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2554
A Combination of Semiquantative Telomerase Assay
and In-Cell Telomerase Activity Measurement Using
Exfoliated Urothelial Cells for the Detection of
Urothelial Neoplasia
Kazuma Ohyashiki, M.D.1
Naoyuki Yahata, M.D.1
Junko H. Ohyashiki, M.D.1
Hiroshi Iwama, M.D.1
Shigefumi Hayashi, M.D.1
Keiko Ando, M.D.1
Taku Aizawa, M.D.2
Takaaki Ito, M.D.2
Makoto Miki, M.D.2
Yoshiro Ebihara, M.D.3
1
First Department of Internal Medicine, Tokyo
Medical College, Tokyo, Japan.
2
Department of Urology, Tokyo Medical College,
Tokyo, Japan.
3
Second Department of Pathology, Tokyo Medical
College, Tokyo, Japan.
Supported in part by a grant from the Private
School Foundation, a Grant-in-Aid from the Ministry of Health and Welfare for the Second Term
Comprehensive 10-Year Strategy for Cancer Control, Japan, and Kyowa Hakko Co. Ltd. (Tokyo).
The authors thank Professor J. W. Shay (Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center,
Dallas, Texas) for his critical reading of this article.
Address for reprints: Kazuma Ohyashiki, M.D., Tokyo Medical College, 6 –7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160, Japan.
Received January 15, 1998; revision received May
11, 1998; accepted May 11, 1998.
© 1998 American Cancer Society
BACKGROUND. Telomerase is a ribonucleoprotein that synthesizes telomeres. It is
detected in more than 85% of samples obtained from cancer tissues, including
urologic neoplasia. The authors determined telomerase activity semiquantatively
and in-cell telomerase activity in exfoliated urothelial cells obtained from urologic
neoplasia specimens. The goal of this study was to provide additional information
regarding a noninvasive approach to the detection of urologic neoplasia.
METHODS. The authors used voided urine from 23 patients with urologic neoplasia,
2 patients with nonmalignant urologic disorders, and 10 normal individuals. Semiquantative determination of telomerase activity was performed using a fluorescence-based telomeric repeat amplification protocol (TRAP), and telomerase activity at the cellular level was determined by an in situ TRAP assay.
RESULTS. The fluorescence-based TRAP assay detected urinary telomerase activity
in samples from 10 of 13 patients with urologic neoplasia before treatment,
whereas urinary cells obtained from 3 of 10 patients (including 1 patient with
relapse) during or after treatment had detectable telomerase activity. In contrast,
the in situ TRAP assay detected telomerase positive cells in samples from 11 of 13
patients before treatment and 6 of 10 patients during or after treatment. Of note
was a dissociation of the results of the fluorescence-based TRAP assay and those of
the in situ TRAP assay for some patients. Some patients for whom telomerase
activity was not detected with the fluorescence-based TRAP assay had a low
frequency of telomerase positive cells in their urine.
CONCLUSIONS. A combination of semiquantative analysis and an in situ TRAP assay
to detect telomerase positive cells might be a useful tool in the identification and
monitoring of patients with urothelial neoplasia. Cancer 1998;83:2554 – 60.
© 1998 American Cancer Society.
KEYWORDS: telomerase, in situ polymerase chain reaction, fluorescence-based
telomeric repeat amplification protocol, urothelial neoplasia.
T
elomerase is a ribonucleoprotein that synthesizes erosive telomere
(TTAGGG) hexametric repeats.1,2 Telomerase activity is detected
in self-renewal stem cells,3 including hematopoietic stem cells,4,5 hair
follicle cells,6 and the cryptic portion of the small intestine,7 whereas
normal adult somatic cells do not carry this enzyme activity. Human
chromosomal ends, known as telomeres, progressively shorten with
cell replication in vivo and in vitro,8 and it is believed that critical
shortening of telomeres without the presence of telomerase activity
results in the cessation of cell division. In somatic cells, the critical
shortening of telomere length may induce cell death.9,10 Although
In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al.
telomeres of cancer cells might also be shortened with
cell replication, up-regulation of telomerase activity
may compensate for telomere length erosion and
maintain cellular proliferation.9,10 It has been demonstrated that approximately 86% of primary cancer tissues carry telomerase activity,11 and therefore telomerase activity is considered a new marker for cancer
cells.12,13
Telomerase activity is detected using a polymerase chain reaction (PCR)– based assay called telomeric
repeat amplification protocol (TRAP).14,15 With the
sensitive TRAP assay, it has been determined that
most bladder carcinomas carry telomerase activity.14,16 –18 Lin et al. demonstrated that most bladder
carcinomas with high telomerase activity were in an
advanced stage and had deep invasion,16 whereas
other researchers have reported no recognizable correlation between tumor stage and the detectability of
telomerase activity,17,18 thus suggesting that telomerase reactivation might occur in the early stages of
carcinogenesis. This finding further indicates that detection of telomerase activity might be useful in the
early detection of bladder carcinoma.
Yoshida et al. reported that natural voided urine
specimens from 16 of 26 patients (62%) with bladder
carcinoma contained detectable telomerase activity,18
whereas Muller et al. failed to detect telomerase activity in urine specimens; they detected telomerase activity in 73% of patients with bladder carcinoma using
bladder washings by physiologic saline.17 To understand the discrepancies among the reports and to
provide further insight into the early detection of
urothelial neoplasia, we identified telomerase positive
cells in the urinary exfoliated cells using a recently
developed in situ TRAP assay.19 This technique allowed us to detect telomerase activity at the single cell
level. The goal of this study was to obtain more information about the detection and noninvasive follow-up of patients with urothelial neoplasia.
METHODS
Samples
Exfoliated cells were collected from 100 mL of freshly
obtained, naturally voided urine from 23 patients with
urologic neoplasia. We also used urine samples obtained
from 2 patients with nonmalignant urothelial disorders
(one each with renal stone and hydronephrosis). Half
the urine was used to detect telomerase activity using
fluorescence-based TRAP assay, and the remaining was
used for in situ TRAP assay. The voided urine was collected in a centrifuge tube (50 mL) and immediately
cooled on ice and centrifuged. To avoid contamination
of red blood cells that interfere with PCR, the pelleted
cells were resuspended in 30 mL of filtrated hypotonic
2555
solution (1.2114 g Tris, 0.7456 g KCl, and 0.2033 g MgCl26H2O to 1 liter H2O) and pipetted 15 times and then 10
mL filtrated hypertonic solution (16 g NaCl, 0.4 g KCl,
5.795 g Na2HPO4-12H2O, and 0.4 g KH2PO4 to 1 liter
H2O) was added immediately.20 Half the suspended cells
were centrifuged, and the cell pellet was stored at – 80°C
until analysis of telomerase activity. The remaining half
of the suspended cells were cytospinned (at 400 rpm for
3 minutes) on silane-coated nonfluorescence glass slides
and air-dried by fan (within 5 minutes). We used COLO
#320DM (the colon carcinoma cell line) as a positive
control.
Fluorescence-Based TRAP Assay
Telomerase activity was assessed according to the
method of Kim et al.,14,15 with minor modifications.21,22 After the addition of 10 –50 ␮L telomerase
assay lysis buffer (0.5% CHAPS [3-cholamidopropyldimethylammonio-1-propane-sulfate; Pierce Chemical Co., Rockford, IL], 5 mM mercaptoethanol [Sigma
Chemical Co., St, Louis, MO], and 0.1 mM AEBSF
[4-(2-aminothyl)-benzensulfonyl-fluoride-hydrochlorine, ICN Biomedicals, Inc., Irvine, CA]), the cells were
lysed on ice. The lysate was incubated on ice for 30
minutes and then centrifuged at 10,000 relative centrifugal force for 30 minutes at 4°C. The supernatant
was collected, and the protein content was determined by standard procedures (BCA protein assay). A
volume of 6 ␮g or 0.6 ␮g protein equivalent was added
to a 48 ␮L reaction solution consisting of TRAP buffer
(Oncor Inc., MD), dNTP’s mix (Oncor), TRAP primer
mix (Oncor), 2 units Taq polymerase (Takara Shuzo,
Shiga, Japan), and 10 pmol FITC-labeled TS primer
(5´-AAT CCG TCG AGC AGA GTT-3´: 5´-end labeling
using FluorePrimeTM (Pharmacia Biotech, Uppsala,
Sweden). The PCR conditions were 30 cycles of 94°C
for 30 seconds, 55°C for 30 seconds, and 72°C for 15
minutes. We used a TRAP-eze detection kit (Oncor)
that included 36 bp internal standard,23 FITC-labeled
TS primer, and an automated laser fluorescence (ALF)
DNA sequencer II (Pharmacia Biotech), and we performed analysis with the Fragment Manager program
(Pharmacia Biotech).21,22 The PCR products (1.5 ␮L)
were subjected to 12% acrylamide denaturing gel electrophoresis. The telomerase activity signals in each
sample were normalized to the signal from the internal standard, then expressed as a relative value.
In Situ TRAP Assay
In situ PCR was performed as described previously.19
Twenty-five ␮L containing 20 mM Tris HCl (pH 8.3),
1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM
ethyleneglycoltetraacetic acid, 50 ␮M deoxynucleoside triphosphates, 1 ␮g of T4 gene 32 protein (Boehr-
2556
CANCER December 15, 1998 / Volume 83 / Number 12
inger Mannheim, Indianapolis, IN), bovine serum albumin (0.1 mg/mL), 2 units of Taq DNA polymerase,
and 10 pmol FITC-labeled TS forward-primer (5⬘-AAT
CCG TCG AGC AGA GTT-3⬘) were placed within each
frame, and the slides were incubated for 30 minutes at
22°C. After TS extension, 25 ␮L of the same solution
but with 10 pmol FITC-labeled CX reverse-primer (5⬘CCC TTA CCC TTA CCC TTA CCC TAA-3⬘) were added,
coverslips were sealed, and the solution was heated to
90°C for 1.5 minutes to inactivate telomerase and then
amplified using a Hybrid OmniSlide System thermocycler (National Labnet Co., Woodbridge, NJ). The
PCR conditions were 30 cycles of 94°C for 30 seconds,
50°C for 30 seconds, and 72°C for 1.5 minutes. Frames
and top liners for the in situ PCR were removed completely, and slides were washed in tap water and then
sealed with a cover glass using MacIlvaine buffer/
glycerin solution (1:1 ⫽ volume:volume). Cells were
observed with a fluorescence microscope using a Bfilter (Nikon, Tokyo, Japan). At least 100 cells of each
specimen were examined, and cells with a fluorescence positive nucleus were considered positive for
telomerase activity. When a cell had brighter fluorescence in the cytoplasm than in the nucleus, the cell
was considered negative for telomerase activity. Because the primers themselves were fluorescent, we
performed in situ TRAP without PCR as a control,
whereas other controls (data not shown) included using only one primer, PCR without primers, or PCR
without Taq polymerase as previously described.19
RESULTS
Fluorescence-Based TRAP Assay to Detect Telomerase
Activity in Exfoliated Urothelial Cells from Urologic
Neoplasia
Fluorescence-based TRAP assay could detect low levels
of telomerase activity semiquantatively.21,22 In the exfoliated urothelial cells, telomerase activity was detected in
10 of 13 untreated patients (relative telomerase activity
ranging from 0.68 to 30.3 in the 10 patients) with various
forms of urologic neoplasia, including 7 of 7 with bladder
carcinoma, 1 of 2 with renal cell carcinoma, 1 of 3 with
renal pelvic tumor, and 1 patient with renal metastasis of
lung carcinoma (Table 1 and Fig. 1). One of the 2 patients with bladder carcinoma after surgery (Patient 11)
showed telomerase activity, and 1 patient (Patient 10)
with bladder carcinoma who received chemotherapy
had telomerase activity in the urine (Table 1). Of the 7
patients with bladder carcinoma before treatment, 2 patients (Patients 3 and 7) had negative results when 6 ␮g
protein were applied to the CHAPS extract, but both of
them had detectable telomerase activity after dilution,
indicating the presence of inhibitors of PCR in the samples (Fig. 1).
In voided urine from 10 normal individuals and 2
patients with nonmalignant urologic disorders (Patients 24 and 25), no telomerase activity was detectable (Table 1 and Fig. 1).
Determination of In-Cell Telomerase Activity Using an In
Situ TRAP Assay
Six patients with bladder carcinoma before treatment
had a high frequency of fluorescence positive cells (ⱖ3
cells with bright fluorescence in each field of ⫻330
magnification) in the urine, and all of them had telomerase activity using fluorescence-based TRAP assay (“positive/frequent” in Table 1 and Fig. 2A). It is
noteworthy that 2 patients (Patients 4 and 7) had Class
II/IIIa cytology, but both fluorescent-based TRAP and
in situ TRAP assay demonstrated telomerase activity
in the urine (Table 1). In these patients, bright fluorescent signals were detected in the nuclear portion.
In Patient 9 (bladder carcinoma during irradiation),
although TRAP failed to detect telomerase activity,
cells with fluorescence signals only in the cytoplasmic
area were detected (Fig. 2B). Cytoplasmic fluorescence
without bright fluorescence in the nucleus is detected
in some normal resting lymphocytes as well;19 thus,
the signal may represent nonspecific fluorescence
commonly observed in normal lymphocytes.
In Patient 15 (prostate carcinoma during irradiation), although the TRAP assay failed to detect periodic 6
bp telomerase signals, a minor population of urinary
cells had fluorescence in the nuclear area (“positive/
rare” in Table 1 and Fig. 2C). The dissociation similar to
that observed in Patient 15 was also noted in 1 patient
with bladder carcinoma at relapse (Patient 8), 2 with
prostate carcinoma who underwent irradiation (Patients
15 and 16), and 1 with renal pelvic tumor before treatment (Patient 20). In patients with renal cell carcinoma,
in-cell telomerase activity was detected only in a patient
in whom telomerase activity was revealed by a fluorescence-based TRAP assay (Table 1).
In 1 patient with ureter carcinoma (Patient 22)
and 1 with renal stone (Patient 25), TRAP assay failed
to detect telomerase activity; however, an in situ TRAP
assay demonstrated granular fluorescent signals in the
cytoplasmic area without bright fluorescence in the
nuclear portion (Fig. 2D): the granular fluorescence
positive cells without bright nuclear fluorescence
might have been neutrophils because they had lobulated nuclei.19 Thus, this fluorescence pattern represented nonspecific staining for neutrophils without
telomerase activity.
We also analyzed 12 samples obtained from normal subjects and subjects with nonmalignant urologic
disorders. Only 1 patient with renal stone had granular
fluorescence in the cytoplasmic area without bright
In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al.
2557
TABLE 1
Clinical Findings and Telomerase Activity in Exfoliated Urinary Cells from Patients with Urologic Disorders
Telomerase
activityb
Diagnosis No.
Bladder carcinoma
1
2
3
4
5
6
7
8
9
10
11
12
Prostate carcinoma
13
14
15
16
Renal cell carcinoma
17
18
Renal pelvic tumor
19
20
21
Ureter carcinoma
22
Lung carcinoma
metastasis
23
Nonmalignant urologic
disorders
Renal stone
24
Hydronephrosis
25
Clinical
stage
Pathology
(6 ␮g)
(0.6 ␮g)
In situ TRAPc
No. of positive
cells/no. of
cells examined
Age/gender
Status
Cytology
(class)a
58/M
56/M
74/M
71/F
65/M
49/M
65/F
75/M
75/M
66/F
56/F
84/M
Preoperation
Preoperation
Preoperation
Preoperation
Preoperation
Prechemotherapy
Prechemotherapy
Relapse
During irradiation
During chemotherapy
Postoperation
Postoperation
V
V
V
II
V
V
IIIa
II
II
V
II
V
T2N0M0
T3N0M0
T3bN0M0
T3bN0M0
T3N0M0
T3N1M0
T3N0M0
T3N0M0
T1N0M0
T3N1M0
T2N0M0
T1N0M0
TCC, G3⬎G2
TCC, G3
TCC, G3⬎G2
TCC, G2
TCC, G1⬎G2
TCC, G3
TCC, G2
TCC, G3
TCC, G3
TCC, G2
TCC, G3
TCC, G3
5.87
ND
0
ND
ND
2.34
0
0
ND
1.35
ND
ND
1.5
2.27
1.08
0.86
25.6
0.68
1.48
0
0
13.2
3.5
0
Positive/frequent
Positive/frequent
Positive/frequent
Positive/rare
Positive/frequent
Positive/frequent
Positive/frequent
Positive/rare
Cytoplasmic
Positive/rare
Positive/rare
Negative
56/200
73/200
35/200
8/200
113/200
68/100
21/100
3/100
0/200
7/100
11/100
0/200
59/M
Postoperation
NA
T2N0M0
0
0
Negative
0/200
72/M
82/M
69/M
Chemotherapy
During irradiation
During irradiation
II
NA
II
T2N0M1
T3N0M0
T2N0M0
Well ⬍⬍ poorly diff.
adeno
Moderately diff. adeno
Poorly diff. adeno
Poorly diff. adeno
ND
ND
ND
2.07
0
0
Positive/frequent
Positive/rare
Positive/rare
22/100
8/100
6/100
68/M
60/M
Preoperation
Preoperation
II
II
T3N0M0V0
T3N0M0
Alveolar clear, G2
Alveolar clear, G2⬎G1
ND
ND
0
30.03
Negative
Positive/frequent
0/200
65/200
44/M
64/M
67/F
Preoperation
Preoperation
Postoperative (Rel)
IIIb
IIIb
II
T2N0M0
T2N0Mx
TaN0M0
TCC, G2⬎⬎G3
TCC, G2⬎G1⬎G3
TCC, G2
ND
0
ND
2.25
0
0
Positive/rare
Positive/rare
Negative
11/200
5/100
0/200
70/F
Preoperation
IIIb
TCC, G3
0
0
Cytoplasmic
granular
0/100
64/M
Preoperation
II
Poorly diff. adeno
ND
3.48
Positive/frequent
33/100
27/M
IIIa
ND
0
Cytoplasmic
granular
0/100
65/M
I
ND
0
Negative
0/100
TRAP: telomeric repeat amplification protocol; TCC: transitional cell carcinoma; CHAP: cyclophosphamide, hexamethylmelamine, doxorubicin, and cisplatin; NA: no available data; ND: not done; diff. adeno:
differentiated adenoma.
a
Cytology: The data at the time of examination of telomerase activity.
b
Telomerase activity: Parentheses indicate relative telomerase activity (the protein content in the CHAP extract).
c
In situ TRAP: Positive: positive cells for nuclear fluorescence; Cytoplasmic: cells with weak positive fluorescence in the cytoplasmic area without bright fluorescence at nucleus; Cytoplasmic granular: cells with
granular positive fluorescence in the cytoplasmic area; Negative: only background fluorescence; Frequent: ⱖ3 cells with bright fluorescence in the field of 330 magnification; Rare: ⬍3 cells with bright fluorescence
in the field of 330 magnification.
nuclear fluorescence, and the remaining 11 had only
weak background fluorescence.
Association between Cytology and Telomerase Activity
As shown in Table 1, 5 patients (Patients 1, 2, 3, 5, and 6)
with bladder carcinoma and Class V cytology before
treatment had telomerase activity in their exfoliated
urothelial cells according to a fluorescence-based TRAP
assay and a high frequency of telomerase positive cells
according to an in situ TRAP assay. In contrast, some
patients (Patients 4, 7, 8, 11, 14, 16, 18, 19, 20, and 23)
with Class II/III cytology had in-cell telomerase activity
2558
CANCER December 15, 1998 / Volume 83 / Number 12
DISCUSSION
FIGURE 1. Representative fluorocurves show telomerase activity (telomerase
signals). Multiple peaks correspond to telomeric repeats that were synthesized
by the presence of telomerase in exfoliated urothelial cells obtained from
patients with urologic neoplasia and in a colon carcinoma cell line,
COLO#320DM. To quantitate the telomerase activity, we used the internal
standard showing its peak at 36 bp. The results analyzed by the Fragment
Manager system and the area of telomerase signals is compared with the area
of internal standard signal.21,22 In each subject, the size markers (50 bp and
100 bp) are shown on top. Lane 2: Patient 6 (bladder carcinoma, Class V
cytology); Lanes 3 and 4: Patient 3 (bladder carcinoma, Class V); Lane 5:
Patient 18 (renal cell carcinoma, Class II); Lane 6: Patient 14 (prostate
carcinoma, Class II); Lane 7: Patient 23 (lung carcinoma metastasis, Class II);
Lane 8: urine sample from a normal individual; Lane 9: COLO#320DM. In
Patient 3, multiple peaks were detected after dilution (Lanes 3 and 4). In Patient
14 (prostate carcinoma, Class II cytology; Lane 6), small but detectable 6 bp
periodic multiple peaks corresponding to telomerase activity were noted.
in the urine, although for 3 of them (Patients 8, 16, and
20) fluorescence-based TRAP assay failed to detect telomerase activity. The dissociation might be due to the
limitation of cytologic examination rather than contaminated lymphocytes, because all the patients with telomerase activity in their urine using a fluorescence-based
TRAP assay had bright fluorescence positive cells with
various frequencies using an in situ TRAP assay.
We demonstrated that a fluorescence-based TRAP assay could detect telomerase activity semiquantatively
in voided urine obtained from various urothelial neoplasia. None of the normal individuals had telomerase
activity detected, indicating that the telomerase activity detected in the urinary sample may be derived
from exfoliated cancers of the patients. Although Műller et al. failed to detect telomerase activity in naturally
voided urine using either original TRAP or fluorescence-based TRAP assay.17 Yoshida et al. reported that
62% of the bladder carcinomas had telomerase activity
in the exfoliated urine,18 supporting our results that in
urinary samples from patients with urologic neoplasias, especially bladder carcinoma, telomerase activity
can be detected. Yoshida et al. used protease inhibitors to determine urinary telomerase,18 in contrast to
the report of Muller et al.17 We used freshly obtained
voided urine and eliminated red blood cells that interfered with PCR by using hypotonic/hypertonic solution; thus, this procedure could be used to determine telomerase activity in the urothelial exfoliated
cells from patients with bladder carcinoma. Our experiments using two different concentrations of protein in the CHAPS extract demonstrated the presence
of PCR inhibitors in some samples, although Lin et al.
did not find any bladder carcinoma that had PCR
inhibitors.16 More recently, Kinoshita et al. reported
that telomerase activity was detected in exfoliated
cells in 23 of 42 voided urine specimens (55%) and in
36 of 43 bladder-washing fluid samples obtained from
patients with bladder carcinoma, and they concluded
that telomerase activity was more sensitive in detecting the presence of cancer than standard urine cytologic examination.24
We then applied an in situ TRAP assay to detect
telomerase positive cells. We demonstrated that exfoliated urothelial cells from patients with various forms
of urologic neoplasia showing telomerase activity using a fluorescence-based TRAP assay usually contained a high frequency of cells with bright fluorescence in the nuclei that may have corresponded to
telomerase activity. In some patients without detectable telomerase activity using a TRAP assay, an in situ
TRAP assay demonstrated a minor population of telomerase positive cells. Thus, an in situ TRAP assay to
detect telomerase positive cells might be a powerful
approach in the early detection and monitoring of
urologic neoplasia during and after treatment.
A high incidence of telomerase activity has been
demonstrated in prostate carcinoma cases, 28 of 31
(90%) by Lin et al.25 and 21 of 25 (84%) by Sommerfeld
et al.26 Although the number of urinary specimens
In-Cell Telomerase Activity in Urothelial Neoplasia/Ohyashiki et al.
2559
FIGURE 2. The fluorescent signals shown correspond to telomerase activity in exfoliated urothelial cells obtained from patients with urologic neoplasia and from
a normal individual. Many urinary cells (“positive/frequent” in Table 1) obtained from a patient with untreated bladder carcinoma (Patient 6) had bright fluorescence
corresponding to telomerase activity (A). In Patient 12 (bladder carcinoma postoperation), only cells with cytoplasmic fluorescence without nuclear bright
fluorescence were detected (B); possibly these cells were lymphocytes. In the urinary sample obtained from a patient with bladder carcinoma during chemotherapy
(Patient 10), a minor population (“positive/rare” in Table 1) of urothelial cells showed bright fluorescent signals at the nuclei (C). Punctate fluorescence in cytoplasm
without bright fluorescence in the nucleus was noted in the urinary specimen obtained from a patient with renal stone (Patient 25) (D). This pattern may represent
nonspecific fluorescence in neutrophils.
from patients with prostate carcinoma in this study
was small, we identified one patient with telomerase
activity by using fluorescence-based TRAP assay and
revealed with an in situ TRAP assay that three of four
patients had telomerase positive cells during treatment, indicating that using a urinary specimen to
monitor prostate carcinoma might also be possible.
This is in accordance with renal cell carcinoma,27 because we found that 1 of 2 patients had telomerase
activity, indicating that these techniques are applicable in not only bladder carcinoma but also in other
forms of urologic neoplasia presenting hematuria.
Our current study demonstrates that the detection
of telomerase activity semiquantatively and of telomerase positive cells using an in situ TRAP assay could
provide additional information regarding the monitoring of patients with urologic neoplasia, because the in
situ TRAP assay might distinguish telomerase positive
cancer cells from infiltrated lymphocytes and detect a
minor population of telomerase positive cells.
REFERENCES
1.
2.
3.
4.
5.
6.
Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989;59:521–9.
Blackburn EB. Telomerases. Annu Rev Biochem 1992;61:
113–29.
Wright WE, Piatyszek MQ, Rainey WE, Byrd W, Shay JW.
Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 1996;18:173–9.
Hiyama K, Hirai Y, Koizumi S, Akiyama M, Hiyama E, Piatyszeck MA, et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol
1995;155:3711–5.
Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S.
Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995;85:2315–20.
Ramirez RD, Wright WE, Shay JW, Taylor RS. Telomerase
activity concentrates in the active segments of in human
hair follicles. J Invest Dermatol 1997;108:113–7.
2560
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
CANCER December 15, 1998 / Volume 83 / Number 12
Hiyama E, Hiyama K, Tatsumoto N, Shay JW, Yokoyama T.
Telomerase activity in human intestine. Int J Oncol 1996;9:
453– 8.
Harley CB. Telomere loss: mitotic clock or genetic time
bomb? Mutat Res 1991;256:271– 82.
Shay JW, Werbin H, Wright WE. Telomere shortening may
contribute to aging and cancer. Mol Cell Diff 1994;2:1–21.
Holt SE, Shay JW, Wright WE. Refining the telomere-telomerase hypothesis of aging and cancer. Nature Biotechnol
1996;14:1–5.
Shay JW, Bacchetti S. A survey of telomerase activity in
human cancer. Eur J Cancer 1997;33:787–91.
Breslow RA, Shay JW, Gadzar AF, Srivastava S. Telomerase
and early detection of cancer: a National Cancer Institute
workshop. J Natl Cancer Inst 1997;89:618 –23.
Ryu MS. Telomeres, telomerase, and immortality. J Natl
Cancer Inst 1995;87:884 –94.
Kim NW, Piatyszeck MA, Prowse KR, Harley CB, West MD,
Ho PLC, et al. Specific association of human telomerase
activity with immortal cells and cancer. Science 1994;269:
2011–5.
Piatyszek MA, Kim NW, Weinrich SL, Hiyama K, Hiyama E,
Wright WE, et al. Detection of telomerase activity in human
cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci 1995;17:1–15.
Lin Y, Miyamoto H, Fujinami K, Umemura H, Hosaka M,
Iwasaki Y, et al. Telomerase activity in human bladder cancer. Clin. Cancer Res 1992;9:929 –32.
Müller M, Heine B, Heicappell R, Ermich T, Hummel M,
Stein H, et al. Telomerase activity in bladder cancer, bladder
washings and in urine. Int J Oncol 1996;9:1169 –73.
Yoshida K, Sugino T, Yahara H, Woodman A, Bolodeoku J,
Nargund V, et al. Telomerase activity in bladder carcinoma
and its implication for noninvasive diagnosis by detection of
exfoliated cancer cells in urine. Cancer 1997;79:362–9.
19. Ohyashiki K, Ohyashiki JH, Nishimaki J, Toyama K, Ebihara
Y, Kato H, et al. Cytological detection of telomerase activity
using an in situ telomeric repeat amplification protocol
assay. Cancer Res 1997;57:2100 –3.
20. Ito Y, Kuratsuji T, Aizawa S, Sai M, Ohyashiki K, Toyama K.
Superoxide anion production and expression of cytochrome
b558 by neutrophils are impaired in some patients with
myelodysplastic syndromes. Ann Hematol 1991;63:270 –5.
21. Ohyashiki JH, Ohyashiki K, Sano T, Toyama K. Non-radioisotopic and semi-quantitative procedure for terminal repeat amplification protocol. Jpn J Cancer Res 1996;87:329 –
31.
22. Ohyashiki JH, Ohyashiki K, Toyama K, Shay JW. A nonradioactive fluorescence-based telomeric repeat amplification protocol to detect and quantitate telomerase activity.
Trends Genet 1996;112:395– 6.
23. Holt SE, Norton JC, Wright WE, Shay JW. Comparison of the
telomeric repeat amplification protocol (TRAP) to the new
TRAP-eze telomerase detection kit. Methods Cell Sci 1996;
18:237– 48.
24. Kinoshita H, Ogawa O, Kakehi Y, Mishina M, Mitsumori K,
Itoh N, et al. Detection of telomerase activity in exfoliated
cells in urine from patients with bladder cancer. J Natl
Cancer Inst 1997;89:724 –30.
25. Lin Y, Umemura H, Fujinami K, Hosaka M, Harada M,
Kubota Y. Telomerase activity in primary prostate cancer.
J Urol 1997;157:1161–5.
26. Sommerfeld H-J, Meeker AK, Piatyszek MA, Bova GS, Shay
JW, Coffey DS. Telomerase activity: prevalent marker of
malignant human prostate tissue. Cancer Res 1996;56:218 –
22.
27. Fiedler W, Dahse R, Schlichter A, Junker K, Kosmehl H, Ernst
G, et al. Telomerase activity and telomere length in different
areas of renal cell carcinoma. Int J Oncol 1996;9:1227–32.
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