Expression of Human Telomerase Subunits and …clincancerres.aacrjournals.org/content/clincanres/4/7/...32 protein (Boeringer-Mannheim), and 2.5 units of Taq DNA polymerase (Wako,

Vol. 4, 1603-1608, July 1998 Clinical Cancer Research 1603

Advances in Brief

Expression of Human Telomerase Subunits and Correlation with

Telomerase Activity in Urothelial Cancer

Hideaki Ito, Satoru Kyo,’ Taro Kanaya,

Masahiro Takakura, Masaki Inoue, and

Mikio Namiki

Departments of Urology [H. I., M. N.] and Obstetrics and Gynecology[S. K., T. K., M. T., M. I.], School of Medicine, KanazawaUniversity, Kanazawa, Ishikawa 920, Japan

Abstract

The activation of telomerase and stabffization of te-

bomeres are thought to be required for cellular immortality

and oncogenesis. Three major components of human tebom-erase-human tebomerase RNA (hTERC), tebomerase-asso-

ciated protein (TEP1), and human tebomerase catalytic sub-

unit (hTERT)-have recently been identified. However, theroles played by these subunits in the regulation of tebomer-

ase activity are still unclear. In the present study, a total of

37 urothelial cancers, including one metastatic lesion, and

adjacent normal tissues as well as cell lines derived from

bladder cancers were examined for the expression of eachtelomerase subunit. Reverse transcnption-PCR analysis re-

vealed that more than 90% of urothelial cancers expressedhTERT mRNA, whereas less than 20% of normal adjacent

tissues did. In contrast, hTERC and TEP1 mRNA were

commonly expressed in both cancers and normal tissues. Allof the three cell lines derived from bladder cancer expressedeach of the tebomerase subunits, whereas the two normalprimary fibroblast cell lines expressed hTERC and TEP1mRNA but not hTERT mRNA. Telomerase activity was

examined using tebomeric repeat amplification protocol as-

say. All of the cancers examined exhibited tebomerase activ-ity, whereas only 2 of 12 normal tissues exhibited weak

activity. There was a significant association of tebomeraseactivity with hTERT mRNA expression but not with hTERC

or TEP1 mRNA expression. These findings provide strong

evidence that the expression of hTERT is a rate-limitingdeterminant of the enzymatic activity of human tebomerase

and that the up-regulation of hTERT expression may play a

critical role in human carcinogenesis.

Received 1/22/98; revised 5/4/98; accepted 5/6/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

I To whom requests for reprints should be addressed, at Department ofObstetrics and Gynecology, School of Medicine, Kanazawa University,

13-1, Takaramachi, Kanazawa, Ishikawa 920, Japan. Phone: 8 1-0-76-

265-2425; Fax: 8 1-0-76-234-4266; E-mail: [email protected].

Introduction

Telomeres are the distal ends of eukaryotic chromosomes

which in humans contain TTAGGG repeats (1). They protect

and stabilize the ends of chromosomes (2). Because cellular

DNA polymerases cannot replicate the 5’ end of linear DNA

molecules, the number of tebomere repeats in normal somatic

cells decreases with cell division (3). The shortening of te-

lomeres results in chromosomal instability, which leads to cel-

lular senescence. Telomerase is a specialized ribonucleoprotein

polymerase containing an integral RNA with a short template

element that directs the de novo synthesis of telomeric repeats at

chromosome ends (4, 5). Studies that use the PCR-based TRAP2

assay (6-9) have reported that telomerase is activated in a

variety of malignant tumors. In contrast, telomerase activity is

usually repressed in normal somatic tissues except in some

self-renewing tissues with high regenerative potential, such as

hematopoietic and endometrial cells (10, 1 1 ). Telomerase reac-

tivation is thus thought to be required for stabilization of te-

lomere length in attaining cellular immortality (12, 13).

Little is known, however, concerning the molecular mech-

anisms by which human telomerase is activated in tumors. This

is due mainly to the lack of findings concerning constituents of

the human telomerase complex. hTERC was identified first and

functions as a template for telomere elongation by telomerase

(14). Disrupting the function of telomerase RNA in Tetrahy-

mena through overexpression of an inactive form of telomerase

RNA has been shown to lead to progressive shortening of

telomeres (5). The introduction of antisense hTERC into tumor

cells results in the loss of telomere sequences with subsequent

cellular senescence (14). These findings suggest that hTERC

plays an essential role in the maintenance of tebomeres. The

level of hTERC expression has also been shown to increase with

tumor progression (15). Three proteins in different species as-

sociated with telomerase activity have also been identified. p80

and p95 were purified from ciliate Tetrahymena (16), and the

gene encoding a mammalian homologue of p80, TEP1, has also

been cloned (17, 18). However, recent studies have demon-

strated that the expression of these telomerase-associated pro-

teins does not reflect the level of telomerase activity (19). The

role of these proteins in the regulation of telomerase activity is

still unclear. Two related proteins, Est2p and p123, were iden-

tified as catalytic subunits of telomerase in the yeast Saccharo-

myces cerevisiae and the ciliate Euplotes aediculatus, respec-

tively. Both proteins contain characteristic sequences in their

genes that are well conserved in catalytic regions of reverse

transcriptases (20, 21). The human homobogue of Est2p and

p123 has most recently been cloned (hTERT) (22, 23). The

2 The abbreviations used are: TRAP, telomeric repeat amplification

protocol; hTERC, human telomerase RNA component; TEP1 , telomer-ase-associated protein; hTERT, human telomerase reverse transcriptase;RT-PCR, reverse transcription-PCR.

Research. on June 1, 2018. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1604 Expression of Telomerase Subunits in Urothelial Cancers

expression of hTERT is observed at high levels in telomerase-

positive cell lines but not in telomerase-negative cell lines,

which suggests that hTERT is a catalytic subunit homologue

protein. However, the expression of hTERT and its correlation

with telomerase activity have not been determined for a large

number of clinical samples.

In the present study, the expression of each telomerase

subunit was examined in urothelial cancers and cell lines as well

as in normal urothelial tissues, and the correlation between

subunit expressions and telomerase activity was determined. We

found that hTERT was expressed in most of the cancers exam-

med but not in normal urothelial tissues, whereas hTERC and

TEP1 were broadly expressed both in cancers and in normal

tissues. A strong correlation was found between telomerase

activity and the expression of hTERT, providing evidence that

hTERT functions as a critical determinant of the enzymatic

activity of human telomerase.

Materials and Methods

Tissue Samples. Thirty-seven specimens of urothelial

tumors, including 20 bladder cancers, 1 metastatic lymph node,

10 ureteral tumors, 5 renal pelvic tumors, and 1 renal cancer

with renal pelvic invasion, as well as 22 specimens of adjacent

normal tissue, were obtained at the time of surgery. The histo-

logical diagnoses were determined using sections of the same

specimens as used for RT-PCR. At the same institutions where

the surgeries and the diagnoses took place, detailed clinicopath-

ological findings were evaluated according to the General Rules

for Clinical and Pathological Studies on Bladder Cancer, Renal

Pelvic, and Ureteral Cancer, with the use of the tumor-node-

metastasis classification system for malignant tumors. All of the

samples were obtained within 2 h after surgical removal and

were then frozen and stored at - 80#{176}Cuntil used for RT-PCR

and TRAP assay.

Urinary sediment tissue samples were also obtained from

patients with and without bladder cancer from 50- and 150-nil

samples of urine, respectively. The patients without bladder

cancer included two with urolithiasis, two with prostatic cancer,

and one with breast cancer. The tissues were soaked in PBS, and

cell pellets were recovered. These samples were stored at

-80#{176}Cuntil use.

Cell Lines. T24, RT4, and KK47 cells (derived from

bladder cancers) were grown in RPM! 1640 (Life Technologies,

Inc.) supplemented with 10% fetal bovine serum in the presence

of 5% CO2 at 37#{176}C.Normal primary fibroblast cells from

embryo and skin were purchased from Clonetics (San Diego,

CA) and grown in accordance with the manufacturer’s protocol.

TRAP Assay. Frozen samples of 50-200 mg were re-

suspended in ice-cold wash buffer [10 mM HEPES-KOH (pH

7.5), 1 .5 mM MgCl2, 10 niivi KC1, and 1 nmi DTT]. After

washing, the pellets were homogenized in 20-100 pA of ice-cold

lysis buffer [10 mM Tris-HC1 (pH 7.5), 1 mr�i MgCl2, 1 mrvt

EGTA, 0.1 mr�i phenylmethylsulfonyl fluoride, 5 m�i f3-mercap-

toethanol, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-

propanesulfonate (Sigma), and 10% glycerol]. After 30 rain of

incubation on ice, the lysate was centrifuged at 15,000 X g for

30 ruin at 4#{176}C,and the supernatant was frozen and stored at

- 80#{176}C.The protein concentration in the extract was measured

by the Bradford assay (24). Five p.g of protein were used for the

TRAP assay. Assay tubes were prepared by sequestering 0.2 p.g

of CX primer (5’-CCCTFACCCTFACCCTFACCCTfAA-3’)

under wax barrier (Ampliwax, Perkin-Elmer Corp., Foster City,

CA). Each extract was assayed in 50 p.1 of reaction mixture

containing 20 mr�i Tris-HC1 (pH 8.0), 1 .5 nmi MgCl2, 60 m�i

KC1, 0.005% Tween 20, 1 mi�i EGTA, 50 p.M dNTPs, 0.2 �i.g of

TS primer (5’-AATCCGTCGAGCAGAG1T-3’), 1 �i�g of T4g

32 protein (Boeringer-Mannheim), and 2.5 units of Taq DNA

polymerase (Wako, Japan). After a 30-mm incubation at 23#{176}C

for the telomerase-mediated extension of the TS primer, the

reaction mixture was heated at 90#{176}Cfor 3 mm and then sub-

jected to 31 cycles of PCR including denaturation at 94#{176}Cfor

45 s, annealing at 50#{176}Cfor 45 s, and extension at 72#{176}Cfor 60 s.

The PCR products were electrophoresed on a 12% polyacryl-

amide gel and visualized with SYBR Green I nucleic acid gel

stain (FMC BioProducts, Rockland, ME).

RNA-PCR Analysis. Total RNA was isolated from the

tissues using Isogen (Nippon Gene, Tokyo, Japan) in accord-

ance with the manufacturer’s protocol. cDNA was synthesized

from 4 �g of RNA using an RNA PCR Kit Version 2 (TaKaRa,

Tokyo, Japan) with random primers. Analysis of the expression

of each telomerase subunit was performed by RT-PCR ampli-

fication as described previously (22) with slight modification.

To amplify the cDNA, l-�i.l aliqots ofreverse-transcribed cDNA

were subjected to PCR in 10 p.1 of 1 X buffer containing 0.2 mrvt

each of dATP, dCTP, dGTP, and d1TP, and 0.6 units of ExTaq

DNA polymerase (TaKaRa). A 0.45-�i.l portion of DMSO (SIG-

MA) was added to the buffer for hTERT. hTERT mRNA was

amplified using the primer pair 5’-CGGAAGAGTGTCTG-

GAGCAA-3’ (LT5) and 5’-GGATGAAGCGGAGTCTGGA-3’

(LT6); TEP1 mRNA was amplified using the primer pair 5’-

TCAAGCCAAACCTGAATCTGAG-3’ (TEP1 . 1) and 5’CCC-

CGAGTGAATC1TFCTACGC-3’ (TEP1.2); and hTERC was

amplified using the primer pair 5’-TCTAACCCTAACTGA-

GAAGGGCGTAG-3’ (F3b) and 5’-GTVFGCTCTAGAAT-

GAACGQTGGAAG-3’ (R3c). The reaction conditions were: 22

cycles of denaturation at 94#{176}Cfor 45 s, annealing at 55#{176}Cfor

45 s, and extension at 72#{176}Cfor 90 s for hTERC; 28 cycles of

denaturation at 94#{176}Cfor 45 s, annealing at 60#{176}Cfor 45 s, and

extension at 72#{176}Cfor 90 s for TEP1 ; and 31 cycles of denatur-

ation at 94#{176}Cfor 45 s, annealing at 60#{176}C45 s, and extension at

72#{176}Cfor 90 s, with a final extension time of 5 mm at 72#{176}Cfor

hTERT. PCR products for hTERC and TEP1 were separated by

elecirophoresis through 2% agarose gels and stained with

ethidium bromide (Nippon Gene). PCR products for hTERT

were electrophoresed in 7% polyacrylamide gel and stained with

SYBR Green I (FMC BioProducts). The efficiency of cDNA

synthesis from each sample was estimated by PCR with 3-actin-

specific primers.

Statistical Analysis. The x2 test was used to evaluate the

significance of differences. Findings of P < 0.05 were consid-

ered significant.

Results

A total of 37 urothelial cancers as well as 22 adjacent

normal tissue specimens were examined for the expression of

hTERC, TEP1 mRNA, and hTERT mRNA (Table 1) and te-



Table I Telomerase activity and the expression of telomerase subunits in urothelial cancer

Case Age Sex Site Histology Grade Stage Recurrence

Cancer Adjacent normal tissue

Multi” Meta” hTERC TEP1 hTERT TRAP hTERC TEPI hTERT TRAP

12

3

4

5

67

8

9101 1

12

13141516171819202122

23

24

2526

2728

29

303 1

32

33

34

35

3637

7971

53

75

50

6269

53

796958

65

68745672697758746753

67

59

5366

6165

71

6669

65

69

79

665768

MM

F

M

M

FM

F

FMM

F

MFMMMMMMMM

M

M

MM

MM

M

MM

M

F

F

FMM

BladderBladderBladder

BladderBladderBladderBladder

BladderBladderBladderBladderBladderBladderBladderBladderBladderBladderBladderBladderBladderInguinal LN’�Renal pelvisRenal pelvis

Renal pelvisRenal pelvisRenal pelvisRenal pelvisUreterUreterUreterUreterUreterUreterUreterUreterUreterUreter

TCCCTCCAdeno

TCCTCCTCCTCC

TCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCC

TCCRCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCC

G2G3

Mod diW

G2G2G2G3

G2G2GlG2G2G2G3G3G2G2G3G3G3G3G2G2

G2G3G2G2G2GlG2G2G2G2G2G3G2G2

Ta PrimaryTa RecurrentT1 PrimaryT1 RecurrentT, PrimaryT, PrimaryT1 PrimaryT, PrimaryT, RecurrentT, PrimaryT, RecurrentT2 PrimaryT2 PrimaryT2 RecurrentT3 PrimaryT3 PrimaryT3 Primary

T3 PrimaryT3 RecurrentT3 PrimaryT4 RecurrentTa PrimaryTa PrimaryT, Primary

T2 PrimaryT3 PrimaryT3 Primary

Ta Primary

T, PrimaryT, PrimaryT2 Primary

T2 Primary

T3 PrimaryT3 PrimaryT3 Primary

T3 PrimaryT3 Primary

M - + + + + ND” �

M - + + - ND ND NDS - + + + + ND ND

M - + + + + ND NDM - + + + + ND NDS - + + + + ND ND

M - + + + + + +

S - - + + ND ND NDS - + + + ND ND ND

M - + + + ND ND NDM - + + + ND + +

S - + + + + ND NDS - + + + + + +

S - ND ND ND + ND NDS - + + + + + +

S - + + - + + +

S - - + + ND ND NDS - + + + ND + +

M - ND ND ND + ND NDM - + + + ND ND NDM + + + + + ND NDS - + + + ND + +

S + + + + ND + +

M - ND ND ND + ND NDS - + + + ND + +

M - + + + ND ND NDM - + - + ND + +

S - + + + + + +

S - + + - + + +

M - - + + + - +

S - + + + + ND NDS - + + + ND + +

M - + + + + ND NDS - ND ND ND + + +

S + - + + + + +

M + + + + + + +

S - + + + ND + +

ND NDND NDND NDND -

ND ND- ND

ND NDND NDND ND

+ NDND ND

- -

ND ND- -

- -

ND ND+ ND

ND -

ND NDND ND

- ND- ND

ND -

- NDND ND

- ND- ND- -

- -

ND -

- NDND ND

+ -

- +

- +

- ND

renal cell carcinoma.

Clinical Cancer Research 1605

a Multi, multiplicity; M, multiple; S. single.b Meta, metastasis.

C TCC, transitional cell carcinoma; Adeno, adenocarcinoma; RCC,d �i-� not determined.e Mod diff, moderately differentiated.

1LN, lymph node.

lomerase activity. On RT-PCR analysis, hTERC expression was

found in 29 (87.9%) of 33 cancers (3 cases were deleted from

RT-PCR analysis because of degradation of the RNA) and in

17 (94.4%) of 18 adjacent normal tissue specimens (4 cases

were deleted from RT-PCR analysis because of degradation

of the RNA). Similarly, TEP1 expression was found in 32

(97.0%) of 33 tumors and in all of the 18 adjacent normal-

tissue specimens. These findings suggest that hTERC and

TEP1 are widely expressed both in cancers and in normal

tissues. In contrast, 30 (90.9%) of 33 cancers expressed

hTERT mRNA, whereas only 3 (16.7%) of 18 adjacent

normal tissue specimens did so, suggesting a strong associ-

ation of hTERT expression with cancer lesions. No signifi-

cant correlation was found between the expression of hTERT

mRNA and clinicopathological features of urothelial cancers,

including age, clinical stage, and histological type. We also

examined three cell lines (T24, RT4, and KK47) derived

from bladder cancers and two normal primary fibroblast cell

lines from human embryo and skin. All of the cancer cell

lines expressed each of the telomerase subunits, whereas the

two normal primary fibroblast cell lines expressed hTERC

and TEP1 mRNA but not hTERT mRNA (Fig. 1).

We next examined telomerase activity in cases for which

extracts were available for the TRAP assay. Some of the results

obtained were reported previously (9). Telomerase activity was

positive in all 22 of the urothelial cancers examined but in only

2 of the 12 normal adjacent tissue specimens. Eighteen of these

22 urothelial cancers and 8 of the 12 normal tissue specimens

were simultaneously examined for hTERT mRNA. Sixteen

(88.9%) of the 18 urothelial cancers were positive for both

telomerase activity and hTERT mRNA, whereas S (62.5%) of

the 8 normal tissue specimens were negative for both. When

cancerous and normal samples were combined, 21 (80.8%) of

26 cases examined were concordant for both results (P <



I I I I I �I I

TT N TN TTNT TNT

.�‘ � �

hTERC [3-actin

-� �“i �o #{246}, .. . * . a � � �

Telomerase - - + + +

activity12345

+ + - + - + + - + + - +

6 7 8 9 10 11 12 13 14 15 16 17


Fig. 1 Representative resultsof RT-PCR analysis for theexpression of each tebomerasesubunit in urothelial cancersand cell lines. Expression ofhTERC, TEP1 mRNA, andhTERT mRNA was examinedby RT-PCR. Lane 1, primaryhuman embryonal fibroblast;Lane 2, primary human skin fi-broblast; Lane 3, T24 cells;Lane 4, RT4 cells; Lane 5,

KK47 cells; Lane 6, case 6;Lane 7, case 12; Lanes 8 and 9,case 13; Lanes 10 and I 1, case14; Lane 12, case 19; Lanes 13

and 14, case 26; Lane 15, case30; Lanes 16 and 17, case 33.Results of the TRAP assay areshown below each lane.

Normalprimary cells Cell line

I II I

hTERT � t.J � �d � �

TEP1

Table 2 Correlation between telomerase activity and the expressionof telomerase subunits

Telomeraseactivity

hTE

+

RC

-

TEP1

+ -

hTE

+

RT

-

Urothelial cancer+ 16 2 18 0 16 2- 0 0 0 0 0 0

Normal tissue

+ 2 0 2 0 0 2

- 5 1 6 0 1 5

0.001). In contrast to the findings for hTERT, there was no

significant correlation between telomerase activity and either

hTERC or TEP1 mRNA expression (Table 2).

We next examined whether hTERT mRNA was detectable

in urinary sediment cells from patients with vesical lesions.

Eight (66.7%) of 12 urine samples from bladder cancer patients

expressed hTERT mRNA, whereas none of those from patients

without bladder lesions did so, suggesting the potential useful-

ness of hTERT mRNA detection for the cytological screening of

urothelial cancers.

Discussion

hTERT has been identified as the catalytic subunit of

telomerase (22, 23, 25, 26). The expression of hTERT is

observed at high levels in telomerase-positive cancer cell

lines but not in normal tissues (22, 23). Recent studies have

demonstrated that hTERT expression is frequently observed

in malignant tumors such as hepatocellular carcinoma and

cervical cancer but not in adjacent normal tissues (26, 27). In

normal tissues, human chorion at early weeks of gestation is

known to exhibit telomerase activity, which is associated

with hTERT expression (28). The findings of this study

clearly showed that hTERT was expressed in most of the

urothelial cancers examined but not in most normal urothelial

tissues. A strong correlation was found between telomerase

activity and hTERT mRNA expression. These findings,

therefore, support the concept that the expression of hTERT

is a critical determinant of the enzymatic activity of human

telomerase. However, we found some cases of discordancy in

which telomerase activation was not associated with hTERT

mRNA expression or in which the presence of hTERT mRNA

was not associated with telomerase activation. The former

cases suggested that other unknown factors may substitute for

the function of the hTERT subunit in conferring full enzy-

matic activity. The latter cases suggest several possibilities:

(a) the level of expression of each subunit (hTERC, TEP1,

and hTERT) or the balance of their levels of expression,

although we did not determine quantitatively the level of

each subunit, may critically determine enzymatic activity; (b)

the posttranscriptional modification of the subunits may reg-

ulate enzymatic activity; and (c) unknown telomerase inhib-

itors may be present in cell extracts in such cases, thus

decreasing telomerase activity.

In contrast, hTERC and TEP1 were broadly expressed in

both cancers and normal urothelial tissue. No significant

correlation was found between telomerase activity and the

expression of hTERC or TEP1 mRNA. The disruption of the

function of telomerase RNA in Tetrahymena through the

overexpression of an inactive form of telomerase RNA has

been shown to lead to progressive shortening of telomeres

(5). Tumor cells transfected with antisense hTERC lost telo-

meric DNA, which resulted in cellular senescence (14). Most

recently, targeting the telomerase RNA gene in mice has been

shown to lead to progressive shortening of telomeres (29).

These findings suggest that hTERC function is absolutely

required for telomerase activity. Our findings, however,

showed that the expression of hTERC is not sufficient by

itself for enzymatic activity.

TEP1 has been identified as a human homologue of Tet-

rahymena p8O, which was shown to associate with telomerase in

vivo (17, 18). The function of TEP1 is not yet clearly under-

stood, but the presence of repeated sequences in its gene, re-

ferred to as WD-40, which functions in the interaction of pro-

teins, suggests that TEP1 may play a role as an interface

between telomerase and the protein member of the telosome. A



Clinical Cancer Research 1607

recent study has also suggested the possibility that posttranscrip-

tional modification of TEP1 may regulate telomerase activity

(18). Although the present study failed to find any association

between the TEP1 mRNA expression and telomerase activity,

TEP1 may regulate telomerase activity through the mechanisms

described above. Additional biochemical analyses will be

needed to determine the function of TEP1.

The findings of the present study suggest that hTERT

mRNA detection may be useful for cytological examination

of urothelial cancers. hTERT mRNA was detected in approx-

imately 70% of urinary sediment samples from patients with

bladder cancer but was never observed in those samples from

patients without bladder lesions, which indicates that it is

specific to cancer lesions. The failure to detect hTERT

mRNA in urinary sediments in cancer patients may have been

due to the small number of cancer cells contained in urinary

sediment samples. However, we detected hTERT mRNA in

some urinary samples in which telomerase activity was not

detected by the TRAP assay (data not shown), although we

do not know at present why these discrepancies arose. These

findings suggest that RT-PCR for hTERT mRNA may be

more sensitive in some cases than the TRAP assay in detect-

ing cancers.

In our study, the expression of hTERT mRNA was

observed not only in cancers but also in adjacent normal

tissue specimens. The biological significance of this expres-

sion in normal tissues remains unclear, but it is possible that

these tissues contained microscopic cancer lesions. It may,

therefore, be important to follow up these cases for detection

of recurrence.

In summary, hTERT expression was commonly ob-

served in the cancers we examined and may be a rate-limiting

determinant of telomerase activity. It will be most important

to determine the onset of hTERT expression during the steps

of carcinogenesis. Understanding the molecular mechanism

through which hTERT is expressed may also provide critical

insights into the molecular basis for cellular immortality and

carcinogenesis.

Acknowledgments

We are grateful to Drs. T. Misaki and S. Nakashima (Department

of Urology, Tonami General Hospital, Toyama, Japan) and Drs. S.Hirano and H. Fuse (Department of Urology, Kouseiren Takaoka Hos-pital, Toyama, Japan) for the sampling of the tissues.

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1998;4:1603-1608. Clin Cancer Res H Ito, S Kyo, T Kanaya, et al. telomerase activity in urothelial cancer.Expression of human telomerase subunits and correlation with

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