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760
Telomerase Activity in Renal Cell Carcinoma
Ken-ichiro Yoshida, M.D.
Shu-ichi Sakamoto, M.D.
Shuhei Sumi, M.D.
Yotsuo Higashi, M.D.
Satoshi Kitahara, M.D.
Department of Urology, Dokkyo University School
of Medicine, Tochigi, Japan.
BACKGROUND. Telomerase activity has been shown to be increased in numerous
tumors and cell lines, although to the authors’ knowledge there has been no
previous assessment of telomerase activity in renal cell carcinoma (RCC). To
examine whether telomerase activity could be used as a biochemical parameter for
predicting the behavior of RCC, telomerase activity was quantified in RCC samples
and correlated with clinicopathologic findings.
METHODS. Telomerase activity was quantified in 50 RCC samples and 21 samples
of normal renal tissue using fluorescence-based telomeric repeat amplification.
RESULTS. Telomerase activity was detected in 28 of 50 RCC samples (mean 6
standard deviation, 35.03 6 18.47 U/mg protein), whereas there was no telomerase
activity in 21 normal renal tissue specimens. Telomerase activity in tumors classified as pT4 was significantly higher than in tumors classified as pT1 to pT3.
Telomerase activity also correlated with patient age.
CONCLUSIONS. Telomerase activity was increased in RCC samples compared with
normal renal tissues. Although significant associations were observed between
telomerase activity and clinicopathologic parameters, further study is required to
determine whether telomerase activity affects the biologic and clinical behavior of
RCC. Cancer 1998;83:760 – 6. © 1998 American Cancer Society.
KEYWORDS: telomerase activity, renal cell carcinoma, TNM classification, clinicopathologic parameters.
R
Address for reprints: Ken-ichiro Yoshida, M.D.,
Department of Urology, Dokkyo University School
of Medicine, 880 Kitakobayashi, Mibu, Shimotsuga-gun, Tochigi 321-02, Japan.
Received September 29, 1997; revision received
January 12, 1998; accepted February 2, 1998.
© 1998 American Cancer Society
enal cell carcinoma (RCC) is a tumor that varies in its biologic and
clinical behavior.1–3 The biologic behavior of RCC is difficult to
predict from histologic findings.4 Because recurrence and metastasis
can occur years after the primary presentation of RCC and there can
be major differences in clinical outcome even within the same tumor
stages, additional prognostic indicators are needed. Several previous
studies have focused on genes believed to be involved in autocrine
and paracrine mechanisms of tumor proliferation and progression.5–9
In this study the activity of the enzyme telomerase was assessed
as a means of better defining the behavior of RCC. This enzyme is a
ribonucleoprotein that elongates telomeric sequences.10 These sequences are specialized structures of characteristic repetitive DNA
sequences that form specialized nucleoprotein complexes at the ends
of eukaryotic chromosomes. Possible functions of telomere sequences include prevention of chromosome degradation, end-to-end
fusions, rearrangements, and chromosome loss.11 Human telomeres
undergo progressive shortening with each cell division through replication-dependent sequence loss at DNA termini.12 It is believed that
dividing cells undergo only a limited number of replication cycles
unless they activate the telomerase enzyme.13 Recent studies have
demonstrated that a variety of cell lines and malignant tumors have
telomerase activity, whereas normal somatic cells do not. This suggests that telomerase activation is a critical step in cell immortaliza-
Renal Cell Carcinoma and Telomerase Activity/Yoshida et al.
tion and oncogenesis.14 –16 Recent studies also have
demonstrated that telomerase activity can be a useful
indicator of clinical outcome.15,17–21
In this study telomerase activity in RCC was quantified using fluorescence-based telomeric repeat amplification (TRAP) and the TRAP-eze telomerase detection kit (Oncor, Inc., Gaithersburg, MD).22–25 The
relation between telomerase activity and clinicopathologic findings then was examined.
MATERIALS AND METHODS
Patients and Tumors
Fifty previously untreated patients with primary RCC
(35 men and 15 women) diagnosed from January 1993
through April 1997 were studied. The median age was
55 years (range, 31–75 years). All patients underwent
radical nephrectomy with lymph node dissection.
Fifty tumor samples, free from necrotic tissues, and 21
normal tissue samples were obtained from the RCC
patients. Normal renal tissues were defined histologically. All patients consented to the use of their renal
tissues for the current study. After removal of the
tumor, a specimen was frozen immediately in the
operating room and stored at 280 °C until use.
TNM staging of the tumors was determined according to the International Union Against Cancer
(UICC) and the American Joint Committee on Cancer
(AJCC) staging systems.26 Microscopic sections were
evaluated by a pathologist without knowledge of patient clinical characteristics. The classification of the
tumor was determined according to the UICC/AJCC.27
Nuclear grade was determined according to Fuhrman
et al. using the worst area identified.28,29 Of 50 patients, 13 (26%) had distant metastasis. Distant metastasis were present in the lung alone in seven patients,
the lung and liver in three patients, and the lung and
other sites in three patients. Eight patients died of
disease with a median survival of 8 months; 42 were
alive at last follow-up. Patient follow-up ranged from
3– 46 months (median follow-up, 17 months).
Determination of Telomerase Activity
Telomerase activity was quantified with the TRAPeze telomerase detection kit (Oncor Inc.) according
to the manufacturer’s instructions. In brief, frozen
samples (50 mg) were homogenized in 200 mL of icecold 3-[(3-cholamidopropyl)dimethylammonio)]-1propanesulfonate (CHAPS) lysis buffer (containing
10 mM Tris-HCl [pH 7.5], 1.5 mM MgCl2, 1 mM
ethyleneglycoltetraacetic acid [EGTA], 0.1 mM benzaminide, 5 mM 2-mercaptoethanol, 0.5% CHAPS,
and 10% glycerol) and were incubated for 30 minutes on ice. After incubation the lysates were centrifuged at 12,000 3 g for 20 minutes at 4 °C. The
761
resulting supernatant fluids were used in the telomerase assay. The protein concentration was determined with the Coomassie Protein Assay Reagent
(Bio-Rad, Hercules, CA), and an extract aliquot containing 1 mg of protein was used for each telomerase
assay. Extracts were incubated with 0.1 ng fluorescence-labeled TS forward primer (59-AATCCGTCGAGCAGAGTT-39; Pharmacia Biotech, Uppsala,
Sweden) in a master mix; the reaction buffer contained 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63
mM KCL, 0.005% Tween-20, 1 mM EGTA, 50 mM
deoxynucleoside triphosphates, 1 mg T4 gene 32
protein (Boehringer-Mannheim Biochemicals, Indianapolis, IN), 0.01% bovine serum albumin, 2 U of
Taq DNA polymerase (Takara Shuzo, Kyoto, Japan),
and 0.5–5 mg of the CHAPS cell extracts. After a
30-minute incubation at 30 °C, 30 cycles of polymerase chain reaction (PCR) were performed (94 °C for
30 seconds, 60 °C for 30 seconds, and 72 °C for 45
seconds). The products were diluted with an equal
volume of formamide dye solution, heated at 94 °C
for 5 minutes, and 5 mL was applied to each lane of
a 10% denaturing gel containing 6 M urea. The gel
was fitted to an automated DNA sequencer (ALF Red
DNA Sequencer II; Pharmacia Biotech). The temperature of the gel was maintained at 45 °C during
electrophoresis at 45 watts. The data from the DNA
sequencer were collected and analyzed automatically using Fragment Manager VI. I software (Pharmacia Biotech). Each peak was quantified in terms
of size, peak height, and peak area. Telomerase positive cell extracts provided in the kit (TSR 8) were
used as a positive control. Telomerase activity was
determined as telomerase values relative to those of
a positive control: (measured total area of telomerase activity [50 base pair (bp), 56 bp, 62 bp, 68 bp,
etc.])/(measured area of internal control [36 bp]) 3
(measured total area of telomerase activity [50 bp,
56 bp, 62 bp, 68 bp, etc.] in positive control)/(measured area of internal control [36 bp] in positive
control) 3 100. Telomerase activity was expressed as
total product generated (TPG) units/mg protein. All
samples were assayed in duplicate and the mean
value was determined.
Statistical Analysis
Values were expressed as the mean 6 standard deviation. Statistical analyses were performed using the
Mann–Whitney U test and chi-square test. Correlations were examined by Spearman’s rank correlation
procedure. P , 0.05 was considered to be statistically
significant. All statistical analyses were performed using Stat View data analysis software (Abacus Concepts
Inc., Berkeley, CA).
762
CANCER August 15, 1998 / Volume 83 / Number 4
FIGURE 1. A representative assay of telomerase activity in renal cell
carcinoma tissues using the TRAP (telomeric repeat amplification)-eze telomerase detection kit. (Oncor, Inc., Gaithersburg, MD) The first peak is the
fluorescence-labeled TS primer. The second peak is the internal control (36
base pair [bp]). The polymerase chain reaction product of telomerase extension
yielded a 6-nucleotide peak profile (50 bp, 56 bp, 62 bp, 68 bp, 74 bp, etc.).
RESULTS
Telomerase Activity and Clinicopathologic Correlations
A representative telomerase activity assay using
pooled RCC tissues is shown in Figure 1. The first peak
is fluorescence-labeled TS. The second peak is the
internal control (36 bp). The telomerase activity was
detected from the third peak in a shark-tooth pattern,
extending from 50 bp with a 6-nucleotide periodicity
(50 bp, 56 bp, 62 bp, 68 bp, 74 bp, etc.). The calculated
telomerase activity in this case was 44.31 U/mg protein.
Table 1 summarizes the clinicopathologic and
telomerase activity data of 50 RCC cases. Telomerase activity was detected in 28 of 50 RCC samples
(56%). Telomerase activity in RCC specimens ranged
from 0 –94.26 U/mg protein, with a mean value of
19.67 6 23.28 U/mg protein. Telomerase activity was
not detected in 21 normal renal tissues. The telomerase activity of these paired samples are shown in
Table 2. The histologic findings in 22 RCC cases
without telomerase activity were as follows: Cases 3,
12, 3, 4, and none were staged as pT1, pT2, pT3a,
pT3b, and pT4, respectively; Cases 12, 5, 5, and none
were staged as pV0, pV1a, pV1b, and pV2, respectively; Cases 20 and 2 were staged as pN0 and pN1,
respectively; and Cases 17 and 5 were staged as pM0
and pM1, respectively. Fifteen were of conventional
type, 4 were of papillary type, and 3 were of chromophobic type. Eight were nuclear Grade 1, 11 were
nuclear Grade 2, 3 were nuclear Grade 3, and none
was nuclear Grade 4. The RCC samples without telomerase activity did not have any distinct differences in histopathologic findings compared with
RCC samples with telomerase activity.
Comparative studies of telomerase activity in various histopathologic states of the tumor are summa-
rized in Table 3. There was no significant difference in
the mean telomerase activity between male (21.30 6
22.03 U/mg protein; n 5 35) and female patients
(15.69 6 23.13 U/mg protein; n 5 15). Telomerase
activity was not detected in the pT1 samples (n 5 2).
The mean telomerase activity in the pT2 samples was
17.55 6 20.82 U/mg protein (n 5 27). The mean telomerase activity in the pT3a samples was 24.02 6
18.38 U/mg protein (n 5 14), whereas the enzyme
activity was not detected in the pT3b samples (n 5 3).
The mean telomerase activity was highest in the pT4
samples (48.66 6 31.37 U/mg protein; n 5 4), and this
was significantly higher than in the pT2 (P 5 0.0275)
and pT3 samples (P 5 0.0285). In contrast, no significant differences were observed among pT1, pT2, and
pT3 samples. The mean telomerase activity in pV0
tumors was 17.31 6 20.65 U/mg protein (n 5 25), and
was 26.35 6 26.48 U/mg protein (n 5 16) and 14.04 6
17.46 U/mg protein (n 5 9) in pV1a and pV1b tumors,
respectively. The mean value of telomerase activity in
pN0, pN1, and pN2 tumors was 19.00 6 22.93 (n 5 45),
32.35 6 10.44 (n 5 3), and 14.39 6 20.34 U/mg protein
(n 5 2), respectively. The mean value of telomerase
activity in pM0 tumors was 20.07 6 24.32 U/mg protein (n 5 37), which was similar to the mean value of
18.33 6 15.84 U/mg protein (n 5 13) for pM1 tumors.
The mean telomerase activity in conventional type
tumors (21.18 6 23.18 U/mg protein; n 5 37) was
highest, followed by papillary type (18.90 6 21.45
U/mg protein; n 5 9) and chromophobic type tumors
(6.81 6 13.62 U/mg protein; n 5 4). The mean value of
telomerase activity in nuclear Grade 1 tumors was
21.14 6 25.38 U/mg protein (n 5 19), and, in decreasing order, was 19.48 6 20.83 U/mg protein for nuclear
Grade 2 tumors (n 5 26) and 14.53 6 20.70 U/mg
protein for nuclear Grade 3 tumors (n 5 5). The mean
telomerase activity in Stage I and II tumors had similar
values (Stage I: 17.13 6 34.25 U/mg protein [n 5 4] and
Stage II: 15.84 6 19.20 U/mg protein [n 5 22]), whereas
the mean telomerase activity of Stage III and IV tumors was slightly higher than that of Stage I and II
tumors (Stage III: 21.02 6 21.18 U/mg protein [n 5 11]
and Stage IV: 25.58 6 25.43 U/mg protein [n 5 13]). No
significant differences in telomerase activity were
noted among these various histopathologic parameters (Table 3).
Relations between telomerase activity and patient
age, nuclear grade, or tumor stage also were studied. A
significant correlation was observed between telomerase activity and patient age (P 5 0.0250) (Fig. 2),
whereas no significant correlation was observed between telomerase activity and nuclear grade or tumor
stage.
At last follow-up eight patients had died of their
Renal Cell Carcinoma and Telomerase Activity/Yoshida et al.
763
TABLE 1
Clinicopathologic Findings and Telomerase Activity in Renal Cell Carcinoma
Case no.
Gender
Age (yrs)
TNM staging
Classification
Nuclear
grade
Stage
Telomerase
activitya
Clinical
outcome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
M
M
F
F
M
M
M
M
M
M
M
F
F
M
M
F
M
F
M
M
F
F
F
M
M
F
M
M
M
M
M
M
M
F
M
M
F
F
M
M
M
M
M
M
F
M
M
M
M
F
66
55
61
73
67
58
64
74
36
55
41
49
54
74
62
53
52
56
62
31
52
72
50
70
38
75
43
49
54
63
60
55
44
48
73
49
61
44
46
62
55
62
69
51
65
76
56
63
66
70
pT2pV1aN0M0
pT3bpV1bN0M1
pT3apV1aN0M0
pT2pV1aN0M0
pT2pV0N0M0
pT2pV0N1M1
pT2pV0N0M0
pT2pV0N0M0
pT3bpV1bN2M1
pT2pV0N0M0
pT2pV1aN0M0
pT3bpV1aN2M0
pT2pV0N0M0
pT3apV0N0M0
pT3apV1aN0M0
pT2pV0N0M0
pT2pV1aN0M0
pT1pV1aN0M0
pT2pV0N0M0
pT2pV0N0M0
pT3bpV1bN0M1
pT4pV1bN1M1
pT2pV0N0M0
pT3apV1aN0M1
pT2pV1aN0M0
pT3apV1aN0M0
pT3apV1aN0M1
pT3apV1aN0M0
pT3apV0N0M1
pT4pV1aN0M0
pT4pV1bN2M1
pT3apV0N0M0
pT3apV1bN0M1
pT2pV0N0M0
pT4pV1bN1M1
pT2pV0N0M0
pT1pV0N0M0
pT2pV1aN0M0
pT1pV0N0M0
pT2pV0N0M0
pT3apV1bN0M0
pT2pV1aN0M1
pT2pV0N0M0
pT3apV1aN0M0
pT2V0N0M0
pT2V0N0M0
pT2V0N0M0
pT2V1aN0M0
pT2V0N0M0
pT2V0N0M1
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Papillary
Papillary
Conventional
Conventional
Conventional
Chromophobic
Chromophobic
Papillary
Papillary
Conventional
Conventional
Papillary
Conventional
Chromophobic
Chromophobic
Conventional
Papillary
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Conventional
Papillary
Conventional
Conventional
Conventional
Conventional
Conventional
Papillary
Papillary
1
2
2
2
1
2
1
2
2
1
2
3
1
2
2
2
2
2
2
3
2
2
3
3
1
2
1
1
2
1
2
1
2
1
3
1
2
1
1
1
1
2
2
1
1
2
2
2
1
2
II
III
III
II
II
IV
II
II
IV
II
III
III
II
III
III
II
II
I
II
II
IV
IV
II
IV
II
III
IV
III
IV
IV
IV
III
IV
II
IV
II
I
II
I
II
III
IV
II
III
I
II
II
II
I
IV
18.52
0
20.21
44.31
56.42
25.45
20.65
26.86
0
0
52.46
0
0
0
61.64
0
0
0
0
0
0
27.23
0
28.31
0
20.74
29.06
21.72
23.68
94.26
28.77
28.40
0
54.28
44.36
0
68.50
0
0
34.40
26.03
31.46
35.40
0
0
11.83
0
28.01
17.90
0
Alive
DOD
Alive
Alive
Alive
Alive
Alive
Alive
DOD
Alive
DOD
DOD
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
DOD
Alive
Alive
DOD
Alive
Alive
Alive
Alive
Alive
Alive
DOD
Alive
Alive
Alive
DOD
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
M: male; DOD: died of disease; F: female.
a
Telomerase activity is expressed as U/mg protein.
disease. Telomerase activity was detected in the tumors from four of these patients. The mean value of
telomerase activity in those who died of RCC was
19.24 6 22.0 U/mg protein (n 5 8) compared with a
mean value of 19.69 6 22.60 U/mg protein (n 5 42)
among the patients still alive at last follow-up. No
764
CANCER August 15, 1998 / Volume 83 / Number 4
TABLE 2
Telomerase Activity in the Paired Samples of Normal and Carcinoma
Tissues of the Same Kidney
TABLE 3
Relation between Telomerase Activity and Clinicopathologic Features
of Renal Cell Carcinoma
Telomerase activity (U/mg protein)
Case no.
a
1
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
37
46
47
48
49
a
Carcinoma Tissue
Normal Tissue
18.52
0
20.21
56.42
25.45
20.65
26.86
0
0
52.46
0
0
0
61.64
0
0
68.50
11.83
0
28.01
17.90
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Case no. is the same as that in Table 1.
significant difference in telomerase activity was observed between the two groups.
Gender Male
Female
TNM staging
pT1
pT2
pT3
pT4
pV0
pV1a
pV1b
N0
N1
N2
M0
M1
Stage I
II
III
IV
Nuclear grade
1
2
3
Tumor classification
Conventional
Papillary
Chromophobic
Telomerase activitya
No. of
cases
Statistical analysis
21.30 6 22.03
15.69 6 23.13
35
15
NS
0
17.55 6 20.82
18.37 6 19.07
48.66 6 31.37
17.31 6 20.65
26.35 6 26.48
14.04 6 17.46
19.00 6 22.93
32.35 6 10.44
14.39 6 20.34
20.07 6 24.32
18.33 6 15.84
17.13 6 34.25
15.84 6 19.20
21.02 6 21.18
25.58 6 25.43
2
27
17
4
25
16
9
45
3
2
37
13
4
22
11
13
21.14 6 25.38
19.48 6 20.83
14.53 6 20.70
19
26
5
NS
21.18 6 23.18
18.90 6 21.45
6.81 6 13.62
37
9
4
NS
pT2 vs. pT4: P 5 0.0275
pT3 vs. pT4: P 5 0.0285
NS
NS
NS
NS
NS: not significant.
a
Telomerase activity is expressed as U/mg protein.
Assessment of Telomerase Negative Tumors
It is possible that the absence of telomerase activity in
a tumor may result from the presence of an inhibitor.
We examined this hypothesis by mixing extracts from
each of 22 telomerase negative tumors with the extract
from positive control cells. In no cases did inhibition
of the telomerase activity of positive control cells
occur.
DISCUSSION
Telomeres are the specialized structures that contain TTAGGG repeats at the ends of all eukaryotic
chromosomes. They are believed to contribute to
chromosome stabilization by protecting genomic
DNA against degradation and deleterious recombination events.10,30 Using a newly developed PCRbased telomerase assay, researchers have found that
a variety of cell lines and malignant tumors express
telomerase activity,14 –21 unlike normal somatic
cells. This suggests that telomerase activation may
be a critical step in cell immortalization and oncogenesis.
The TRAP method generally has been used for the
FIGURE 2. Relation between telomerase activity and patient age. A significant correlation was observed between telomerase activity and patient age in
renal cell carcinoma (correlation coefficient 5 0.317; P 5 0.0250).
detection of telomerase activity. However, it is a timeconsuming procedure that uses a radioisotope labeling system and has difficulties with regard to quantitation. To overcome these problems we used the
Renal Cell Carcinoma and Telomerase Activity/Yoshida et al.
TRAP-eze telomerase detection kit in the current
study. In this method telomerase activity is represented by fluorescence curves, and the peak area is
calculated automatically. The introduction of an internal standard allows telomerase activity to be measured in real time.21–25
The current study demonstrated telomerase activity in 28 of 50 RCC samples but no activity in normal
renal tissues. These findings suggest that activation of
telomerase may be a sign of immortalization and an
important feature of RCC oncogenesis. Our results
also show that telomerase is not always activated in
RCC. Because we did not demonstrate telomerase activity inhibitors in telomerase negative RCC samples,
it appears that telomerase activity is not the only factor regulating telomere length and stability. Murnane
et al.31 and Bryan et al.32 have reported a telomeraseindependent mechanism in immortalized human cell
lines that do not contain detectable telomerase activity. Recently, the experimental data from telomerase
knock-out mouse suggest that telomerase is not required for oncogenic transformation or tumor formation.33 Some other organisms also are known to have
telomerase-independent means of lengthening their
telomeres. The yeast Saccharomyces cerevisiae has the
ability to utilize recombination as a backup mechanism for telomere repair. Nonreciprocal recombination at the boundary between telomeric and nontelomeric DNA resulted in the acquisition of telomeric
repeats by very short telomeres in this organism.34 The
telomeres of Drosophila are maintained by transposition of specialized retrotransposons to broken or natural chromosome ends.35–37 None of these mechanisms as yet have been demonstrated in RCC.
Recent studies have reported a positive correlation between telomerase activity and clinicopathologic findings for certain types of tumors.14,15,17,19,38
Lin et al.19 reported that telomerase activity was associated with pathologic grade and clinical stage in
bladder carcinoma. Chadeneau et al.14 reported an
association between telomerase activity and the acquisition of malignancy in colorectal carcinoma, and
Hohaus et al.38 reported an association with disease
stage. In the current study we observed an association
between telomerase activity and pT. This suggests that
telomerase activity in RCC may occur as a later event
in cancer progression, similar to a report in nonsmall
cell lung carcinoma.16 Moreover, recent studies have
reported associations between telomerase activity and
patient outcome in gastric carcinoma15 and neuroblastoma.20 In the current study we did not find an
association between telomerase activity and patient
outcome. However, the follow-up period in the current study was too short to draw definitive conclusions
765
regarding a possible relation between telomerase activity and patient outcome in RCC.
In the current study we also demonstrated that
RCC tumors in older patients have higher telomerase
activity than those in younger patients. This finding
may be related to an intriguing observation that
younger patients with RCC have a better outcome
than older patients.39 – 42 Further studies are warranted
to determine whether telomerase activity may underlie the unpredictable biologic and clinical behavior
of RCC.
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