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Int. J. Cancer (Pred. Oncol.): 84, 529–532 (1999)
r 1999 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
TELOMERASE ACTIVITY CORRELATES WITH HISTO-PATHOLOGICAL FACTORS
IN UTERINE ENDOMETRIAL CARCINOMA
Yasuhiko EBINA1*, Hideto YAMADA1, Takafumi FUJINO1, Itsuko FURUTA1, Noriaki SAKURAGI1, Ritsu YAMAMOTO1, Motonobu KATOH2,
Mitsuo OSHIMURA2 and Seiichiro FUJIMOTO1
1Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo, Japan
2Department of Molecular and Cell Genetics, Tottori University Faculty of Medicine, School of Life Science, Yonago, Japan
Telomerase activity has been implicated in the progression
of various human tumors. Our aim was to evaluate telomerase activity and to compare it with histo-pathological factors
in uterine endometrial carcinoma, to look for possible correlations. Telomerase activity was measured by dilution analysis using a PCR-based telomeric repeat amplification method
and detected in 31 of 35 primary endometrial carcinoma
tumor specimens. High telomerase activity, detected after
100-fold dilution of extracts, was identified in 15 specimens.
There was no significant correlation between the positive
telomerase activity and tumor surgical stage or histopathological factors. However, high telomerase activity was
significantly correlated with advanced surgical stage and with
pelvic lymph node metastasis. Our findings suggest that an
increase in telomerase activity may be associated with tumor
progression and that its level may have a prognostic value in
endometrial carcinoma. Int. J. Cancer (Pred. Oncol.) 84:529–
532, 1999.
r 1999 Wiley-Liss, Inc.
Human telomeres consist of 4 to 15 kb of hexameric DNA
repeats (TTAGGG) located at the ends of chromosomes. Due to the
inability of DNA polymerase to replicate the ends of doublestranded DNA, telomeres progressively shorten with each round of
cell division. Telomeric ends are considered to play an important
role in the protection of the chromosomal ends against aberrant
recombination and end fusion. The progressive shortening of
telomeres eventually results in chromosomal instability, leading to
cellular senescence.
Telomerase is a ribonucleoprotein that synthesizes telomeric
DNA onto chromosomal ends using a segment of its RNA
component as a template. The development of a highly sensitive
PCR-based assay for telomerase activity has made it possible to
study a wide variety of tumors and normal tissues (Kim et al.,
1994). Using this method, telomerase activity has been detected in
85% of gastric carcinomas (Hiyama E., et al., 1995), 85% of
hepatocellular carcinomas (Tahara et al., 1995) and 93% of breast
carcinomas (Hiyama et al., 1996). In gynecological malignancies,
telomerase activity has been detected in 83% to 100% of uterine
cervical carcinomas (Kyo et al., 1996; Garham et al., 1997), 82% to
100% of uterine endometrial carcinomas (Kyo et al., 1996, 1997;
Brien et al., 1997; Garham et al., 1997; Saito et al., 1997; Shroyer
et al., 1997; Yokoyama et al., 1998) and 86% to 96% of ovarian
carcinomas (Kyo et al., 1996; Garham et al., 1997; Yokoyama et
al., 1998).
Correlations between telomerase activity and clinico-pathological factors such as tumor stage and grade in gastric and breast
cancer have also been described (Hiyama E., et al., 1995, 1996).
Use of telomerase activity as a prognostic marker of clinical
outcome was proposed because it has been related to the malignancy potential of the above-mentioned tumors. In endometrial
carcinoma, however, telomerase activity has never been correlated
with histo-pathological and prognostic factors (Brien et al., 1997;
Kyo et al., 1997; Shroyer et al., 1997).
In the present study, using dilution analysis and the PCR-based
telomeric repeat amplification method, we have analyzed telomerase activity in endometrial carcinoma tissues and looked for
correlations between telomerase activity and histo-pathological
factors.
MATERIAL AND METHODS
Tissue specimens
Endometrial carcinoma tissues taken from primary tumor
sites were obtained from 35 Japanese patients (age range 23
to 77 years) at the time of surgery. Eleven patients (31.4%)
were pre-menopausal and 24 (68.6%) were post-menopausal.
These patients had never been treated with irradiation, chemotherapy or hormonal therapy before tissue sampling. Specimens derived from primary lesions of endometrial carcinomas
were frozen immediately after sampling and stored once at ⫺80°C
until analysis. The presence of malignant cells in the sampled
tissues was confirmed before analysis by microscopy, using
cryostat sections. Histo-pathological data, including histological
subtype, histological and nuclear grade, myometrium and cervical
invasion, adnexal metastasis, pelvic lymph node and para-aortic
lymph node metastasis, lymph-vascular space invasion and
extra-uterine disease, were collected and compared with
telomerase activity. Histological grade was determined as follows:
well-differentiated (G1), moderately differentiated (G2) or poorly
differentiated (G3) according to FIGO criteria. Lymph-vascular
space invasion was considered present when tumor cells
were within or attached to the wall of a vascular or lymphatic space
lined by flattened endothelial cells. Cervical invasion was diagnosed when tumor cells were within or adhered to endocervical
tissue.
Telomerase assay
Telomeric repeat amplification protocol (TRAP) assay was
performed according to the methods described by Kim et al.
(1994), using an internal telomerase activity standard (ITAS), to
exclude the possibility of Taq DNA polymerase inhibitory activity
in the tumor extracts. Each tissue specimen (approx. 100 mg,
frozen at ⫺80°C) was washed in ice-cold buffer [10 mM HEPESKOH (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT], pelleted,
then sufficiently homogenized in 200 ml ice-cold lysis buffer [1
mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 60% glycerol,
0.5% CHAPS, 5 mM 2-mercaptoethanol, 0.1 mM AEBSF]. After
30-min incubation on ice and centrifugation at 16,000 g for 20 min
at 4°C, the supernatant was collected and stored at ⫺80°C. The
protein concentration in the extract was measured using a BCA
protein assay kit (Pierce, Rockford, IL). A portion of the supernatant was included in each TRAP assay tube containing 50 µl of
reaction mixture composed of 20 mM Tris-HCl (pH 8.3), 1.5 mM
MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 50 µM
dNTPs, 0.1 µg TS primer, 1 µg T4 gene 32 protein (BoehringerMannheim, Mannheim, Germany), 2 U Taq DNA polymerase
(Takara Shuzo, Kyoto, Japan) and 0.1 µg CX primer at the tube
bottom sequestered by Ampliwax (Perkin Elmer, Norwalk, CT).
Telomeric repeat was added to TS primer and maintained at 23°C
for 30 min. The assay tube was then processed through 31
temperature cycles at 95°C for 30 sec, 50°C for 30 sec and 72°C for
*Correspondence to: Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, N15 W7, Kita-Ku, Sapporo 060,
Japan. Fax: 81-11-706-7711.
Received 31 March 1999
EBINA ET AL.
530
FIGURE 1 – Telomerase activity in endometrial carcinoma. Each extract prepared from tumor tissue was subjected to serial dilutions and
analyzed by TRAP assay at 6 µg protein (standard condition), at 0.6 µg (10-fold) and at 0.06 µg (100-fold). Lysis buffer was used as a negative
control and a uterine cervical cancer cell line (Siha) as a positive control for telomerase signals from 10, 100 and 1,000 cells. The ITAS band is an
internal control for PCR amplification. Case C, which remained positive after 100-fold dilution, was classified as high. Case A, which was positive
after 10-fold dilution and became negative after 100-fold dilution, was classified as low. Case B showed no telomerase activity.
90 sec. The final products were visualized by non-denaturing
10% PAGE and visualized by SYBR green I nucleic acid gel
stain (FMC, Rockland, ME). The sensitivity of the non-RI assay
was as high as that of the conventional RI-TRAP assay.
We classified a sample as telomerase-positive when a 6-bp DNA
ladder was observed and its intensity was equivalent to or stronger
than that obtained using 10 cells of a uterine cervical cancer cell
line (SiHa). Because there was variation in the intensity of the
TRAP signals in the tumor specimens, we estimated telomerase
activity using serially diluted extracts containing 6 µg, 0.6 µg
(10-fold) and 0.06 µg (100-fold) protein. Telomerase activity
detected after 100-fold dilution was classified as high, whereas
that which disappeared in diluted extracts was classified as low
(Fig. 1).
Statistical analysis
Statistical analysis was performed using Fisher’s exact test; p ⬍
0.05 was considered to be statistically significant.
RESULTS
Telomerase activity was detected in 31 (88.6%) of 35 endometrial carcinoma tissues. Extracts from all telomerase-negative
samples gave a positive signal with the internal telomerase assay
standard, excluding the possibility of Taq polymerase inhibition.
After semi-quantification of telomerase activity by serial dilution
of each extract, the 31 tumors showing positive telomerase signals
were divided into 2 groups: 15 tumors had high telomerase activity
(i.e., retained a TRAP signal after 100-fold dilution of the extract)
and 16 had low telomerase activity (i.e., expressed TRAP signals
before dilution or after 10-fold dilution but not after 100-fold
dilution of the extract).
The incidence of positive telomerase activity was not statistically significant in regard to surgical stage. However, the incidence
of high telomerase activity was significantly high in stage III or IV
tumors (62.5%, 10 of 16) when compared with that in stage I or II
tumors (26.3%, 5 of 19; p ⬍ 0.05; Table I).
TABLE I – TELOMERASE ACTIVITY AND SURGICAL STAGE
IN ENDOMETRIAL CARCINOMA
Surgical
stage
Number
I
II
III
IV
I ⫹ II
III ⫹ IV
Total
16
3
12
4
19
16
35
Telomerase activity
Negative
Low1
High2
3
1
0
0
4
0
4
9
1
5
1
10
6
16
4
1
7
3
5
10
15
1Positive using 6 or 0.6 µg of extract but negative using 0.06 µg of
extract.–2Positive using 6, 0.6 or 0.06 µg of extract.
The correlation between histo-pathological factors and telomerase activity is shown in Table II. There was no significant
correlation between the frequency of positive telomerase activity
and histological subtype, histological grade, nuclear grade, myometrial invasion, cervical invasion, adnexal metastasis, pelvic node
metastasis, para-aortic node metastasis, lymph-vascular space
invasion or extra-uterine disease. Tumor lesions from all 4
telomerase-negative patients were strictly confined to the uterus.
All specimens with adnexal, pelvic and para-aortic lymph node
metastases showed positive telomerase activity. The incidence of
high telomerase activity was significantly high in patients with
pelvic lymph node metastasis ( p ⬍ 0.05). Although all 7 specimens of extra-uterine disease had positive telomerase activity and
tended to have high telomerase activity, this was not significant
( p ⫽ 0.10).
When only post-menopausal patients were included in the
statistical analysis, 4 of 15 (26.7%) tumors from stage I or II
patients exhibited high telomerase activity and 7 of 9 (77.8%)
tumors from stage III or IV patients showed high telomerase
activity with statistical significance ( p ⬍ 0.05). Pelvic lymph node
metastasis was also significantly correlated with high telomerase
activity (presence of metastasis 66.7% vs. absence of metastasis
38.9%, p ⬍ 0.05). Thus, high telomerase activity significantly
TELOMERASE ACTIVITY IN ENDOMETRIAL CANCER
TABLE II – CORRELATION BETWEEN HISTO-PATHOLOGICAL FACTORS AND
TELOMERASE ACTIVITY IN ENDOMETRIAL CARCINOMA
Histo-pathological factors
Histological subtype
Endometrioid carcinoma
Serous adenocarcinoma
Histological grade
G1
G2
G3
Nuclear grade
1
2
3
Myometrial invasion
Tumor limited to endometrium
Invasion to ⬍1/2 myometrium
Invasion ⬎1/2 myometrium
Serosal invasion
Cervical invasion
Negative
Positive
Adnexal metastasis
Negative
Positive
Pelvic node metastasis
Negative
Positive
Para-aortic node metastasis
Negative
Positive
Lymph-vascular space invasion
Negative
Positive
Extra-uterine disease
Negative
Positive
Number
Telomerase activity
Negative
Low1
High2
33
2
4
0
15
1
14
1
19
11
5
1
3
0
9
6
1
9
2
4
16
14
5
1
3
0
8
7
1
7
4
4
6
15
9
5
1
3
0
0
4
5
5
2
1
7
4
3
28
7
3
1
14
2
11
4
29
6
4
0
14
2
11
4
26
6
4
0
14
1
8
5
28
3
4
0
14
1
10
2
21
14
3
1
10
6
8
7
28
7
4
0
14
2
10
5
1Positive using 6 or 0.6 µg of extract but negative using 0.06 µg of
extract.–2Positive using 6, 0.6 or 0.06 µg of extract.
correlated with pelvic lymph node metastasis and advanced
surgical stage, even in post-menopausal patients.
DISCUSSION
Uterine endometrial carcinoma is one of the common invasive
malignancies of the female genital tract, and its prevalence is
steadily increasing in Japan as well as in Western countries.
However, information regarding the molecular mechanisms of
endometrial carcinogenesis remains limited. Mutations of ras
(Enomoto et al., 1990; Ignar-Trawbridge et al., 1992) and p53
(Okamoto et al., 1991; Risinger et al., 1992) genes are involved in
the development and progression of endometrial carcinoma, but the
incidence of mutation in these 2 genes is low, estimated at 10% to
46% and 13% to 14%, respectively (Enomoto et al., 1990;
Okamoto et al., 1991; Ignar-Trawbridge et al., 1992; Risinger et
531
al., 1992). Our relatively new approach of the molecular investigation of telomere–telomerase interactions may provide additional
insight into the mechanisms of endometrial carcinogenesis.
Most normal human somatic cells exhibit undetectable levels of
telomerase activity, whereas a majority of tumor cells express
telomerase activity and shortened telomeres (Kim et al., 1994). In
normal cells, telomerase is particularly expressed in embryonic
cells (Wright et al., 1998), adult male germ-line cells (Wright et al.,
1998), proliferative cells of renewable tissues such as basal cells of
the epidermis (Harle-Bachor and Boukamp, 1996), circulating
peripheral blood leukocytes (Hiyama K. et al., 1995) and normally
cycling endometrium (Brien et al., 1997; Kyo et al., 1997; Saito et
al., 1997). Kyo et al. (1997) have evaluated telomerase activity by
semi-quantitative analysis and have shown that normal endometrium expresses telomerase activity, the level of which is regulated
during the menstrual cycle. In contrast, atrophic endometrial
samples from post-menopausal women exhibit no or only weak
telomerase activity (Brien et al., 1997; Kyo et al., 1997; Saito et al.,
1997; Shroyer et al., 1997), suggesting that residual telomerase
activity in post-menopausal endometrium reflects loss of function
in the basal endometrial lining, which is no longer capable of
regeneration.
Various investigations correlating telomerase activity with prognostic factors have yielded conflicting results. Studies of human
gastric (Hiyama E., et al., 1995) and breast (Hiyama E., et al.,
1996) cancer have shown that the frequency of lymph node
metastasis in patients with positive telomerase activity in primary
tumors was higher than that in patients without telomerase activity.
In pancreatic carcinoma, the level of telomerase activity in primary
tumors obtained surgically from patients with distant metastases is
significantly higher than in primary tumors without metastases
(Hiyama E., et al., 1997). In contrast, in hepatocellular (Tahara et
al., 1995) and renal cell (Mehle et al., 1996) carcinoma, no
relationship between telomerase activity and clinico-pathological
parameters, such as histo-pathological grade, tumor size, DNA
ploidy, stage and clinical outcome, has been noted.
To our knowledge, in human endometrial carcinomas no correlation between telomerase activity and any histo-pathological prognostic factors has been observed (Brien et al., 1997; Shroyer et al.,
1997). However, we have found here that high telomerase activity
significantly correlates with the presence of pelvic lymph node
metastasis and advanced surgical stage. These correlations were
confirmed when only post-menopausal patients were analyzed. The
presence of pelvic node metastasis and advanced surgical stage are
indeed adverse factors that determine poor prognosis in patients
with endometrial cancer. Endometrial carcinomas with high telomerase activity may be more aggressive than those without or with
low telomerase activity.
ACKNOWLEDGEMENTS
We thank Drs. H. Hareyama (Sapporo City Hospital), S.
Shimada (Kushiro Red Cross Hospital), M. Morikawa (Ohji
General Hospital) and M. Oikawa (Hokkaido University) for the
collection of samples.
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