[CANCER RESEARCH 58. Ml2-3818. September I. 1998]

Telomerase Activity Exclusively in Cervical Carcinomas and a Subset of CervicalIntraepithelial Neoplasia Grade III Lesions: Strong Association with ElevatedMessenger RNA Levels of Its Catalytic Subunit and High-Risk HumanPapillomavirus DNA1

Peter J. F. Snijders,2 Mark van Duin, Jan M. M. Walboomers, Renske D. M. Steenbergen, Elle K. J. Risse,

Theo J. M. Helmerhorst, RenéH. M. Verheijen, and Chris J. L. M. Meijer

Departments of Pathology ¡P.J. F. S., M. v. D., J. M. M. W., R. D. M. S., E. K. J. R., C. J. L. M. M.] and Obstetrics and Gyneco/ogy ¡R.H. M. V.J. University Hospital VríjeUniversiteit, 1081 HV Amsterdam, and the Department of Obstetrics and Cynecology, University Hospital Rotterdam. 3000 CA Rotterdam IT. J. M. HJ. The Netherlands

ABSTRACT

In this study, we investigated telomerase activity and human telomerasereverse transcriptase (hTERT) mRNA expression in relation to high-risk

human papillomavirus (HPV) DNA presence in the spectrum of cervicalpremalignant lesions. Reconstruction experiments revealed that telomerase activity determined by the telomeric repeat amplification protocolassay and hTERT mRNA by reverse transcriptase-PCR could be detected

in down to 100 and 1 Sill» cervical cancer cells, respectively. Telomericrepeat amplification protocol analysis on cervical tissue specimens revealed that none of the histomorphologically normal cervical samples(n = 8) and cervical intraepithelial neoplasia (CIN) grade I (n = 10) andgrade II (n = 8) lesions had detectable telomerase activity. However,telomerase activity was shown in 40% of CIN grade III lesions (n = 15)and 96% of squamous cell carcinomas (n = 24). Despite the fact that

hTERT mRNA was found at much higher frequencies, semiquantitativereverse transcriptase-PCR revealed that elevated hTERT mRNA levels

were strongly correlated with detectable telomerase activity. Furthermore, telomerase activity and elevated hTERT mRNA levels were onlydetected in cases that contained high-risk HPV DNA. In contrast, low orundetectable hTERT mRNA levels were demonstrated in both high-risk

HPV positive and negative cases. These data indicate that telomeraseactivity detectable with the assay used and concomitant elevated levels ofhTERT mRNA reflect a rather late step in the CIN to squamous cellcarcinoma sequence, which follows infection with high-risk HPV.

INTRODUCTION

Infection with high-risk HPV1 types, particularly HPV 16 and HPV

18, plays a pivotal role in cervical carcinogenesis (1). It has beenreported that cervical SCCs develop from a continuum of premalignant cervical lesions, so-called CIN lesions, graded (grades I-III) on

the basis of mildly to severely dysplastic features (2). Analyses of thenatural history of CIN lesions have revealed that persistence ofhigh-risk HPV types is a likely prerequisite for the development ofCIN HI lesions (3). Moreover, high-grade CIN lesions display the

greatest potential of progression to invasive cancer. Still, CIN lesionsof all grades represent a heterogeneous disease, as they can spontaneously regress, persist, or, in a small subset of cases, progress toinvasive cancer. This indicates that HPV infection, albeit apparentlynecessary, is insufficient for malignant growth. Presently, no markers

Received 3/17/98; accepted 7/2/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Grani VU96-1I51 of the Dutch Cancer Society. The research of

P. J. F. S. has been made possible by a fellowship of the Royal Netherlands Academy ofArts and Sciences.

2 To whom requests for reprints should be addressed, at the Department of Pathology.De Boelelaan 1117. 1081 HV Amsterdam, The Netherlands. Fax: 31-20-4442964: E-mail:

pjf.snijders@azvu.nl.3 The abbreviations used are: HPV. human papillomavirus; CIN. cervical intraepithe

lial neoplasia; SCC, squamous cell carcinoma; hTERT, human telomerase reverse transcriptase; TRAP, telomeric repeat amplification protocol; RT-PCR, reverse transcriptase-

PCR.

are available that can indicate which of the CIN lesions have reacheda point-of-no-return in terms of malignant potential.

In vitro studies have revealed that high-risk HPV genotypes can

induce immortalization of primary human keratinocytes by means oftheir E6 and E7 oncogene functions (4, 5). However, the process ofimmortalization, considered an important step toward malignancy,requires host gene alterations in addition to the expression of theseviral oncoproteins (1). Although the identity of these genes is stillunknown, immortalization mediated by full-length HPV 16 or HPV

18 has been shown to be associated with reactivation of the telomerelengthening enzyme telomerase and arrest of telomere shorteningupon culturing (6, 7). In contrast to immortal cells, mortal precursorsexhibit telomere shortening with each cell division, a process that hasbeen considered to represent a molecular clock for their finite prolif-

erative capacity (8). Once telomeres become critically short, thiswould trigger entry into senescence. Telomerase is a ribonucleopro-

tein complex that can add six bp repeats to telomere ends, therebycompensating for telomere shortening and allowing cells to bypass thesenescence barrier. Telomerase is normally not active in mortal cells,but strongly activated in the majority of immortal cell populations.Consequently, activation of this enzyme is likely to be the mostcommon mode by which telomere shortening is compensated and animmortal state is acquired. The potential importance of telomeraseactivity for cervical carcinogenesis has been underlined by the observation of telomerase activity in the far majority, if not all, cervicalcarcinomas analyzed, but in none or only a minority of paired histomorphologically normal uterine tissue specimens or in normal cervical samples (9-17).

Recently, several components of human telomerase have beencloned (18-21). There is strong evidence that among these subunits,

the catalytic subunit (20, 21), recently named hTERT according to theHuman Genome Organization Nomenclature Committee of the Genome Database, is the limiting component for telomerase activity; itsexpression at the mRNA level is strongly associated with enzymeactivity and concomitant immortalization, and its introduction intonormal human epithelial cells and fibroblasts was sufficient to reconstitute telomerase activity, arrest telomere shortening, and extend thelife span in vitro (20-24). Moreover, hTERT mRNA expression has

been found to be strongly correlated with telomerase activity incervical cancer (17).

The goal of this study was to find out to what frequency and at whatstage telomerase activity and hTERT mRNA expression can be detected in the spectrum of HPV-containing cervical premalignant le

sions. For this reason, consecutive sections of frozen CIN and cervicalcancer specimens were used for measurement of telomerase activityby the TRAP assay (25), hTERT mRNA expression analysis byRT-PCR, and histomorphological assessment, respectively. In con

trast to cervical carcinomas, 96% of which revealed telomerase activity, telomerase activity was undetectable and hTERT mRNA wasabsent or present at low levels in histomorphologically normal cervi-

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TELOMERASE AND hTERT mRNA STATUS IN CERVICAL LESIONS

cal epithelium, as well as in CIN I and CIN II lesions. However, CINIII lesions showed heterogeneity for telomerase parameters: only 40%of them displayed detectable enzyme activity and elevated hTERTmRNA. These data suggest that telomerase activity detectable withthe TRAP assay used and concomitant elevated hTERT mRNA levelsreflect a rather late stage during cervical carcinogenesis and mayprovide a marker for progressive high-grade CIN lesions.

MATERIALS AND METHODS

Tissue Specimens and Cell Lines. Biopsy specimens were selected froma random group of CIN lesions and SCCs of which both formalin-fixed and

frozen tissue biopsies were available. Generally, the stored frozen biopsies hadbeen snap-fro/en in liquid nitrogen within half an hour after they were taken.

Selection of the samples was based on both of the following criteria: (a) anoptimal preservation of morphology of the frozen tissue, as determined byhistopathological examination of tissue sections: and (/;) the capability to yieldsufficient protein of proper quality for TRAP analysis (see below). At the end.65 samples that fulfilled both criteria were used in this study, comprising 8histomorphologically normal cervical specimens from patients with CIN disease, 10 CIN I (mildly dysplastic) lesions. 8 CIN II (moderately dysplastic)lesions. 15 CIN III (severely dysplastic) lesions, and 24 SCCs. Of the frozenspecimens, consecutive sections were cut. the first and last of which werestained with H&E for careful histomorphological examination. This examination also included a crude estimation of the percentage of dysplastic/neoplasticcells relative to histomorphologically normal epithelial and underlying stromalcells, as well as the relative degree of lymphocytic infiltrate. Series of in-

between sections were used for extracting protein. RNA, and DNA for TRAP.RT-PCR. and HPV DNA PCR purposes, respectively.

The immortal cervical carcinoma cell lines SiHa. HeLa. and CaSki wereobtained from the American Type Culture Collection. A cell lineage of primarykeratinocytes named EK94-2 has been described before, by Steenbergen et al.(7). EK94-2 cells showed neither telomerase activity nor hTERT mRNA

expression, exhibited a finite proliferative capacity, and underwent senescenceat passage 7. Cells were grown either in DMEM (Life Sciences. Inc.) supplemented with 10% PCS (SiHa. HeLa. CaSki) or in serum-free keratinocytegrowth medium (EK94-2: Life Sciences. Inc.). From subconfluent cultures,

cells were harvested by trypsinization, washed with PBS. and pellets wereresuspended in TRAP lysis buffer or RNAzol B (Campro Scientific), forextraction of protein or RNA, respectively.

Measurement of Telomerase Activity. Telomerase activity was measuredby the TRAP assay as described (25). except that the PCR was carried out for31 cycles in a reaction volume of 25 /xl. For this purpose. 8-10 tissue sections

or pellets of cultured cells were suspended in 50 /¿Iof TRAP lysis buffer, andthe suspension was homogenized by mixing vigorously. The suspension wasplaced on ice for 30 min and subsequently spun down in a microcentrifuge atmaximum speed for 30 min at 4°C.The supernatant was transferred to a newtube, snap-frozen in liquid nitrogen, and stored at —¿�80°C.The amount of

protein was calculated using the Bio-Rad protein detection kit. Before TRAP

analysis, proteins were first tested for the presence of PCR inhibitors. Therefore, 500 ng of protein extract was mixed with 10 ng of human placenta! DNAand subjected to PCR specific for the ß-globingene using primers PC03 andPCO5 spanning a 209-bp fragment, as described before (26). Only samples that

showed a clear amplification signal, indistinguishable in intensity from thesignal obtained from unmixed human placental DNA. were considered to beappropiate for TRAP analysis. During the course of the study, samples wereadditionally subjected to a TRAP assay using the TRAPe/.e kit (Oncor),provided with an internal PCR control, according to instructions of the manufacturer.

TRAP analysis was performed on 500 ng of protein extract. To determinethe specificity of the assay, all protein samples were preheated for 10 min at70°Cto inactivate telomerase and were tested in parallel in the TRAP assay.

No stepladder patterns were observed after this preheating step. Only clinicalsamples showing an unequivocal regular stepladder pattern after TRAP werescored positive for telomerase activity. For reconstruction experiments, serialdilutions of cell line protein in a background of 500 ng of BSA were used.After separation of TRAP products on polyacrylamide gels, autoradiographywas performed during 24 h at -80°C using intensifying screens.

Primer Selection and RT-PCR for hTERT. On the basis of the cDNAsequences conserved between hTRT (20) and hEST2 (21), hTERT oligonu-cleotides to be used for RT-PCR were selected using the PCRPLAN program

of the PC/Gene software (IntelliGenetics. Inc.). The nucleotide sequence of theselected oligonucleotides were: Forward PCR primer hTERTIF: 5'-GAAG-GCACTGTTCAGCGTGCTCAAC-3' (nt position 2029/2030-2054/2055 ofhTRT/hEST2); reverse PCR primer HTERT2R: 5'-GGTTTGATGATGCTG-GCGATGACC-3' (nt position 2236/2237-2259/2260 of hTRT/hEST2>: internal oligonucleotide probe hTERT3P: 5'-GCCGCCTGAGCTGTACTTTGT-CAAGGTGGA-3' (nt position 2161/2162-2190/2191 of hTRT/hEST2).

Primers hTERTIF and hTERT2R direct the amplification of a 230-bp cDNA

fragment in the PCR. These primers were selected in such a way to flankputative splice junctions, allowing a discrimination between amplification ofspliced cDNA and exemplification of traces genomic DNA eventually presentin the RNA sample. Indeed, hTERTIF and hTERT2R directed the amplification of a genomic DNA fragment approximately 800 bp in size, indicating thatat the mRNA level a fragment(s) of about 570 bp is spliced out.

For RNA quality analysis RT-PCR was performed using intron Hankingprimers specific for the mRNA encoding the Ul small nuclear ribonucleopro-

tein specific A protein (snRNP U1A mRNA: 27). The following oligonucleotides were used for this target: Forward PCR primer U1A1: 5'-CAGTATGC-CAAGACCGACTCAGA-3'; reverse PCR primer U1A2: 5'-GGCCCGG-CATGTGGTGCATAA-3': internal oligonucleotide probe U1A3 5'-AGAA-GAGGAAGCCCAAGAGCCAGCATAA-3'. Primers U1A1 and UIA2 direct

the amplification of a 215 bp cDNA fragment.Total RNA was extracted using RNA/ol B, according to instructions of the

manufacturer (Campro Scientific). First strand cDNA was synthesized usingantisense primers for both hTERT and the housekeeping gene xnRNP UÃŒAina single reaction. Subsequent RT-PCR reactions for hTERT and snRNP UÃŒA

were performed separately, the latter serving as a control for both proper RNAquality and a successful cDNA synthesis. First strand cDNA synthesis wasperformed on 100 ng of total RNA. The conditions of cDNA synthesis. PCRamplification for 40 cycles, and oligonucleotide hybridization were essentiallyas described before (27). After PCR. 10 ¡i\of product was electrophoresed ona 1.5% agarose gel, blotted onto a nylon membrane (Qiabrane). and hybridizedwith (y-12P)ATP end-labeled hTERT3P and U1 A3 oligoprobes for hTERT and

xnRNP UIA targets, respectively, as described previously. (27). Autoradiography was performed for 8 h (snRNP UIA) up to 24 h (hTEtíT)at -80°C. using

intensifying screens.For semiquantitative assessment of hTERT mRNA levels, the CIN lesions

and histologically normal samples as well as 100 ng of SiHa RNA weresubjected to another RT-PCR round for 30 cycles. At this number of PCR

cycles the amplification reaction showed linearity, as was determined on serialdilutions of SiHa RNA. All PCR products were run on the same agarose geland blotted to the same nylon membrane, before hybridization with thehTERT3P and U1A3 oligonucleotide probes, respectively. Signal intensitieswere measured after exposure of the hybridized filters to a phosphor imager(Molecular Dynamics). Levels of hTERT mRNA. normalized to the hTERTlevels from 100 ng SiHa RNA were calculated according to the followingformula: intensity ratio hTERT:snRNP UIA of the sample: intensity ratiohTERT:snRNP UIA of 100 ng SiHa RNA X 100%.

Statistical comparison of hTERT mRNA levels was carried out using theMann Whitney (/test. Differences were considered significant when P < 0.05.

HPV Detection and Typing. HPV DNA was detected by general primer-

mediated PCR using the GP5+/GP6+ primer combination (28). Typing wasperformed on GP5+/GP6+ PCR products for 14 high-risk (HPV 16. 18, 31,33, 35, 39, 45, 51, 52, 56. 58, 59. 66. and 68) and 6 low-risk (HPV 6, 11, 40,

42, 43, and 44) HPV types using a nonradioactive enzyme immunoassay asdescribed (29).

RESULTS

Analysis of Telomerase Activity and hTERT mRNA Expressionin Cervical Carcinoma Cell Lines. Telomerase activity was measured by TRAP analysis on dilution series of protein extracted from theHPV containing cervical cancer cell lines CaSki, HeLa, and SiHa.Telomerase activity, characterized by a regular stepladder pattern, wasclearly detectable in down to 5 ng of protein of all cell lines in a

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TELOMERASE AND hTERT mRNA STATUS IN CERVICAL LESIONS

background of 500 ng of protein (Fig. 1/4). Only the SiHa cell linerevealed a weak, though irregular stepladder pattern when 500 pg ofprotein was tested. Occasionally, anomalous bands were seen thatwere inconsistent with a stepladder pattern (Fig. \A, Lanes SiHa 50pgand 5 pg). These bands were considered nonspecific rather thanreflecting telomerase activity. Assuming that 1 cell contains approximately 50 pg of protein, this indicates that the detection limit fortelomerase activity is at the level of at least 100 cervical cancer cellswhen using a 500-ng protein background. In addition, we investigated

telomerase en/.yme stability by incubating pellets of SiHa cells atdifferent temperatures during varying time intervals before freezing.Subsequent TRAP analysis on 500 ng and 50 ng of protein lysates

B 4°C 20°C 37°C 55°C

0' 30' 60' O/N 30' 60' O/N 30' 60' 10' 30'

CIN I CIN II

Fig. 1. Telomerase activity measurements using the TRAP assay. A. TRAP analysis onserial prolein dilutions of SiHa. CaSki. and HeLa cell lines in a background of 500 ng ofBSA. The amounts ot cell line protein are indicated above the lanes. B. stability analysisof telomerase complex by preincubating pellets of SiHa cells at several temperaturesduring various time intervals. TRAP results on 500 ng (left lanes) and 50 ng (right lunes)of SiHa protein are shown. Temperatures and incubation times are indicated above thelanes. O/N. overnight. C. representative results of TRAP analysis on one CIN I. two CINII. tour CIN 111.and tour SCC samples. Reaction products without (-) and with ( + ) a

prior preheating step to inactivate telomerase are shown. POS. the positive control,comprising 5<X)ng of SiHa protein.

revealed no reduction in TRAP signals after overnight incubations atboth 4°Cand 20°C.Moreover, no reduction in signal intensity wasevident after incubations up to l h at 37°C(Fig. Iß).However, TRAPsignals disappeared completely after preincubating the cells at 55°C.

Subsequently, hTERT mRNA expression was analyzed by RT-PCR

for 40 cycles on dilution series of SiHa RNA in a background of 100ng of RNA from primary foreskin keratinocytes (EK94-2, passage 6).

Amplified hTERT cDNA was detectable in down to 10 pg of SiHaRNA (Fig. 3, right upper panel). Assuming that 1 cell containsapproximately 10 pg of RNA, this indicates that the RT-PCR method

allowed the detection of hTERT mRNA in a single carcinoma cell ofthe SiHa cell line in a background of 10,000 hTERT mRNA negativecells.

Telomerase Activity in Cervical Lesions. Protein extracts of atotal of 65 samples did not reveal any evidence for the presence ofPCR inhibitors and were considered appropiate for TRAP analysis(data not shown). During the course of this study, samples wererescreened with an internal PCR control in the TRAP assay using thecommercially available TRAPeze kit (Oncor), which gave consistentresults.

TRAP analysis revealed that none of the histomorphologicallynormal (n = 8), CIN I (n = 10), and CIN II (n = 8) samples had

detectable telomerase activity. However, telomerase activity was detectable in 6 of 15 CIN III lesions (40%). Histomorphological examination revealed no correlation between telomerase activity and thedysplasia:stroma ratio nor the degree of lymphocytic infiltrate in CINlesions. Examples of H&E-stained sections of CIN III lesions with

and without detectable telomerase activity are shown in Fig. 2. Furthermore, 23 of 24 SCC samples (96%) showed telomerase activity.Also in these cases, the relative percentage of neoplastic cells ordegree of lymphocytic infiltrate did not correlate with detectableactivity. TRAP results on cervical samples are exemplified in Fig. 1C.A summary of the telomerase data is given in Table 1.

hTERT mRNA Expression in Cervical Lesions. A total of 56cervical samples revealed amplifiable cDNA, as determined bysnRNP U l A RT-PCR, and were subjected to hTERT mRNA expression analysis by RT-PCR for 40 cycles (Table 1). These included six

histomorphologically normal samples, two (33%) of which werehTERT RT-PCR positive. In addition, hTERT mRNA positivity wasscored for 4 CIN I (n = 10) lesions (40%), 4 CIN II (n = 6) lesions

(67%), all 10 CIN III samples (100%), and 23 of 24 SCCs (96%).Comparative analysis of data on telomerase activity and hTERTmRNA expression learned that all samples devoid of detectablehTERT mRNA also did not show telomerase activity. These includedthe single SCC sample that was negative for both parameters. Nocorrelation was evident between the hTERT mRNA score and thepercentage of dysplastic/neoplastic cells or degree of lymphocyteinfiltrate. Representative results of hTERT and snRNP U1A RT-PCR

are shown in Fig. 3 (left panels). Because amplified hTERT cDNAsignals tended to be much stronger in cases that displayed detectabletelomerase activity (see Fig. 3, left upper panel), we argued that notthe presence of hTERT mRNA per se, but elevated levels of thistranscript may be correlated with detectable telomerase activity.

This prompted us to perform a semiquantitative RT-PCR for 30

cycles on the 26 CIN lesions and 6 morphologically normal samples.The hTERT:snRNP U1A RT-PCR signal intensity ratios, normalized

to those obtained with 100 ng of SiHa RNA (set to 100%), variedconsiderably from 0-78%. A hTERT:snRNP ratio value of more than0% was scored for 1 of the 6 normal samples (17%), 3 CIN I (n = 10)lesions (30%), 4 CIN II (n = 6) lesions (67%), and all CIN III(n = 10) lesions (100%). Among the cases showing telomeraseactivity (n = 6) these ratios ranged from 19-78% (mean, 45%),whereas cases without telomerase activity (n = 26) displayed a range

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TELOMERASE AND hTERT mRNA STATUS IN CERVICAL LESIONS

»ran

.-

Fig. 2. Examples of H&E-stained sections of the frozen tissue from C1N III lesionswith (A} and without (A) defedatile telomerase activity. Magnification: X 400 (A}and X 200 (fl).

from 0-7% (mean, 2%). The difference in ratio values between

samples with and without detectable telomerase activity was statistically significant (P = 0.0002). Semiquantitative RT-PCR results of

the 18 CIN lesions that initially revealed a hTERT mRNA signal after40 cycles of amplification are shown in Fig. 4.

HPV DNA in Relation to Telomerase Activity and hTERTmRNA Expression in Cervical Lesions. As determined by GP5+/GP6+ general primer-mediated HPV PCR, HPV DNA was detectablein 2 normal (n = 8), 5 CIN I (;i = 11), 2 CIN II (n = 8), and all CINIII (n = 15) samples and all SCCs (n = 24). All HPV-positivesamples contained DNA of one or more of the high-risk HPV types

HPV 16, 18, 33, 35, 45, 56, 58, or 59. Except for 1 normal, 1 CIN I.2 CIN II, 2 CIN III, and 3 SCC samples, all HPV-positive cases

contained DNA of HPV types 16 and/or 18. Comparative analysisrevealed that telomerase activity was not detectable in cases that werehigh-risk HPV negative (Table 1). On the other hand, a significantnumber of high-risk HPV-positive, cases did also not show telomeraseactivity. These comprised all high-risk HPV containing normal, CIN

I, and CIN II samples, 9 of 15 CIN III, and 1 of 24 SCC samples.Conversely, hTERT mRNA expression was not only limited to

HPV-positive cases (Table 1). However, elevated hTERT messages,

characterized by hTERTrsnRNP U1A ratios of 19% or more, wereonly detected in high-risk HPV-positive cases (Fig. 4). Moreover,

when considering only the CIN lesions and normal samples withoutdetectable telomerase activity (n = 26), a significant difference in theratio value was evident between those that were HPV-positive (range.

0-7%; mean, 3.1%) and the HPV-negative (range, 0-3%; mean,0.5%) cases (P = 0.017).

DISCUSSION

This study aimed at the analysis of telomerase activity and hTERTmRNA expression in the spectrum of cervical premalignant lesions.For this purpose, the utility of both TRAP and RT-PCR methods was

first tested in reconstruction experiments on immortal cells of cervicalcancer cell lines. It seemed that in our hands the hTERT RT-PCR

method was approximately 100 times more sensitive than TRAP indetecting immortal cancer cells, reaching a sensitivity of approximately 1 SiHa cell in a background of 10.000 keratinocytes withouttelomerase activity. Moreover, we showed a rather high stability ofthe telomerase complex after incubating SiHa cells at temperatures upto 37°during varying time intervals. This feature potentially allows a

reliable examination of telomerase activity in clinical samples, notwithstanding the time span between biopsy taking and freezing.

The results on telomerase activity in cervical carcinomas are inaccordance with the data obtained by other groups (9-17). The single

SCC devoid of enzyme activity, which was encountered in this study,is likely to represent one of the uncommon cases in which telomerecompensation is achieved by an alternative, telomerase-independent

mechanism (30). In contrast, telomerase data on premalignant lesionsand normal cervical epithelium seem to contradict results obtained bysome other investigators. Several groups showed already telomeraseactivity in a subset of normal cervical samples and often a progressiveincrease in frequency from low-grade to high-grade lesions (10-13,

15, 16). The discrepancy may, in part, be explained by differences insensitivity of the TRAP protocols used. In our study, 500 ng of inputprotein was used for TRAP, and autoradiography was performed for24 h. Kyo et al. (13) have reported that telomerase activity in normalcervical samples and low-grade lesions, when present, was onlydetectable when using 1-5 /ig of input protein, but not 10-fold

dilutions of these amounts. Moreover, using 6 ;ug of input protein,Shroyer et al. (16) could detect telomerase activity in a significantsubset of normal and low-grade cervical samples only after 7 days of

autoradiography. In addition, differences in morphological assessmentmay effect the outcome of telomerase studies. In the majority ofstudies reported, histomorphological assessment was performed onsamples different from those used for TRAP analysis. In our study,histomorphological assessment was most optimal, because this wasperformed on adjacent tissue sections flanking the sections that wereused for protein and RNA analysis. Reconstruction experiments weperformed on frozen pellets of SiHa cells revealed that sectioningbefore cell lysis does not lead to a decreased sensitivity in detectingtelomerase activity, due to possible air-drying artifacts.4 Finally, some

studies have used exfoliated cervical cells rather than tissue for TRAPanalysis (10, 11, 13, 15). Pilot studies we performed on accompanyingcervical scrapes learned that TRAP scores in the scrapes do notalways correspond with the scores obtained from the biopsy specimens. Apparently, exfoliated superficial epithelial cells do not alwaysrepresent a reliable indicator of telomerase activity in the underlyinglesion, as has been found by Gorham et al. (Il), as well.

Recent studies have shown that in vitro the HPV 16 E6 oncoproteinis capable to induce telomerase activity in monolayer cultures ofproliferating keratinocytes (31). Consequently, the observed patternsof telomerase activity in CIN lesions, the majority of which containedHPV 16 DNA, may be due to differences in levels of E6 expression.Moreover, because telomerase activity in immortal cells is influencedby factors like the phase of the cell cycle or status of differentiation

4 Unpublished observations.

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Table I Telomerase activity and hTERT mRNA expression in relation in HPV [)NA presence in cen'ical premali/inani lesions

Tclomerase aclivily" hTERT mRNA expression

Telomerase positive Telomerase negative hTERT mRNA positive hTERT mRNA negative

LesionNormalCIN

1C1NIICINIIIsec/I81081524HPV

positive___623HPV negative HPV positive24291HPV negative666_-n6IO61024HPVpositive_221023HPVnegative222_-HPV positive22__1HPV negative242_-

" Telomerase activity was determined on a total of 65 samples.'' hTERT RT-PCR for 40 eycles was applied to a total of 56 samples.

(32-34), also topologica! differences of E6 expression within the

stratified squamous epithelium may underlie the observed heterogeneity in telomerase activity. Particularly in low-grade CIN lesions,

transcription of the F6 and E7 oncogenes is primarily observed indifferentiated cells (35, 36). Because these cells have withdrawn fromthe cell cycle, telomerase complex, when present, is likely to be silent.In contrast, high-grade CIN lesions often display elevated E6/E7transcripts in the proliferating basal-like cells that occupy much or all

CINi CINil CIN III SiHa RNA

8 è

hTRT

snRNPUlA

Fig. 3. RT-PCR results of hTERT and snRNP UIA after 40 cycles of amplificationfollowed by Southern hlol h\hridi/ation of the PCR products, l^'ft panel. RT-PCR on a

representative group of seven CIN lesions. The same CIN lesions were used as shown inFig. 1C'. Note that the CIN III lesion in the far right lane represents the only sample in this

panel with telomerase activity. Right ptinel. RT-PCR results on a dilution series of SiHaRNA in a background of EK94-2 RNA. The amounts of input SiHa RNA are indicated

above the lanes.

of the epithelium. In these cases, the eventual telomerase induction byE6 may become manifest. However, pilot E6/E7 mRNA in situhybridization experiments on a small series of HPV 16 containingCIN III lesions with and without detectable telomerase activity revealed no topologica! differences in E6/E7 transcription: the presenceof E6/E7 transcripts in the proliferating cell layers could be demonstrated in both cases with and without detectable telomerase activity.4

Consequently, it is unlikely that expression of telomerase activity inCIN lesions is solely a reflection of E6 expression in proliferatingcells. Instead, it is more likely that E6/E7 transcription in proliferatingcells is a prerequisite for induction of telomerase activity, a processthat apparently requires additional alterations within the host cell. Thishypothesis fits very well with recent data on HPV-mediated immor

talization in vitro (37).Furthermore, we showed that the presence of hTERT mRNA per se

was not indicative for telomerase activity. However, when considering the amounts of hTERT mRNA, it became clear that relativelyelevated hTERT transcript levels are strongly correlated with detectable telomerase activity. Similar results have been obtained afteranalysis of human liver tissue samples (24). Therefore, the results mayreflect a higher sensitivity of the RT-PCR method compared with the

TRAP method we used. One caveat to this conclusion is the possibility that hTERT mRNA presence does not necessarily indicatetelomerase activity, because posttranscriptional regulation may takeplace or. alternatively, a certain threshold level of hTERT mRNA maybe required for enzyme activity.

An additional tendency became evident that even after exclusion ofcases with telomerase activity, hTERT mRNA levels were significantly higher in the HPV-positive group than in the HPV-negativegroup of samples. Whether this would be due to any effect of HPV-

100

Fig. 4. Semiquanlilative hTERT RT-PCR resultsof IS hTERT mRNA containing CIN lesions. Levels of hTERT mRNA are indicated in columns asthe percentage of hTERT:snRNP UIA signal intensity ratio relative to that of I(X) ng ( IO.(XX)cells) ofSiHa RNA (set to IfXM I after 30 cycles of amplification. Results of hTERT mRNA levels are shownin relation to telomerase activity and HPV DNApresence. The HPV genotypes that were detectedare indicated above the columns.

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encoded genes (e.g., E6), eventually in combination with the topologyof its expression, remains to be examined. One aspect that should beconsidered, particularly in the case of low-level hTERT expression, is

that it is still unknown to what extent the detected mRNAs are derivedfrom the atypical cells present in the lesion. It has been described thatboth activated lymphocytes and epithelial stem cells may expresstelomerase activity at low levels (38-41), and consequently also

hTERT. Although in this study no correlation was found between thedegree of lymphocytic infiltrate and detectable telomerase activity orhTERT transcripts, it cannot be excluded that low hTERT levelsrepresent low quantities of these cell types expressing telomeraseactivity. This may result in telomerase activities that fall beyond thedetection level of TRAP but not of the hTERT RT-PCR. Therefore, in

situ techniques are required to address hTERT expression in CINlesions in more detail. Currently, we are in the process of developingan RNA in situ hybridization assay for hTERT. Moreover, work is inprogress to generate monoclonal antibodies against hTERT, whichmay have implications for future immunohistochemical approaches.

Assuming that in addition to high-risk HPV functions host cell

alterations are necessary to gain telomerase activity of CIN lesions,one would expect a different genetic make-up of CIN lesions with and

without telomerase activity. In vitro, telomerase reactivation andimmortalization of primary keratinocytes mediated by HPV 16 orHPV 18, was associated with several cytogenetic and moleculargenetic alterations (7, 42). The alterations included allelic losses at 3p,10p, 11, 13q, and/or 18q, each of which may potentially underlieactivation of telomerase. Interestingly, allelic losses at several of theseloci have been found in both cervical carcinomas and to differentdegrees in their precursor lesions (43-45). Loss of heterozygosity

studies with a panel of markers specific for these loci are currentlyunderway in an attempt to discover molecular genetic differencesbetween CIN lesions with and without telomerase activity.

Also, specific cytogenetic changes may mark CIN lesions withtelomerase activity. Of particular interest are comparative genomichybridization data reported by Heselmeyer et al. (46), showing that again at 3q defines the transition from CIN III to invasive carcinoma.More recent studies have revealed that the gene encoding humantelomerase RNA resides at 3q, and chromosomal gains at this locuswere associated with overexpression of the human telomerase RNAgene (47). Overexpression of hTR was detectable in 43% of cervicalcarcinomas (48). Another potentially intriguing finding is that approximately one-third of cervical carcinomas are characterized by a gain,

if not amplification, at 5p (49), the locus where the hTERT generesides (21). Consequently, also future comparative genomic hybridization studies may yield interesting genetic markers that allow todistinguish CIN lesions with telomerase activity.

Finally, it is tempting to speculate that telomerase activity detectable with our TRAP protocol and concomitant elevated hTERTmRNA levels may mark CIN lesions that have gained an immortalphenotype and as such have reached a point-of-no-return in terms of

malignant potential. If this would be true, immortalization of cervicalepithelial cells in vivo would represent a rather late event in the CINto SCC sequence that follows the presence of high-risk HPV sequences. Depending on the time of follow-up and the degree ofdyskaryosis of cervical cells, percentages ranging from 10-80% have

been given for women with CIN lesions who ultimately developcarcinomas (50, 51). Still, the significance of telomerase activityand/or elevated hTERT messages in predicting the biological behaviorof CIN lesions needs further attention. Of particular value in thiscontext can be telomerase studies on tissue specimens containing CINlesions adjacent to cervical carcinomas and cervical biopsies ofwomen with incompletely removed CIN III, who later developedcarcinoma. The results from such studies may ultimately contribute to

the establishment of telomerase parameters as late progression markers for CIN lesions.

ACKNOWLEDGMENTS

We are indebted to E. Klaassen and H. Schrijnemakers for technical assistance; Dr. L. Rozendaal for helpful assistance in the statistical analysis; andDrs. Q. Sun, L. T. Chow, and T. R. Broker for the pilot RNA in situ hybridization experiments.

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1998;58:3812-3818. Cancer Res   Peter J. F. Snijders, Mark van Duin, Jan M. M. Walboomers, et al.   Catalytic Subunit and High-Risk Human Papillomavirus DNAStrong Association with Elevated Messenger RNA Levels of ItsSubset of Cervical Intraepithelial Neoplasia Grade III Lesions: Telomerase Activity Exclusively in Cervical Carcinomas and a

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