
MKN28 0.206 GC-N2 0.102
Colon cancer GC-T3 0.396
SW1417 0.632 GC-N3 0.056
SW480 0.446 GC-T4 0.338
SW837 0.441 GC-N4 0.052
SW948 0.303 GC-T5 0.463
LS513 0.231 GC-N5 0.032
Normal ®broblast Colon cancer
CSC 2F0-C25 0.113 CC-T1 0.662
ACBRI #469 0.106 CC-N1 0.052
CCD 1076Sk 0.086 CC-T2 0.712
CC-N2 0.067
aT, tumor tissue; N, non-tumor tissue.

Figure 3. Relationships between E-values and concentration of telomerase
extracts or injection-time. (A) Relationship between E-values and concentra-
tion of telomerase extracts. A colon cancer cell line, SW480 (open circles)
and a normal ®broblast cell (CSC 2F0-C25, closed circles). (B)
Sensorgrams of SW480 for 1- (a), 5- (b), 10- (c) and 30- min (d) injections
at 0.1 mg/ml. (C) E-values with several different injection times, used in
human cancer cell lines (open circles, T47-D; open squares, MCF7; open tri-
angles, SW480) and normal ®broblast cell lines (closed squares, CSC 2F0-
C25; closed triangles, ACBRI #469).
elongation of the TS-5¢B oligomers occurred in the absence of
telomerase extracts (Fig. 2A). Oligomers biotinylated at the
3¢ end (TS-3¢B) were also used as a negative control:
telomerase extracts bound to the TS-3¢B oligomers, but no
5¢ to 3¢ elongation occurred since the 3¢ end was immobilized
on the sensor chip (Fig. 2B). To eliminate the activity of any
polymerase, we also immobilized non-telomeric oligomers
[biotin-5¢-AATCCGTCGAGCAGAGTGTTGGTCAGGCT-
GATCTCAAA: estrogen response element (ERE)] which
were potential estrogen response elements of hTERT promotor
sequences. No signi®cant increase of E-value above the
background level was obtained (<5 RU, Fig. 2A).
To con®rm oligomers elongation on the sensor chip surface,
we recovered the TS-5¢B oligomers and used these as a
template for PCR with primers from the TRAP assay. Six-base
pair ladders were detected from 44 to 80 bp (Fig. 2C).
E-values of SW480 for a 10-min injection increased in a
concentration-dependent manner from 0.025 to 0.150 mg/ml
of cell extract (total amounts of telomerase extracts, 1.25±
7.5 mg), but those of a normal dermal ®broblast cell line (CSC
2F0-C25) were below the background level (<5 RU) until 0.05
mg/ml of cell extract was used (Fig. 3A). We examined
several cancer and ®broblast cell lines by TRE assay for 1-, 5-,
10- and 30-min injections, using 0.1 mg/ml of cell extract, and
found that their E-values increased linearly (Fig. 3B and C).
The coef®cient of variation at each time point was below 5%.
The increased value in RU following immobilization was used
to determine moles of DNA at the chip surface, using an
empirical relationship in which 1500 RU was equivalent to
1.8 ng of DNA (1.276 3 10±13 mol of TS-5¢B oligomer) (9).
An extension of one base on all TS-5¢B oligomers would thus
result in an increase of ~33.8 RU. This value is well above the
background level and should be observable. The elongation
rate was constant in each cell line during any injection time
(Fig. 3C and Table 1).
We could not systemically determine the lower limit of
detection of tumor cells by the dilution method, since the
normal ®broblast cells used in the present study exhibited
weak telomerase activity. But when we dilute 100 T47 breast
cancer cells in 1 3 105 normal ®broblast cells (CCD 1076Sk)
and 0.1 mg/ml of extract were injected for 10 min (total
amounts of telomerase extracts, 5 mg), we found a 10 RU
increase of E-value in comparison with extracts without tumor
cells. No signi®cant increase of E-value was con®rmed when
PAGE 5 OF 6
Figure 4. Correlation between the results of telomerase activities, as
measured by the TRE and TRAP assays.
10 tumor cells were diluted in 1 3 105 CCD 1076Sk cells. The
limit of detection of tumor cells was 1 tumor cell/1000
background cells.
TRE assay using biological samples and comparison
with TRAP assay
We examined elongation rates for 18 cancer cell lines and
three normal human ®broblast cell lines. Tissues from human
carcinomas and matching normal mucosa were obtained from
12 patients (®ve esophageal, ®ve gastric and two colonic
carcinomas), and examined for telomerase activity using the
TRE assay. The elongation rates of cancer cells were found to
Figure 5. Inhibitory effects of telomerase activity for AZT (A) and TAM (B) in two breast cancer cell lines (MCF-7 and T47-D).
Nucleic Acids Research, 2003, Vol. 31, No. 2 e4
be two to 10 times higher than those of normal mucosa and
normal ®broblast cell lines (Table 1). The ®broblast cell
lines gave elongation rates ranging from 0.086 mer/min to
0.133 mer/min, giving total elongations of 2.4 mer to 3.9 mer
for a 30-min injection.
We then examined the same samples of cancer and normal
®broblast cell lines using a standard TRAP assay with the
TRAPeze telomerase detection kit (Intergen). Telomerase
activities as measured by both the TRE and TRAP assays
showed a good correlation between the two assays in cancer
cell lines (Fig. 4). Telomerase activities of ®broblast cell lines
could be evaluated quantitatively by the TRE assay, but failed
to amplify a 6-bp ladder by the TRAP assay. Because the
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minimum length of TRE for successful visualization of
TRAP-laddering is 6 nt, the elongation rates of ®broblast
cell lines (2.4±3.9 mer in 30 min), which were detected by the
TRE assay, might be too low to be ampli®ed in the TRAP
assay.
Inhibitory effects of anti-telomerase agents in breast
cancer cell lines
AZT inhibited telomerase activity by 50±70% in two breast
cancer cell lines (MCF-7 and T47-D) according to TRE and
TRAP assays. 17b-Estradiol (E2) elevated the telomerase
activity (120±150%) and TAM down-regulated it at the non-
treatment level. Results of both assays were well correlated
(Fig. 5).
e4 Nucleic Acids Research, 2003, Vol. 31, No. 2
DISCUSSION
In this paper, we have described how we implemented a
Biosensor chip assay for direct measurement of telomerase
activity, using elongation rate and avoiding PCR-related
artifacts and troublesome post-PCR procedures associated
with various biological samples. The TRE assay gives many
advantages over previous methods (i.e. no labeling is required;
reproducibility and rapidity; PCR-related artifacts are elimin-
ated; easy handling using an automated one-step analysis).
The most signi®cant advantage of the TRE assay is that the
telomerase activity of somatic cells or tissues can be evaluated
quantitatively.
The TRAP assay is currently the most sensitive assay
available for detecting telomerase activity in low cell
numbers, which would be possible, for example, using fewer
than 10 cells with high telomerase expression (16). However,
in the case of low telomerase expression (for example in
somatic cells), the concentration of protein extracts used in the
TRAP assay must be increased. High protein concentration in
the reaction mixture inhibits Taq DNA polymerase in the
conventional TRAP assay, which means that the assay cannot
be used effectively to detect low levels of telomerase
expression. Our present results show that the telomerase
activity of somatic cells may be more than 10 times less than
that of carcinomas. We have found that the TRE assay is
suitable for the precise quantitative comparison of telomerase
activity, in a wide range of human cells from somatic cells to
carcinoma cells.
Most human somatic cells or tissues express low or
undetectable levels of telomerase activity. Hence, continual
cell renewal seen in liver cirrhosis leads to telomere shorten-
ing, resulting in cellular senescence and end-stage organ
failure (1,17). Thus, in theory, external intervention to
increase telomerase activity may prolong the life span of
target cells and avoid organ failure (1,17). On the other hand,
up-regulation of telomerase activity would increase the risk of
cancer development. Precise assessment of telomerase
activity, from levels found in somatic cells to those found in
cancer cells, is needed to allow safe clinical trials of telomere/
telomerase therapeutic agents.
The application of the TRE assay would naturally extend to
the screening for modulators of telomerase activity as
therapeutic agents, but the BIACORE apparatus and sensor
chips are expensive. Telomerase down-regulation and telo-
mere shortening would compel cancer cells to undergo
apoptosis, while up-regulation and elongation might be able
to prolong the life span of target cells and thus preserve organs
(1,17). The TRE assay is intended for practical use in the
assessment of telomerase activity in pre-clinical and clinical
trials of such telomerase-based therapies, because of its
rapidity and simplicity.
PAGE 6 OF 6
ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid 193671332,
13770705 and 13770093 from the Japanese Ministry of
Education, Science, Sports and Culture of Japan.
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