relative contribution of normal and neoplastic cells determines … · coma, and one...

Vol. 3, 1849-1857. October 1997 Clinical Cancer Research 1849

Relative Contribution of Normal and Neoplastic Cells Determines

Telomerase Activity and Telomere Length in Primary

Cancers of the Prostate, Colon, and Sarcoma1

Monika Engelhardt, Juan Albanell,Pamela Drullinsky, Wei Han, Jose Guillem,Howard I. Scher, Viktor Reuter, and

Malcolm A. S. Moore2

James Ewing Laboratory of Developmental Hcmatopoiesis [M. E..J. A., P. D., W. H.. M. A. S. M.j, Genitourinary Oncology Service

IH. I. S.], and Departments of Surgery [J. G.] and Pathology [V. RI,Memorial Sloan-Kettering Cancer Center. New York, New York

10021; and Department of Medicine, Cornell University MedicalCollege, New York, New York [H. I. S.l

ABSTRACT

Telomerase and telomere length are increasingly inves-

tigated as potential diagnostic and prognostic markers in

human tumors. Among other factors, telomerase and te-

lomere length may be influenced by the degree of tumor cell

content in tumor specimens. We studied telomerase activity

and telomere length with concomitant integration of his-

topathological data to determine whether both were influ-

enced by the amount of tumor cells. We measured telomer-

ase in 153 specimens: in 51 solid tumor blocks; in 51 cryostat

sections; and in 51 adjacent normal tissues from patients

with sarcoma (n 10) and colorectal (n 11) and prostate

cancer (n = 30) using the sensitive and rapid detection

telomeric repeat amplification protocol assay. Telomere

length was determined by telomere restriction fragment

Southern blot analysis. From cryostat sections, tumor cell

infiltration was assessed. Telomerase activity was detected in

all colorectal tumors and sarcomas, as expected. In primary

prostate cancer, however, telomerase activity was less fre-

quently observed (14 of 30, 47%). Moreover, a decreased

intensity compared to colon cancer and sarcoma was evident

(P < 0.001). The median tumor cell infiltration was signifi-

Received 3/12/97; revised 6/1 1/97; accepted 7/2/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by NIH Grant U9 CA 67842-01 (to

M. A. S. M.), grants from the Gar Rcichman Fund of the Cancer Re-

search Institute (to M. A. S. M.), Byrne Foundation (to M. A. S. M.),Martin Himmel Fund (to H. I. S. and M. A. S. M.), and CaPCurc Foun-

dation (to H. I. S.), Grant 96/5706 from the Fondo de lnvcstigacion

Sanitaria (to J. A.), and Grant 95/319/1-I from the Deutsche Forsch-

ungsgcmcinschaft (to M. E.).

2 To whom requests for reprints should be addressed. at Laboratory of

Developmental Hcmatopoicsis, Memorial Sloan-Kettering Cancer Cen-ten, RRL 717-C, 1275 York Avenue, Mailbox 101, New York. NY10021 . Phone: (212) 639-8171; Fax: (212) 717-3618: E-mail: m-moore@

ski.mskcc.org.

cantly higher in sarcoma (65%) and colon (30%) compared

to prostate cancer (5%; P < 0.001). There was a positive

correlation between tumor cell infiltration and telomerase

activity (r = 0.89; P < 0.001). Telomere restriction frag-

ments in tumors were shorter compared to the normal

tissues with peak differences in colon, sarcoma, and prostate

of 1.8, 2.8, and 1 kilobase pairs, respectively (P < 0.002).

Our data suggest the presence of a positive correlation

between the degree of tumor cell content in human solid

tumors and the level of telomerase activity detected. We

demonstrated that the amount of tumor cells also affects

telomere restriction fragment analysis. Therefore, with the

predominance of normal cells in tumor specimens, telomer-

ase activity measured may not reflect the malignant pheno-

type, and telomere loss may be underestimated. This phe-

nomenon was most evident in prostate cancer. Our results

will have implications for the future when telomerase activ-

ity and telomere lengths may be used for early screening,

diagnosis, and prognosis determinations and when telomer-

ase inhibitors are applied to clinical practice.

INTRODUCTION

Telomeres are specialized chromosomal end structures

composed of TT’AGGG repeats in humans. They function to

protect chromosomes from degradation, fusion, and recombina-

tion events (1). In the absence of compensatory mechanisms,

telomeres shorten with each cell division because the termini of

linear molecules are not replicated by conventional DNA poly-

merases (2, 3). To date, telomere shortening has been observed

in most dividing somatic cells. This suggests that when a critical

telomere length is reached, cell senescence occurs (4, 5). Te-

lomerase has been identified as a ribonucleoprotein enzyme that

can compensate for telomene loss by adding TTAGGG nucleo-

tide repeats onto chromosomal ends ( 1-4). Low levels of te-

lomenase activity have recently been detected in human primi-

tive hematopoietic cells (5-7). However, in most other normal

somatic cells, telomerase has not been detected, and conse-

quently telomere shortening occurs after a limited number of

population doublings (8-10). In contrast, in germ cell tissue,

spontaneously immortalized tumor cell lines, and the majority of

tumors, high telomenase activity, stable telomere length, and

unlimited proliferative potential have been observed (4, 1 1-26).

Previous studies have suggested an association of tumor

cell infiltration and telomerase activity ( 1 8, 2 1 , 27). but to date

no study has conclusively correlated the effect of tumor infil-

tration on telomenase level and telomere length. In this study. we

present a detailed analysis of three different types of solid

tumors in an attempt to clarify this phenomenon. We selected

sarcoma and colon cancer, both considered to be aggressively

proliferating tumors, and prostate cancer, which in comparison

to the former two is thought to be more indolent. Our results

Research. on August 24, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

1850 Telomerase and Tclomeres in Solid Tumors

demonstrate the value of the careful integration of pathological

evaluation when interpreting telomerase activity and telomere

length measurements.

MATERIALS AND METHODS

Tissue Samples. Frozen tissues from S 1 solid tumors and

5 1 normal adjacent tissues from patients with sarcoma (ti 10)

and colon (ii - 1 1 ) and prostate cancer (n = 30) were retrieved

from our tissue bank. All cases were reviewed by pathologists.

Fifteen prostate tumors were staged T2, 12 were T3, and 3 were

1’4. Twenty-four prostate cancer specimens were moderately

differentiated, 4 were poorly differentiated, and 2 were well-

differentiated. Fourteen prostate cancer patients had tumors with

Gleason grade 5/6 compared to 16 with Gleason grade 7/8 (n =

12) and 9/10 (n 4). In colon, two specimens were staged T1,

three were T,, four were T3, and two were T4. Four colon

specimens were moderate, five were poor, and two were well

differentiated. Nine high-grade sarcomas (three synovial sarco-

mas, two fibrosarcomas, two leiomyosarcomas, one liposar-

coma, and one neurofibrosarcoma) and one low-grade sarcoma

(fibrosarcoma) were evaluated. From each tumor sample, one-

half was used immediately for protein and DNA preparation

(solid tumor block preparation); the other half was embedded

for cryostat sections using tissue Tek II OCT (Mios Ink; Tissue

Tek Diagnostios, Mishawaka, IN). The embedded tissue was

completely frozen in liquid nitrogen, and successive 5-, 60- and

5-jim cryostat sections were obtained in a “sandwich tech-

nique.” Top and bottom 5-�i.m sections were mounted on glass

slides and were stained with H&E for light microscopy inter-

pretation of tumor cell infiltration. The quantitation of the tumor

cell infiltration was assessed by visual estimation from both top

and bottom 5-jim sections by a pathologist in a blind fashion.

The middle 60-�.tm section was assayed for telomerase.

TRAP Assay. Cells from 153 specimens: from 51 solid

tumor blocks; from 5 1 cryostat sections; and from 5 1 normal

adjacent tissues from sarcoma and colorectal and prostate cancer

patients were processed as described previously (4, 28). Tissue

samples were thinly sliced, homogenized with ice-cold lysis buffer

( 106 cells/lOO p.!): 0.5% 3-[(3-cholamidopropyl)-dimethylammo-

nio]-l-propane-sulfonate, 10 msi Tns-HC1 (pH 7.5), 1 mrsi MgC12.

I mM EGTA, 10% glycerol, S msi �3-mercaptoethanol, and 1 ng/ml

leupeptin; and disposable pestles notated at 450 rpm by a drill until

the tissue was dispersed. Tissues were incubated on ice for 30 mmand centrifuged at 12,000 X g for 30 mm at 4#{176}C.The supernatant

was collected, and the protein concentration was determined by the

Bradford assay (Bio-Rad Laboratories, Richmond, CA). Aliquots

were diluted to 1 �ig pnotein/p.l and stoned at -80#{176}C.In brief, the

TRAP assay was performed as follows (4, 1 1 , 28). Two �ag of

protein extract were assayed in a 38-�.tl reaction mixture containing

lOX TRAP buffer [0.2 M Tris-HCI (pH 8.3), 0.5 mrvi MgC1,, 630

mM KCI, 0.05% Tween 20, 10 mrsi EGTA, and 1 mg/mI BSA], 2.5

mM deoxynucleosides, and 0. 1 mg TS primer at 25#{176}Cfor 20 mm.

Ten p.1 of PCR mix containing lOX TRAP buffer, 0.1 mg ACX

primer, Taq polymenase, and [32P]dCTP were then added to each

sample. M2R3 (5’-GAGCAGAGTFAGGGTfAGGGTfAGG-

GTTAG-3 ‘), a synthetic oligonucleotide containing the sequences

of the TS and RP primers, was included in every TRAP assay to

control the efficiency of the PCR reaction. The products were

amplified by 30 cycles at 94#{176}C,60#{176}C,and 72#{176}Cfor 30 s each and

were separated by electrophoresis on a 10% polyacrylamide gel.

The gel was dried for I h at 80#{176}Cand exposed to an image plate.

Using ImageQuant software (Molecular Dynamics, Sunnyvale,

CA), we quantified the signal intensity by determining the radio-

activity of each repeat ladder corrected for the background and by

expressing the total count per reaction (total product generated) as

the percentage of activity in a cell line as described previously (5,

1 1 , 18, 29, 30). For consistency, we assayed an extract from a

neuroblastoma cell line (SK-N-SH) in parallel on each gel, which

shows maximum telomerase activity. The level of specific telom-

erase activity in SK-N-SH was set to 100%, and the relative

specific telomerase activities of each extract were expressed as

percentage of the SK-N-SH standard. All results were determined

from at least three to six independent TRAP assays, and average

activity was calculated. The quantitation method is semiquantita-

tive but sufficient for comparative analysis (5, 1 1 , 29, 30), espe-

cially when the results of at least three independent experiments are

combined. A sample was scored as telomerase positive when the

telomerase-specific, 6-bp DNA ladder was observed. Negative

samples were reexposed for 72 h to exclude weak enzyme activity.

Specimens showing no telomerase were reassayed at 0.2 and 0.02

�i.g of protein, because inhibition of telomerase activity at high

protein concentrations has been reported (14, 23, 27, 28). To

exclude Taq polymerase inhibitors, which may account for telom-

erase negativity, negative samples were reassayed using a PCR

control TSNT (5’-AATCCGTCGAGCAGAGTFAG[GGTfAG]7-

3’) oligonucleotide and RP plus NT primers under standard TRAP

assay conditions.3 In all, the TSNT band presence and lack of

telomerase activity were confirmed.

Alkaline Phosphatase Activity. The stability of alkaline

phosphatase appears to be similar to the stability of telomerase

(17, 28). Therefore, alkaline phosphatase activity was analyzed

in all 153 samples to assess the possibility of protein degrada-

lion as a cause of negative telomerase activity. Thirty p.g of

protein extract, assayed in a 200-p.l reaction mixture [0.75 m�i

4-methyl-umbelliferyl phosphate, 1 12 msi 2-amino-2 methyl-I

propanol buffer (pH 10.4). 3.75 mrvt MgSO4, and 1 mg/mI

polyvinyl pyrrolidone], were incubated at 37#{176}Cfor 1 h, and the

reaction was stopped by adding 20 pi of 1 M NaOH. The

fluorescence of the product was read on a fluorescence plate

reader (CytoFluor 2350). Alkaline phosphatase activity was

found present in all solid tumor and normal adjacent tissue

preparations and 60-p.m sections, with no significant difference

in tumor and normal tissue control specimens.

TRF Assay. Telomene length was determined by TRF”

Southern blot analysis as described previously (4, 6, 9, 16).

Genomic DNA from tumor and normal adjacent tissues was

digested with 400 p.1 of DNA extraction buffer [100 nmi NaCl,

40 ms’i Tris (pH 8.0), 20 msi EDTA (pH 8.0), and 0.5% SDS]

and proteinase K (0. 1 mg/ml). Extraction was performed using

3 N. W. Kim and F. Wu. Advances in quantitation and characterizationof telomerase activity by the telomeric repeat amplification protocol(TRAP), submitted for publication.4 The abbreviations used arc: TRF, telomere restriction fragment; kbp,kilobase pair(s); PD. population doubling.



Table 1 Telomerase activity and tumor cell infiltration in solid tumors (median and ranges)

60-p.m sections

Solid tumor preparation Histologically verified tumor Histologically normal tissue

Colon

Sarcoma

Prostate

0

11

10

30

Telomerase +

11/Il

10/10

14/30

Telomerase

activity” %

23 (9-49)

19 (9.5-45.2)

1.2 (0-15.6)”

Telomerase

9/96/8

10/10

Tumor cellinfiltration %

30(10-100)65 (20-100)

5 (2-15)”

‘I Telomerase activity expressed as a percentage of the neuroblastoma cell line (SK-N-SH standard) control.

1� p < 0.001 compared to telomerase activity in colon and sarcoma.

0/2

0/2

5/20 2.1 (0.5-8)

Clinical Cancer Research 1851

+

Telomerase

Telomerase + activity” %

phenol/chloroform. Five to ten �ig of extracted DNA were

digested with 10 units of MSPI and RSAI (Boehringer Mann-

heim, Indianapolis, IN) for 12-24 h at 37#{176}C.Integrity of the

DNA before digestion and completeness of digestion were mon-

itored by gel electrophoresis. Electrophoresis of digested

genomic DNA was performed in 0.5% agarose gels in 45 m�i

TBE buffer (pH 8.0) for a total of 660-700 V/h. After electro-

phoresis, gels were depurinated in 0.2 N HCL, denatured in

0.5 M NAOH/l.5 M NaCl, neutralized in 0.5 M Tris/l.5 M NaCl,

and transferred to a nylon membrane (Schleicher & Schuell,

Keene, NH) using 20x SSC and dried for 1 h at 70#{176}C.The

telomeric probe (TTAGGG)3 (Genset, San Francisco, CA) was

5’-end labeled with [�y32P]ATP using T4 polynucleotide kinase

(Boehringer Mannheim). Prehybridization and hybridization

were performed at 50#{176}Cusing 5X Denhardt’s solution, sxSSC/0.l M Na2HPO4/0.Ol M Na4P2O7, and 30 �ag of salmon

sperm DNA per ml/0. 1 m�vi ATP. Membranes were washed in

o.sx SSC/0.l% SDS solution. Telomeric smears were visual-

ized by exposing the membranes to an image plate. The mean

lengths of TRFs were analyzed with a Phosphonlmagen (Molec-

ular Dynamics). Mean TRF lengths were defined as

�(OD,) where OD is the densitometer output and L1 is the

length of DNA in position � (3, 31). Sums were calculated over

the range of 2-23 kbp. Telomere peak values were measured by

estimating the band size corresponding to the point with the

highest absorbance (16). Differences in TRF peak values were

defined as a divergence of at least 2 kbp.

Calculation of Telomere Length Changes by Varying

Admixture of Normal Cells. The accuracy of the mean and

peak TRF values to estimate telomere length in tumors can be

affected by the ratio of normal versus tumor cells. We created

computer-based simulations to assess the effects of tumor het-

erogeneity on TRF values. Based on these simulations, we

estimated the influence on telomere length measurements by an

admixture of normal cells in tumor samples. We used the

following formula to recalculate TRF length for all 51 tumor

specimens: Length of TRF

Length of TRF =

�(oD, /L�)/�(OD, )Turnor ( �(OD,/L,)/�(OD,)NormaI

x (admixture of normal cells in %Il00))

Calculation of Number of Cell Divisions. An evalua-

tion of the number of PDs in tumor specimens, based on

differences in mean and peak TRF length between normal

and tumors, was performed. The following formula was

used:

Mean or peak TRF (kbp) of normal specimen

- tumor specimen:O.O5 kbp = number of PDs

Statistics. Comparisons among groups were made with

standard statistical tests. Results are expressed as median and

range except when stated otherwise. The relationship between

tumor cell infiltration and telomerase activity, and telomere

length and telomerase activity, were estimated by linear negres-

sion and correlation analysis. Statistical significance of the data

obtained was analyzed by the Wilcoxon rank sum test and

Student’s I test (Statworks, Cricket Software, Philadelphia, PA).

P less than 0.05 was considered statistically significant.

RESULTS

Telomerase Activity Levels in Solid Tumor Block Spec-

imens. From solid tumor block specimens, all I I colon and 10

sarcoma specimens displayed telomerase activity (Table I ; Fig.

1). Forty-seven % of prostate cancer specimens (14 of 30) had

telomerase activity with a median activity 20-fold lower than

found in colon and sarcoma (P < 0.001 ; Table I ; Fig. IA).

Downtitnating the protein 10-fold (0.2 p.g of protein) and 100-

fold (0.02 � of protein) demonstrated persistent telomerase

activity in colon and sarcoma. With the dilution of prostate

samples already at limited levels, no telomerase activity in 10-

and 100-fold dilutions was detectable. In all S I normal adjacent

tissues, telomerase activity was undetectable, in agreement with

the reported absence of telomerase in most somatic tissues (2, 4,

13-15).

Telomerase Activity Levels in Cryostat Sections. In SI

tumor specimens, 5-p.m cryostat sections were evaluated his-

topathologically. A high tumor cell content in sarcoma (median,

65%) and in colon (median, 30%) was found compared to

significantly lower tumor cells in prostate sections (median, 5%;

P < 0.001; Fig. 2). From all 51 tumors embedded for cryostats,

not all had tumor cells detectable in the 5-p.m section. Tumor

cells were visualized in 9 of I I colon, 8 of 10 sarcoma, and 10

of 30 prostate sections (Table 1 ). Assays of nine of nine colon

(100%) and six of eight sarcoma (75%) sections showed telo-

menase activity in the 60-p.m cryostat section (Table 1 ). Two

sarcoma sections, despite verified tumor cells, failed to reveal

telomerase from the 60-�j.m preparation. These, however, also

had weak telomerase activity in the larger solid tumor block

preparations. In 2 of I I colon and 2 of 10 sarcoma sections, only



A

B

I

*

�:;4

*

TNTN123TN

Colon

TNTNTNTNTNT

Sarcoma Prostate

TTTTTTNTTTNT NIT

Colon Sarcoma Prostate

1852 Telomerase and Telomeres in Solid Tumors

I��.

I$111

Fig. I Telomerase activity in solid tu-

mon block preparations (A) and in

60-�i.m tissue sections (B). Using 2 p.g of

protein, telomerase activity was detected

in solid tumor (1) block preparations inall colon and sarcoma samples and 47%of prostate specimens. In normal (N)adjacent colon, sarcoma, and prostatetissue, tclomerasc was undetectable. Ic-lomerase, assayed from 60-p.m cryostatsections with histologically verified tu-mon cells, showed activity in all colonand prostate sections (100%) and in sixofeight sarcoma sections (75%). Lane 1,

neuroblastoma cell line; Lane 2, M2R3control (5’-GAGCAGAGTTAGGGT-TAGGGTTAGGGTTAG-3’. a syntheticoligonucleotide containing the se-quenccs of the TS and RP primers, in-cluded in every TRAP assay to controlthe efficiency of the PCR); Lane 3, neg-ative control (using buffer alone).

normal tissue was detectable and revealed no telomerase activity

(Fig. lB). In prostate, all sections assayed with verified tumor

cells (10 of 10) showed telomerase activity in the 60-jam cry-

ostat section (Fig. lB). In the remaining 20, only normal tissue

was detectable (Fig. 1B). Of these 20, in 15 no telomenase

activity was present, but in 5 �‘normal” prostate sections, low

telomerase activity was unexpectedly found (Table I ). In these,

a histological reevaluation was performed once telomerase was

known, which showed tumor cells in one retrospectively (Table

I ). The detectability of telomerase in four of five prostate

specimens without histological tumor cell verification was most

likely due to the lack of tumor cell infiltration in 5-p.m sections

as opposed to the 60-rim section.

Correlation Analysis of Tumor Cell Infiltration and

Telomerase Activity. A correlation analysis of tumor cell

infiltration and average telomerase activity, calculated from at

least three independent TRAP assays, demonstrated a positive

correlation (r = 0.89; P < 0.001; Fig. 3). High telomerase

activity was observed in specimens that consisted predomi-

nantly of tumor cells. The same trend of high telomerase activity

in samples with increased tumor cell content was found when

each tumor type was analyzed separately. Increased tumor cell

content was defined as tumor infiltration over the median infil-

tration present in each tissue: over 65% in sarcoma, 30% in

colon, and 5% in prostate cancer. In sarcoma, median telomer-

ase activity was higher with 31% (range, 20-45.2%) in high

tumor infiltration specimens versus 10.3% (range, 9.5-13%) in

specimens with low tumor cell infiltration (P = 0.008). In colon,

telomerase activity was 31 % (range, 20-49%) in high tumor

infiltration specimens versus 15.9% (range, 9-16.4%) in low

tumor cell infiltration specimens (P = 0.02); and in prostate

cancer, 8.5% (range, 5.3-15.6%) versus 0% (range, 0-8.8%;




Fig. 2 Tumor samples were used for cryo-stat sections of 5, 60, and 5 p.m obtained in a“sandwich technique.” Thin top and bottomsections (5-�i.m) were stained with H&E forlight microscopy interpretation of tumor infil-tration. A, section of sarcoma in which over90% of cells arc tumor (X200). B, sectioncontaining normal colonic mucosa, stroma,and colonic adenocarcinoma (arrow). Ap-proximately 50% of cells are tumor (X200).C, section containing predominantly benignprostatic glands and stroma. A few glands ofprostatic adenocarcinoma representing lessthan 5% of the tissue sampled is also present(arrow; X200).



.

S.

0

1854 Telomerase and Telomeres in Solid Tumors

0Ce

C)CeC)

C

C)

r=0.89p<0.001

10 20 30 40 50 60 70 80 90 100

Tumor cell infiltration (%)

Fig. 3 Histological evaluation of tumor cell infiltration using cryostatsections was performed in 5 1 tumor specimens to determine tumorversus noncancerous stroma cells. Median tumor infiltration was high insarcoma (65%) and in colon (30%) compared to significantly lowertumor infiltration in prostate (5%). A correlation analysis of tumorinfiltration and telomerase activity given in percentage of the neuroblas-toma control showed a positive correlation (r = 0.89; P < 0.001).Increased telomerase activity was found in specimens that consistedpredominantly of tumor cells.

P < 0.001). Tumor differentiation (well, moderate, and poor) or

tumor size (T1-T4) did not correlate with telomerase activity in

colon samples. In sarcoma, telomerase activity in nine high-

grade tumors was more than twice as high (median activity,

20%) compared to one low-grade sarcoma (9%). In prostate

cancer, telomerase activity in poorly differentiated tumors (me-

dian activity, 5%), in T3 and T4 tumors (8.7%), and in speci-

mens with higher Gleason grades (Gleason grade 7-10, 6%) was

higher than in well- and moderately differentiated tumors (1%),

T1 and T2 tumors (1 %), and low Gleason grade tumors (grades

1-6, 1.1%).

TRF Length Measurements and Effect of Tumor Cell

Infiltration on Telomere Length Analysis. In colon and

sarcoma, all tumors contained shorter telomeres than the

adjacent normal tissue. In prostate cancer, shorter and equal

TRF lengths compared to normal tissue were observed (Fig.

4). Mean values are shown in Table 2. In 7 of 1 1 colon

samples, 3 of 10 sarcoma samples, and 7 of 30 prostate

samples, two distinct peak TRF values of at least >2 kbp

difference were observed, with the higher peak correspond-

ing to that of the adjacent normal control and the lower peak

most likely representing the tumor (see “Materials and Meth-

ods”). Utilization of peak TRF values demonstrated shorter

telomere lengths in tumor specimens compared to mean TRF

values (Table 2). Therefore, peak values seemed to more

accurately define tumor telomeres than mean TRF values,

and the resultant differences between tumor and normal tis-

sue peak TRFs were 1.8 kbp (P = 0.001), 2.8 kbp (P =

0.001), and 1 kbp (P = 0.002) in colon, sarcoma, and

prostate, respectively (Table 2).The accuracy of the mean and

peak TRF values to estimate telomere length in tumors can be

affected by the ratio of normal versus tumor cells. We created

computer-based simulations to assess the effects of tumor

heterogeneity on TRF values. Our simulations assumed that

tumors are composed of two distinct cell populations, A and

B. A is the population of normal cells, and B is the tumor cell

population. Arbitrary mean and peak TRF values were as-

signed to both A and B in the Microsoft Excel program used

to calculate TRF values. A and B were artificially mixed in

the following manner: 100% A; 90% A, 10% B; 75% A, 25%

B; 50% A, 50% B; 25% A, 75% B; 10% A, 90% B; and 100%

B. The resulting mean and peak TRF values suggested that:

(a) if the TRF values of A and B are close (i.e., A = 6 kbp,

B = 5 kbp) and both populations are mixed, only a single

peak TRF will be detectable. This value will be different than

the peaks of A and B and closer to the predominant popula-

tion. Mean TRF values are also closer to the predominant

population; and (b) if the TRF values of A and B are very

different (i.e., A = 10 kbp, B = 4 kbp) and both populations

are mixed, two peaks will be detectable. These two peaks are

similar or identical to the peaks of A and B. This suggests

that when two peaks are observed in a tumor specimen, they

probably reflect the tumor versus normal cell population.

In terms of mean TRF analysis, only one value is generated

that will be closer to the predominant cell population. These

models suggest that telomere length loss in tumor speci-

mens is underestimated, especially when the degree of tumor

cell infiltration is low. Based on these simulations, we esti-

mated the influence on telomere length measurements by

admixture of normal cells in tumor samples. We used the

formula to recalculate TRF length for all 5 1 tumor speci-

mens: �(ODi/Li)/�(ODi)Tumor (I(ODi1’1�i)1’�(O1)i)Normal X

(admixture of normal cells in %/100)). This formula accu-

rately predicted the TRF values of either A or B from the

simulations in which both were mixed. This mathematical

model used to calculate possible TRF changes (“corrected”

TRF) and applied for tumor cell infiltration of 90, 80, 50, 10,

and 5%, demonstrated TRF length changes of 0.1, 0.2, 0.5,

0.9, and 0.95 kbp. According to these calculations, “correct-

ed” telomere lengths in colon, sarcoma, and prostate cancer

specimens were 5.8, 5.25, and S kbp, respectively (Table 2).

Therefore, corrected for their tumor cell content, colon, sar-

coma, and prostate cancer displayed similar telomere lengths

(Table 2).

Correlation Analysis of Telomere Length versus Telo-

merase Activity. When evaluating telomerase-positive and

-negative samples, we found telomere lengths that were signif-

icantly longer in telomcrase-positive samples compared to those

with no or weak (< 10%) telomerase activity (Table 3). Mean

TRF lengths were 6.7 kbp, and peaks were 6.1 kbp in all

telomerase-positive tumor specimens, compared to mean TRFs

of 5.2 kbp and peaks of 4.8 kbp in specimens with no or low

telomerase activity (P = 0.001). Telomere lengths in each tumor

subgroup displayed the same trend (Table 3).

There was a weak positive correlation between telomere

length and telomerase activity. Analyzing TRFs that were un-

corrected and corrected for their tumor cell infiltration showed

correlation coefficients of r = 0.54 and r 0.45 (P < 0.05),

respectively, implying that elongated telomercs may correlate

with higher telomerase activity.



Colon

121110987

kb 6

5

4

3

2

Sarcoma

�

�!

TNT NTNTN

12111098

kb 76

5

4

3

2

12111098

7

kb 6

5

4

3

2

Prostate

�. � �:

� �

TNTNT NTN

Fig. 4 Results of a representative Southern blot analysis depicting telomere restriction fragments in colon, sarcoma, and prostate tumor samples (7)and adjacent tissue (PT). In colon and sarcoma, all tumors showed shorter telomeres than their normal adjacent tissue. In prostate. shorter and equalTRF lengths compared to normal tissue were observed. Mean TRF differences between tumor and normal in colon, sarcoma, and prostate were 1 kbp(P = 0.04), 1 .9 kbp (P = 0.007). and 0.5 kbp (P = 0.08), respectively.

TNTNT NTNTN


Table 2 Mean and pe ak TRF values in turn or samples and in normal adjacen t tissue

n

Mean TRF (kb) Peak TRF (kb)

Tumor Normal DifferenceTumor Normal Difference

Colon“corrected” TRF”Sarcoma“corrected” TRFb

Prostate“corrected” TRFb

1 1

10

30

6.5 ± 1.1

5.85.5 ± 1.1

5.255.9 ± 0.9

5

7.5 ± 1.2

7 ± 1.5

6.3 ± 0.9

1 ± 0.9”1.7

1.9 ± 1”

1.750.5 ± 0.7c

1.3

5 ± 1.2

4.4 ± 1.5

5.4 ± 1.2

6.7 ± 0.9

7.2 ± 1.8

6.4 ± I

1.8 ± 1.4”

2.8 ± 1.3”

1 ± 1.3”

ap <

b Corrected for admixture of normal cells in tumor specimens using the formula: �(ODj/L,)/�(ODj)Tufl�)r (�(OD,/L,)/�(OD,)N,)rmaI x

(admixture of normal cells in %/lOO)).cP = 0.08.

DISCUSSION

Telomerase activity has been reported in a wide variety of

tumor types (12-26). Telomerase activation is thought to cor-

respond to a late event in the process of multistage tumorigen-

esis, which results in stabilization of telomeres and subsequent

inmiortalization (2). Recently, high telomerase activity has been

correlated with a worse clinical prognosis (12, 14). However,

conflicting results have been reported concerning telomerase

activity with regard to clinical stage and pathological grade,

suggesting that telomerase may be influenced by parameters

other than clinicopathological features (12, 14, 32-34). Integra-

tion of histopathological analysis in telomerase activity and

telomere length measurements of S 1 solid tumors and S 1

matched normal specimens was performed in this study to

clarify these conflicts. Although previous studies have sug-

gested an association of tumor cell infiltration and telomerase

activity (18, 21, 27), no study to date has conclusively investi-

gated this phenomenon. Sarcoma and colon cancer were used as

representatives of more aggressively growing tumors, and pros-

tate cancer was selected as a representative tumor with a more

indolent growth pattern.

We determined that telomerase and telomere lengths have

different values in various tumor types and that both are influ-

enced by the degree of tumor cell infiltration. Our data are in

agreement with previous reports that showed telomerase to be

highly expressed in colon cancer (23). In sarcoma, which has

not been well studied, a similarly high telomerase expression

was observed. However, in primary prostate cancer, undetect-

able or substantially lower telomerase activity was found, which

contrasts to prior findings detecting telomerase in 85% of spec-

imens tested (25). Histological evaluation of S 1 cryostat sections

revealed that tumor infiltration in sarcoma and colon was sig-

nificantly higher compared to prostate cancer. This finding

indicates that low telomerase activity in our prostate cancer



1856 Telornerase and Telomeres in Solid Tumors

Table 3 Mean and peak telomere length differences in specimenswith high (>10%) compared to samples with low (<10% of

neuroblastoma control) or no telomerase activity

Mean TRF Difference Peak TRF Difference(kb) (kb) (kb) (kb)

ColonTelomerase > 10% 6.8 ± 1.1 5.4 ± 1.08Telomerase <10% 5.6 ± 0.6 1.2 3.8 ± 0.1 1.6

Sarcoma

Tclomerase >10% 6.6 ± 0.5 5.8 ± 1.9Telornerase < 10% 4.7 ± 0.6 1.9 3.8 ± 1 .5 2

Prostate

Telomerase >10% 6.1 ± 1 6.1 ± 1.2

Telomerase <10% 5.7 ± 0.8 0.4 5.3 ± 1 0.8

specimens reflects a decreased tumor cell burden and the pre-

dominance of a normal cell population. Correlation analysis

demonstrated that the degree of tumor cell content correlated

with telomerase activity. Thus, the quantitatively different pres-

ence of tumor cells accounts for our results in prostate cancer

and cautions future interpretation of telomerase levels without

the concomitant analysis of tumor cell content. Using cryostat

sections and solid tumor block preparations for tclomerase eval-

uation illustrated well the value of the highly sensitive TRAP

assay as a diagnostic marker for malignancy. Both in small

cryostat sections and in specimens with less than 5% tumor cell

infiltration, telomerase activity was detectable. This was sup-

ported by our histological reevaluation, which revealed tumor

cells retrospectively in at least one case. Therefore, telomenase

may be used as an adjunct to histopathological evaluation,

especially in tumors with low tumor cell infiltration such as

prostate cancer, where it may be possible for a pathologist to

miss the presence of only a few cancer cells.

In malignant cells, telomene length measurements have

been used as a marker for cell proliferation and PDs that occur

prior to telomerase activation at a level sufficient to stabilize

telomeres (I, 4, 9, 12, 16, 18, 21, 22, 25, 35). There are several

theoretical mechanisms accounting for telomere shortening in

malignancy. Short telomeres are thought to result from an in-

creased number of PDs in tumors or because telomerase is

turned on late in tumorigenesis (6, 8-10, 12). Telomenase may

also elongate only some tumor telomeres, thus preventing cell

crisis, on there may be a growth advantage for cells with short

TRFs (6, 12, 15, 16, 22). Finally, unstable tumor telomeres may

lose TTAGGG repeats more readily, accounting for the short

telomenes found in tumors (6, 13, 22, 35).

We used a mathematical model that adjusts telomere length

changes by varying the admixture of normal cells, which dem-

onstrated that mean TRF values in tumor specimens were in-

creasingly effected by the predominance of normal cells. We

also observed two peak values in some tumor specimens: the

higher corresponding to that of the adjacent normal control, and

the lower peak that of the tumor. This observation was sup-

ported by our histopathological evaluations showing a mixture

of normal and tumor cells in every tumor specimen. We illus-

trated that in tumors with scarce tumor cells like in our prostate

cancer specimens, tumor telomeres mistakenly appeared to have

shortened very little and were almost the same length as their

normal controls. Therefore, our calculation model demonstrated

that the acquisition of mean TRF values alone may be maccu-

rate, especially when specimens have very different tumor and

normal telomere lengths. For this reason, both mean and peak

values were analyzed in this study. To our knowledge, compar-

ative analysis between different types of tumors with respect to

the significance of mean and peak TRFs and the effect of tumor

cell content have not been reported. We found on average 1-2

kbp shorter mean TRF values in colon and sarcoma compared to

their normal adjacent control. In prostate with low tumor infil-

tration, shorter or equal TRFs were observed, revealing less

striking differences between tumor and normal controls. Anal-

ysis of peak TRF values more accurately revealed telomere

lengths in tumors that were on average 1 kbp shorter compared

to mean values and demonstrated more impressive differences

between tumor and normal specimens.

In line with previous studies that have reported high telo-

merase activity frequently associated with altered TRF length

(6, 12, 13, 15, 36, 37), we observed 1-2 kbp longer telomeres in

strongly telomerase-positive samples compared to specimens

with low or undetectable telomerase activity. This indicates that

telomerase may successfully stabilize telomeres. We found,

however, that the longest telomeres did not always correlate

with highest telomerase activity, which suggests that once te-

lomerase is activated, telomeres can be stabilized at any length.

Using “uncorrected” TRFs values, calculations of the approxi-

mate number of PDs (assuming a base pair loss of 50 bp/cell

division) occurring in the “premalignant” phase prior to telo-

merase up-regulation revealed more than double the number of

PDs in sarcoma and colon compared to prostate specimens.

However, when telomere length values were used that were

corrected for their tumor cell infiltration, PDs of 34, 35, and 27

in colon, sarcoma, and prostate were found, suggesting that

telomerase is turned on after approximately 30 PDs in solid

tumors, when telomeres have shortened to approximately 5-6

kbp.

In summary, our results demonstrate that telomerase activ-

ity and telomeres in solid tumors vary according to the degree of

tumor cell infiltration. Therefore, when there is a high predom-

inance of normal cells within a tumor, telomerase may not be a

sensitive marker for the malignant phenotype and telomere loss

may be underestimated. Moreover, our study demonstrates the

importance of analyzing both mean and peak TRF values be-

cause mean values reflect the admixture of normal and tumor

cells rather than the malignant population alone. Therefore,

integration of tumor histology and tumor cell infiltration may be

important for future interpretation of telomerase and telomere

length investigations. This will have implications for the future

when telomerase activity and telomere lengths may be interest-

ing targets for early screening, diagnosis, and prognosis deter-

minations and when telomerase inhibitors are used clinically.

ACKNOWLEDGMENTS

We thank Dr. K. R. Prowse (Geron Corporation, Menlo Park, CA)

for expert help in establishing the TRF assay and Dr. N. W. Kim (Geron

Corporation) for providing the alkaline phosphatase protocol.




REFERENCES

1. Blackburn, E. H. Structure and function of telomercs. Nature

(Lond.), 266: 569-573, 1991.

2. Harley, C. B., Kim, N. W., Prowse, K. R., Weinrich, S. L., Hirsch,

K. S., West, M. D., Bacchetti, S., Hirte, H. W., Counter, C. M., Greider,C. W., Wright, W. E., and Shay, J. W. Telomerase, cell immortality, andcancer. Vol. 66, pp. 1-9. Cold Spring Harbor. NY: Cold Spring Harbor

Laboratory, 1994.

3. Landsdorp, P. M. Tclomere length and proliferation potential of

hematopoictic stem cells. J. Cell Sci., 108: 1-6, 1995.

4. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West,

M. D., Ho, P. L. C., Coviello, G. M., Wright, W. E., Weinrich, S. L., andShay, J. W. Specific association of human telomerase activity with

immortal cells and cancer. Science (Washington DC), 266: 201 1-2015,

1994.

5. Broccoli, D., Young, J. W., and de Lange, T. Telomerase activity in

normal and malignant hematopoictic cells. Proc. Natl. Acad. Sci. USA.

92: 9082-9086, 1995.

6. Counter, C. M., Gupta, J., Harley, C. B., Leber, B., and Bacchctti, S.Telomerase activity in normal leukocytes and in hematologic malignan-

cics. Blood, 85: 2315-2320, 1995.

7. Engelhardt, M., Kumar, R., Albanell, J., Pettengell, R., Han, W., and

Moore, M. A. S. Telomerase regulation, cell cycle and telomere stability

in primitive hematopoictic cells. Blood, 90: 182-193, 1997.

8. Vaziri, H., Dragowska, W., Allsopp, R. C., Thomas, T. E., Harley,

C. B., and Landsdorp, P. M. Evidence for a mitotic clock in humanhematopoietic stem cells: loss of telomeric DNA with age. Proc. Natl.

Acad. Sci. USA, 91: 9857-9860, 1994.

9. Counter, C. M., Avilion, A. A., LeFcuvrc, C. E., Stewart, N. G.,Greider, C. W., Harley, C. B., and Bacchetti, S. Telomere shorteningassociated with chromosome instability is arrested in immortal cells

which express telomerase activity. EMBO J., 11: 1921-1929, 1992.

10. Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai,E. V., Futcher, B. A., Greider, C. W., and Harley, C. B. Telomere lengthpredicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci.

USA,89: 10114-10118, 1992.

11. Albanell, J., Han, W., Mellado, B., Gunawardanc, R., Scher, H. 1.,

Dmitrovsky, E., and Moore, M. A. S. Telomerase activity is repressed

during differentiation of maturation-sensitive but not resistant human

tumor cell lines. Cancer Res., 56: 1503-1508, 1996.

12. Hiyama, E., Hiyama, K., Yokoyama, I., Matsuura, Y., Piatyszck,

M. A., and Shay, J. W. Correlating telomerase activity levels withhuman neuroblastoma outcomes. Nat. Med., 1: 249-255, 1995.

13. Counter, C. M., Hirte, H. W., Bacchetti, S., and Harley, C. B.Telomerase activity in human ovarian carcinoma. Proc. Natl. Acad. Sci.USA, 91: 2900-2904, 1994.

14. Hiyama, E., Yokoyarna, I., Tatsurnoto, N., Hiyama, K., Imamura,Y., Murakami, Y., Kodama, I., Piatyszek. M. A., Shay, J. W., andMatsuura, Y. Telomerase activity in gastric cancer. Cancer Res., 55:

3258-3262, 1995.

15. Hiyama, K., Hiyama, E., Ishioka, S., Yamakido, M., Inai, K.,

Gazdar, A. F., Piatyszek, M. A., and Shay, J. W. Telomerase activity in

small-cell and non-small-cell lung cancers. J. Nail Cancer Inst., 87:

895-902, 1995.

16. Mehle, C., Ljungberg, B., and Roos, G. Telomere shortening inrenal cell carcinoma. Cancer Res., 54: 236-241, 1994.

17. Mehle, C., Piatyszck, M. A., Ljungberg, B., Shay, J. W., and Roos,

G. Telomerase activity in human renal cell carcinoma. Oncogenc, 13:

161-166, 1996.

18. Gupta, J., Han, L-P., Wang, P., Gallic, B. L., and Bacchctti, S.Development of retinoblastoma in the absence of telomerase activity.

J. Natl. Cancer Inst., 88: 1 152-1 157, 1996.

19. Langford, L. A., Piatyszek, M. A., Xu, R., Schold, S. C., Jr., andShay. J. W. Tclomcrase activity in human brain tumors. Lancet, 346:

1267-1268, 1995.

20. Zhang, W., Piatyszck, M. A., Kobayashi. I., Estey, E., Andreeff,

M., Deisseroth, A. B.. Wright, W. E., and Shay, J. W. Telomeraseactivity in human acute myelogenous leukemia: inhibition of telomerase

activity by differentiation-inducing agents. Clin. Cancer Res., 2: 799-

803, 1996.

21. Shay, J. W., Werbin, H., and Wright, W. E. Telomeres and telomcra.sein human Icukemias. Leukemia (Baltimore), 10: 1255-1261, 1996.

22. Hastic, N. D., Demster. M., Dunlop, M. G.. Thomson. A. M..

Green. D. K.. and Alshine, R. C. Telomere reduction in human colorectalcarcinoma and with aging. Nature (Lond.), 346: 866-868, 1990.

23. Tahara, H., Kuniyasu, H., Yokozaki, H., Yasui, W., Shay, J. W.,

Ide, T., and Tahara, E. Telomerase activity in prencoplastic and neo-

plastic gastric colorectal lesions. Clin. Cancer Res., 1: 1245-1251, 1995.

24. Li, Z. H., Salovaara, R., Aaltoncn, L. A., and Shibata, D. Telo-

rnerasc activity is commonly detected in hereditary nonpolyposis cob-

rectal cancers. Am. J. Pathol., 148: 1075-1079, 1996.

25. Sommerfeld, H. J., Meeker, A. K., Piatyszek, M. A., Bova, G. S.,Shay, J. W., and Coffey, D. S. Telomerase activity: a prevalent marker

of malignant human prostate tissue. Cancer Res.. 56: 218-222, 1996.

26. Brousset, P., Saati, T. A., Chaouche, N., Zenou, R-C., Schlaifcr, D.,

Chittal, S., and Delsol, G. Tebomerase activity in reactive and neoplastic

lymphoid tissues: infrequent detection of activity in Hodgkin’s disease.

Blood, 89: 26-31, 1997.

27. Holt, S. E., Shay, J. W., and Wright, W. E. Refining the tebomere-

tebomerase hypothesis of aging and cancer. Nat. Biol., 14: 836-839,

1996.

28. Piatyszek, A. M., Kim, N. W., Weinrich, S. L., Hiyama, K.,

Hiyama, E., Wright, W. E., and Shay, J. W. Detection of tebomerase

activity in human cells and tumors by a tebomenic repeat amplification

protocol (TRAP). Methods Cell Sci., 17: 1-15, 1995.

29. Norrback, K-F., Dahlcnborg, K., Carlsson, R., and Roos, G. Te-

bomerase activation in normal B lymphocytes and non-Hodgkin’s lym-

phomas. Blood. 88: 222-229, 1996.

30. Broccoli, D., Godley, L. A., Donchower, L. A., Varmus, H. E., and

de Lange. I. Tebomerase activation in mouse mammary tumors: lack of

detectable tebomere shortening and evidence for regulation of tebomer-

ase RNA with cell proliferation. Mol. Cell. Biol., 16: 3765-3722, 1996.

31. Harley, C. B.. Futcher, A. B., and Grcider, C. W. Tebomeres

shortening during ageing of human fibroblasts. Nat. (Lond.). 345: 458-

460, 1990.

32. Bednarek, A. K., Sahin, A., Brenner, A. J., Johnston, D. A., andAldaz. C. M. Analysis of tebornerase activity levels in breast cancer:

positive detection at the in situ breast carcinoma stage. Clin. Cancer

Res., 3: 11-16, 1997.

33. Kim, N. W., Levitt, D., Huang, G., Wu, F., Osborne, K., and Clark.

G. Correlation of tebomerase with prognostic indicators of breast cancer.Proc. Am. Assoc. Cancer Res., 37: 562, 1996.

34. Kyo, S., Kanaya, I., Ishikawa, H., Ueno, H., and Inouc, M. Tebo-

merase activity in gynecological tumors. Clin. Cancer Res., 2: 2023-2028, 1996.

35. Albsopp. R. C.. Chang, E., Kashefi-Aazam, M., Rogaev, E. I.,

Piatyszck, M. A., Shay, J. W., and Harley, C. B. Tebomere shortening is

associated with cell division in vitro and in vivo. Exp. Cell Res., 220:

194-200, 1995.

36. Bryan, T. M., Englczou, A., Gupta, J., Bacchetti, S., and Rcddel,

R. R. Telomere elongation in immortal human cells without detectabletelomerase activity. EMBO J.. 14: 4240-4248, 1996.

37. Rhyu, A. S. Tebomeres. telomerase, and immortality. J. NatI. Cancer

Inst., 87: 884-894, 1995.



1997;3:1849-1857. Clin Cancer Res M Engelhardt, J Albanell, P Drullinsky, et al. the prostate, colon, and sarcoma.telomerase activity and telomere length in primary cancers of Relative contribution of normal and neoplastic cells determines

Updated version

http://clincancerres.aacrjournals.org/content/3/10/1849

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/3/10/1849To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



relative contribution of normal and neoplastic cells determines … · coma, and one...

Documents