telomerase activity in premalignant and malignant lesions of human oral...

Received 10/2/96; accepted 2/24/97.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

I The present work was done as part of the Research Training Fellowship

Programme of Dr. S. Kannan awarded by the IARC, at the First Department of

Pathology, Hiroshima University School of Medicine, Hiroshima, Japan. The

present study was also supported in part by a Grant-in-Aid for Cancer Research

from the Ministry of Health and Welfare, Japan, for a comprehensive 10-year

strategy for cancer control.2 To whom requests for reprints should be addressed. Fax: 81-82-257-5149.

3 The abbreviations used are: TRAP, telomeric repeat amplification protocol:

WDSCC, well-differentiated squamous cell carcinoma; HPV, human papilloma

virus.

Vol. 6, 413-420, June 1997 Cancer Epidemiology, Biomarkers & Prevention 413

Telomerase Activity in Premalignant and Malignant Lesions of

Human Oral Mucosa1

S. Kannan, Hidetoshi Tahara, Hiroshi Yokozaki,

Babu Mathew, K. R. Nalinakumari, M. Krishnan Nair,and Eiichi Tahara2

Regional Cancer Centre, Thiruvananthapuram 695 01 1 , Kerala State, India

[S. K., B. M., K. R. N., M. K. N.]; and Department of Cellular and MolecularBiology [H. T.l and First Department of Pathology [H. Y., E. T.], Hiroshima

University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734,

Japan

Abstract

Recent studies have demonstrated a strong associationbetween carcinogenesis and re-activation of telomerase invarious human tumors. In the present study, we haveanalyzed the telomerase activity in 105 oral mucosalsamples, including normal mucosa and premalignant andmalignant lesions, by using the telomeric repeatamplification protocol assay. The telomerase activity wasdetected in normal oral squamous epithelium and in 75%

of the oral leukoplakias and oral carcinomas. Althoughthe telomerase activity was observed in normal andhyperplastic squamous epithelium, it showed somerelationship with certain clinico-pathological factors inmalignant lesions. Telomerase activity was found to havea relationship with the grade of tumor differentiation. Of34 well-differentiated squamous cell carcinomas, only 10(30%) exhibited high telomerase activity, whereas inmoderately or poorly differentiated squamous cellcarcinomas, all seven (100%) tumors displayed highactivity. In addition, the level of telomerase activity hadan inverse correlation with the treatment response in theearly-stage tumors, and the activity differed significantlybetween the tumors in the following intraoral sites:nonkeratinizing mucosa (buccal mucosa, alveolus, andfloor of mouth) and tongue. This preliminary resultshows that telomerase activity is present in normal oralsquamous epithelium, as it is in normal hematopoeticcells and in carcinomas, and that telomerase activity hasa relationship with degree of tumor differentiation andtreatment response. Thus, assessing the telomeraseactivity may be a useful prognostic marker in oralsquamous cell carcinomas.

Introduction

Cancer of the oral cavity is the most common neoplasm in Indiaand in most southeast Asian countries (I ). The high incidenceof this disease has been attributed to the prevalence of tobaccochewing among the populations of these countries. Cancer datafrom India show that approximately 30% of all cancers occur inoro-pharyngeal regions and account for about 56,000 deathsannually (2). Recently, the incidence of oral cancer has in-

creased in Western countries, also due to the wide use of

smokeless tobacco (3). Oral cancer has a well-defined precan-cerous lesion, oral leukoplakia, which serves as a good modelto study the processes of oral carcinogenesis (4). However,unlike the other common cancers, the molecular mechanism of

oral carcinogenesis is not well understood. Only a few frag-mentary reports are available regarding the molecular carcino-genic pathway of oral cancer (5-7). These studies showed a

difference in genetic aberrations between the oral cancers fromWestern and southeast Asian countries, which may be due tothe differences in the etiology. In Indian oral cancers, a higher

incidence of ras mutations and a lower incidence of p53 mu-tations were reported, compared with Western cases (5).

In the process of transformation of a cell from normal stateto malignant, one of the key steps is the immortalization ofcells. Recent evidence indicates that telomerase plays an im-

portant role in cell immortalization (8, 9). Telomerase is aRNA-dependent DNA polymerase that synthesizes telomericDNA fragments de novo, using its RNA moiety as a template,

and compensates for the loss of telomere during the cell divi-sion (10-12). The current hypothesis is that, during immortal-ization of a somatic cell, the telomere length will be maintained

by the reexpression of telomerase (I 3). Recently, a simple andhighly sensitive PCR-based “single tube” method, called theTRAP3 assay, was developed for telomerase detection (14, 15).Thereafter, a number of studies were reported regarding the

telomerase biology of normal, immortalized, and malignantcells. These studies have shown that somatic cells in general are

devoid of telomerase, whereas germ-line, immortalized, andmalignant cells express telomerase in varying degrees (8, 13,14). Telomerase activity was found in the majority of human

primary tumors, by us and others. It was detected in 85% ofgastric cancers and 95% of colorectal cancers (16), 85% of livercancers (17), 94% of neuroblastomas (18), 80% oflung cancers(19), 84% of prostate cancers (20), and 93% of breast cancers(21). Telomerase activity was not found in the correspondingnormal tissues; however, its expression was observed in somepreneoplastic lesions of gastric, colorectal, and liver cancers(16, 17). In addition, the telomerase activity was well correlated

with certain clinicopathological factors and proposed some

on June 16, 2018. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

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414 Telomerase Activity in Oral Lesions

Table 1 Relationship between telomerase ac tivity and histopath ology in oral cancers

Histopathology No. of casesLevel of telomer ase activity

Strong Moderate Weak Negative

WDSCC 34 1 (3%) 9 (27%) 13 (38%) 1 1 (32%)

Moderately differentiated squamous cell carcinoma 6 4 (67%) 2 (33%) 0 0

Poorly differentiated squamous cell carcinoma 1 1 0 0 0

Verrucous carcinoma 7 1 (13%) 2 (29%) 2 (29%) 2 (29%)

Atypical squamous epithelium 4 1 (25%) 1 (25%) 2 (50%) 0

Total 52 8 (15%) 14 (27%) 17 (33%) 13 (25%)

prognostic significance. Although the incidence of squamous

cell carcinomas of head and neck is high, telomerase activity inthese tumors has been reported by only two groups (14, 22). Arecent study has demonstrated telomerase activity in 88% ofhead and neck squamous cell carcinomas and 39% of oral

leukoplakia samples examined (22). In the present study, wehave examined the telomerase activity in normal oral mucosa,

oral leukoplakia, and oral squamous cell carcinoma tissuesobtained from Indian patients by using TRAP assay. We havealso studied the relation between the telomerase activity andclinico-pathological features of the malignant lesions.

Materials and Methods

Cell Lines. Six cell lines established from the human oralsquamous cell carcinoma, namely, HO-l-u-l (floor of themouth), HO-i-N-i (buccal mucosa), Ca9-22 (gingiva), HSC-2(buccal mucosa), HSC-3 (tongue), and HSC-4 (tongue), wereexamined for telomerase activity. All of these cell lines were

obtained from Japanese Cancer Research Resources Bank,

Tokyo, Japan, and were maintained in RPMI 1640 supple-mented with 10% fetal bovine serum, 100 units/mi penicillin,

and 100 �tg/ml streptomycin sulfate at 37#{176}Cwith 5% CO2.Confluent cultures were trypsinized and washed with sterilePBS, and the cell pellets were snap frozen in liquid nitrogen and

stored at -80#{176}Cuntil use. Before pelleting, small aliquots ofculture suspension were taken from each dish, and cell numbers

were counted.

Tissue Sample Collection. A total of 105 oral mucosal sam-ples, including 52 incision biopsies of oral cancer, 5 wideexcised (surgical cases) oral squamous cell carcinoma tissues,

36 punch biopsies of oral leukoplakia, and 2 normal oral mu-cosae, were examined for the telomerase activity. All of these

tissue samples were collected prior to any form of treatment forcancer. From each excised tissue, samples were taken macro-scopically from three representative areas: the tumor, the im-mediately adjacent mucosa, and the uninvolved distal mucosa.

All of the tissue materials were briefly rinsed in ice-cold sterilePBS to remove the adherent blood and immediately snap frozenin liquid nitrogen and stored at -80#{176}Cuntil use. Simultaneouswith the collection of tissue samples for the TRAP assay, bits

from each of the tissue samples were fixed in buffered formalinand processed for routine histopathological examination. Per-sonal data and clinical details of each patients were also col-lected and recorded pro forma.

TRAP Assay. TRAP assay in cell lines and tissues was done

essentially the same way as we have described previously (16).Briefly, cell pellets were suspended in 400 �tl of TRAP lysis

buffer and incubated on ice for 30 mm. Similarly, small bits oftissues were homogenized with TRAP lysis buffer (50-250 jd,depending upon the size of the tissue bit) in Kontes tubes withmatching pestles rotated with a Micromix at 450 rpm. After

incubation on the ice, both the extracts were centrifuged at

16,000 x g for 20 mm at 4#{176}C.The supernatants (cell extracts)were aliquoted and immediately stored at - 80#{176}C.Protein con-

centrations of tissue extracts were determined using Coomassiebrilliant blue assay. TRAP analyses were done in three con-centrations oftissue extract, 6 pg (1 X), 0.6 �tg (lOX), and 0.06,.#{227}g(lOOX) protein/assay, in all of the samples except cell lines.In cell lines, extracts equivalent to 1 X l0� cells were used for

TRAP assay. The TRAP assay was done according to the

method described earlier (15, 16), and it consisted of two steps.In the first step, the telomerase in the extract was allowed to

synthesize telomeric oligonucleotides on TS primer (5’-AATC-CGTCGAGCAGAGTT-3’). In the second step, the telomerase-synthesized new oligonucleotides were amplified using PCR by

including reverse CX primer (5’-CCTTACCClTACCCITAC-CCTAA-3’) in the presence of [32P}dCTP. Then, the PCR-amplified products were resolved on 12% nondenaturing poly-acrylamide gel, and the reaction products were finallyvisualized by autoradiography. All of the gels were autoradio-

graphed for short (14 h) and long (48 h) exposure times. The

telomerase activity was graded as strong (dark and prominentladders in both 14- and 48-h exposure autoradiograms), mod-erate (weak ladder in 14-h exposure and dark ladder in 48-hexposure), weak (detectable only in 48-h exposure), and neg-ative (not detectable in either 14- or 48-h exposure). We have

also confirmed the specificity of the telomerase activity byRNase treatment of the tissue extract prior to TRAP assay and

have run a negative control (instead of tissue extract, lysisbuffer alone was added) with all sets of TRAP assay to avoid

false postivity.We have analyzed the relationship between the level of

telomerase activity and clinico-pathological factors such as

histopathology, stage of disease, treatment response, and in-traoral site of the lesions in malignant cases. For this, sampleswith strong and moderate activity of telomerase were taken

together as high-activity group, and samples with negative orweak telomerase activity were taken as a negative group be-

cause we observed a weak telomerase activity in samples fromnormal oral mucosa. f test was applied to validate the signif-icance of the difference between groups.

Results

Telomerase Activity in Oral Cancer Biopsies and CellLines. Telomerase activity in oral cancer tissues is summa-rized in Table 1. Of 52 oral cancers, 39 (75%) exhibited

telomerase activity, whereas the remaining 13 (25%) tumorswere negative. Strong telomerase activity was seen in 8 (15%),

moderate activity was seen in 14 (27%), and weak activity wasseen in 17 (33%) cases. All of the positive tumors were sensi-tive to RNase treatment (Fig. 1). Initially, 16 samples were

negative for TRAP assay with 6-pg tissue extracts. But on



2 3 4 5 16 17 18 19 21 47

RNase - -I- - .4-. - .4.._ .4-.- -I- - -1- - -I- - -I- - �L -

�&b � � . ;l�Il�. �

Fig. 1. Telomerase activity in various histological types of oral cancers. Nu,,,.

bers. case numbers. (�ase 2. verrucous carcinoma: case 3. moderately differen-

tiated squamous cell carcinoma: case 47. poorly differentiated squamous cell

carcinoma. All other cases were WDSCCs. Tissue extracts without ( - ) or with( + ) RNase treatment were assayed for telomerase activity.

‘. 0�� , ...� � . �

Fig. 2. Telomerase activity in oral cancer tissue extracts with different protein

concentrations. Numbers. case numbers.

Cancer Epidemiology, Biomarkers & Prevention 4/5

3 13 18 30 48

I’rotcin (rig) so C).6 O.()6 6.0 0.6 0.06 (‘.0 0.6 0.06 6.0 0.6 0.06 6.0 O.(,

dilution analysis, of 16 negative cases, 3 expressed weak te-lomerase activity with 0.6 p.g of tissue extract. However, wehave not found much processivity or ladder extension in dilutedoral cancer tissue extracts (Fig. 2), as we observed in colorectaland gastric cancers ( I 6).

Histopathological analysis of the tumor samples showed

that majority of them (34 samples, 65%) were WDSCCs, 6were moderately differentiated squamous cell carcinomas, and1 was poorly differentiated squamous cell carcinomas. Of theremaining tumors, seven were verrucous carcinomas, and four

specimens had no frank malignant cells, only atypical squa-mous epithelium. The grade of differentiation of tumors exhib-

ited a relation with the level of telomerase activity. Of 34 casesof WDSCC, 1 1 (33%) were negative and 13 (38%) had onlyweak telomerase activity. However, all moderately and poorlydifferentiated squamous cell carcinomas displayed high telom-erase activity. Verrucous carcinomas showed a pattern of te-lomerase activity similar to that of WDSCCs (Table 1 ). Thestatistical analysis also showed a significant difference betweentelomerase activity and grade of differentiation in oral squa-mous cell carcinomas (x2 = 9.19; P < 0.005).

Lesions were classified as either early (including stage Iand II cancers) and late (including stage III and IV cancers), on

the basis of their composite clinical stage of disease, as de-

scribed by the Union Internationale contre le Cancer (23). Of 52cases, 34 (65%) were late-stage tumors and I 3 were early-stagetumors. With respect to telomerase positivity, no significantdifference was observed between the early and late stage of the

disease (x2 0.00; P > 0.9). All these cases were treated withradiotherapy, except for three early-stage verrucous carcino-mas, which were surgically excised. We have grouped the

patients into three categories: good (no clinical evidence of

disease after treatment), bad (partial or no response to treatment

and persistence of disease clinically), and no follow-up, on thebasis of the responsiveness of the tumor to treatment, judged in

the first follow-up visit (1-2 months) after the completion of thetherapy. On assessing the responsiveness of tumors to radio-therapy, it was found that 26 cases (50%) had only poorresponse and 1 1 cases (21%) had responded well to the treat-ment. No follow-up details were available in the remaining 15

cases. By correlating the telomerase activity with treatmentresponse of tumors, it was found that 50% ( 1 3 of 26) of the

cases with poor response to radiotherapy had a high expressionof telomerase. In the group that responded well, only 28% (3 ofI 1) showed high levels ofthe telomerase activity (Table 2), butit was not statistically significant (x2 = 0.83: P > 0.3). Re-

garding the total telomerase positivity (strong, moderate, andweak), no significant difference was found between good

(73%) and bad (81%) groups for treatment response.Dissection of the relationship between the stage of the

,c � disease and treatment response revealed that 86% ( I 9 of 22) of� � the late-stage cases responded poorly, whereas 55% (6 of 1 1 ) of

the early-stage cases showed good response to the treatment(Table 2). This difference in the treatment response was alsostatistically significant (x2 6.19; P < 0.025). With regard to

the telomerase activity, we found that the majority of the

early-stage cases (5 of 6) revealed weak or negative activity. Ofseven early cases with weak or negative telomerase activity,

five responded well to the treatment, whereas of four earlylesions with moderate or strong telomerase activity, only one

responded well to the treatment (Table 2). However, this dif-

ference was statistically not a valid one (x2 = 2.21 : P > 0.05).It is well known that intraoral localization of the cancer

has a significant effect on the response of radiotherapy or

patient prognosis. To examine whether any such relation ex-isted with respect to the telomerase activity, the tumors were

classified into two groups, tongue cancer and buccal cancer(inclusive of tumors located in the nonkeratinizing oral epithe-

hum, such as buccal mucosa, alveolus, and others). Surpris-ingly, the majority (13 of 16, 81%) of the tongue cancers

exhibited weak or negative telomerase activity, irrespective ofstage. Conversely, in buccal tumors, the majority (27 of 3 1) of

tumors expressed various levels of telomerase activity (x2 =

4.24: P < 0.05). We have also found some conspicuous dif-

ferences in treatment response between these two groups. Al-though most of the tongue cancers were negative or weak intelomerase activity, one negative case has responded well to the

treatment. However, 28% (10 of 36) of buccal cancers re-sponded well to the treatment, of which 7 (70%) had onlynegative or weak telomerase activity, but 71% (10 of 14) of the

poorly responded buccal tumors exhibited high telomerase ac-tivity. This difference in the telomerase activity and treatmentresponse in buccal cancers was also statistically significant (x2= 4.03; P < 0,05). By analyzing all of the variables. such as

stage of the disease, treatment response, and intraoral site of thelesion, together with telomerase activity, we demonstrated that,in early-stage buccal cancers, all (four of four) having negative

or weak telomerase activity responded well to the treatment,whereas two of the three cases with high activity of telomerase



NI

(�)ci)

,-4 -

rl) � I t NI� I � � I

C,) U I Ici) ci) 0 0� � � � U

RNase-+-+-+-�+ +

Fig. 3. Telomerase activity in cell lines established from the oral squamous cell

carcinomas. Details are given in “Materials and Methods.” Cell extracts without

( - ) or with ( + ) RNase treatment were assayed for telomerase activity.


Table 2 Relationship betw een telomerase activity, stage of disease, and treatment resp onse in oral cancers’

Telomerase activity

Treatment response No follow.up

Early stage Late stageEarly stage Late stage

Total

Good Bad Total Good Bad Total

Strong 1 1 2(18%) 0 4 4(18%) 1 t) I (7%)

Moderate 0 2 2(18%) 1 5 6(28%) 0 5 5(35%)

Weak 2 1 3(27%) 2 6 8(36%) 1 3 4(29%)

Negative 3 1 4 (37%) 0 4 4 (18%) 0 4 4(29%)

Total 6(55%) 5(45%) II 3(14%) 19(86%) 22 2(14%) 12(86%) 14

a The disease stages of five cases were not available. The treatment responses in these cases were: two bad (telomerase activity was strong in one case and weak in the

other case), two good (telomerase activity was moderate in one case and weak in other case), and one no follow-up (telomerase activity was negative).

Table 3 Relations hip between telomeras e activity and treatment response, stage of disease , and intraoral site of the cancers

Treatment response

TelomeraseTongue cancer Buccal cancer

Early stage Late stageNo FU”

Early stageTotal

Late stageNo FU Total

Good Bad Good Bad Good Bad Good Bad

Strong 0 1 0 0 0 1(6%) 1 0 0 4 1 6(19%)

Moderate 0 0 0 2 0 2(13%) 0 2 1 3 5 11(35%)

Weak 0 1 0 3 1 5(31%) 2 0 2 3 3 10(33%)

Negative I I 0 4 2 8 (50%) 2 0 0 0 2 4 (13%)

Total I (6%) 3(19%) 0 9(56%) 3(19%) 16 5(16%) 2(7%) 3(10%) 10(32%) Il (35%) 31”

a p�j 5-fluorouracil.

h The disease stages of five buccal cancers were not available. The treatment response in these cases were: two bad (telomerase activity was strong in one case and weak

in other case), two good (telomerase activity was moderate in one case and weak in other case), and one no follow-up (telomerase activity was negative).

responded poorly to the treatment (Table 3). Statistical analysisalso showed a significant (x2 = 3.73; P = 0.05) relationshipbetween the level of telomerase activity and the treatmentresponse in early buccal cancers. In the late stage, irrespectiveof the level of telomerase activity, the treatment responses were

poor in majority of the lesions (88% in high telomerase activitycases and 67% in weak or negative cases). We have alsoexamined whether this difference in treatment response be-

tween buccal and tongue cancers has any relationship with thehistological differentiation in tumors. But we could not foundany significant difference, and the majority of them were thewell-differentiated type in both the sites.

Because we found a conspicuous difference in telomerase

activity between tongue and buccal cancers, we furthersearched for telomerase activity in cell lines established from

squamous cell carcinoma of the various sites of oral cavity, as

detailed in “Materials and Methods.” But we could not find anydifference in telomerase activity in any of the cell lines, becauseall of them displayed identically a very high activity (Fig. 3).

However, these cell lines were not established from the oralcancers of Indian origin.

Telomerase Activity in Oral Leukoplakias. Of 36 oral leu-koplakias examined, 27 (75%) showed telomerase activity.Among them, 63% (17 of 27) expressed only weak telomerase

activity. All of the positive cases were sensitive to RNasetreatment, and negative cases did not exhibit any telomerase

activity, even in diluted extracts (Fig. 4). Histopathologicalexamination of these leukoplakia revealed that none of thelesions contained any notable dysplastic features. With regardto the relationship between telomerase activity and the clinicaltype of the leukoplakia, only 3 of 20 (15%) showed high

telomerase activity in homogenous group, whereas in the non-

homogenous group, 7 of 16 (44%) showed strong or moderate

telomerase activity (x2 3.66; P < 0.06; Table 4).

Telomerase Activity in Normal Oral Mucosa. As we exam-med telomerase activity in oral leukoplakia, we also examined

telomerase activity in normal oral mucosa adjacent to the ma-



A

57 58 59 60 61 62 90 91

RNase

-V.,’ �W �

a�I

�. �..

B

I..-

Fig. 4. Telomerase activity in oral leukoplakia tissues and in normal oral mu-

cosa. Numbers. case numbers. Cases 57-62. leukoplakias: cases 90 and 9/.normal oral mucosae. A. RNase sensitivity analysis in the tissue extracts. -.

without RNase treatment: +. with RNase treatment. B, dilution analysis of tissue

extracts. Lane C, negative control.

Cancer Epidemiology, Biomarkers & Prevention 4/7

lignant lesions. For this, we collected samples from tumor area,

the adjacent mucosa, and the distal uninvolved mucosa of thesame patient from five surgically excised tissues. Surprisingly,telomerase activity was detected in adjacent and distal mucosa,

as well as in cancers of all samples except one, which wasnegative in all areas, even in diluted extracts (Fig. 5). The

histopathological examination of these tissues did not revealany malignant cells in adjacent or distal mucosae. However, thedistal mucosa of one case (patient 36) had dysplastic changesshowing strong telomerase activity. Because adjacent normal

mucosae exhibited telomerase activity, we also examined thetelomerase activity in two normal mucosal samples obtainedduring nononcological maxillofacial reconstructive surgery.Weak telomerase activity was found in both of the normal

- - �Yw�.- w

59 60 61 90 91. C

Protein (�.Lg ) 6.0 0.6 0.06 6.0 0.6 0.06 So 0.6 0.1)5 I,.() 0.6 0.06 6.0 0.1. I).O(.

mucosae, which were also sensitive to RNase treatment (Fig.4A). On dilution analysis, one of them showed more activity in

the 10-fold diluted tissue extract (Fig. 4B).

In addition to that observation, another interesting obser-vation was found in one case of the carcinoma of tongue. AWDSCC located in the right side of the tongue was telomerase

negative, and the second biopsy from the left side ofthe tongue,which was a homogenous leukoplakia, exhibited high telomer-

ase activity. On dilution of the tumor extract, a very weaktelomerase activity was found in the 10-fold diluted sample(Fig. 6).

Discussion

In the present study, we made a distinctive observation that

differs from other available reports on telomerase activity invarious solid tumors. Normal oral mucosa, hyperplastic epithe-

hum, and oral leukoplakias showed a weak telomerase activity.One important reason for this difference may be that, with the

exception of a few reports (14, 22, 23), all telomerase studiesthus far performed have focused on telomerase biology inhuman cancers that originated in nonsquamous epithelium.Squamous epithelium is unique in its stratification and complex

differentiation pattern, and patients with squamous epitheliumcarcinomas also greatly vary in their treatment response (3, 4).Another peculiar feature of squamous epithelium, especially of

the oral mucosa, is that a patient with oral cancer has a 100-foldprobability of developing a second primary tumor in oral epi-thelium: the “field cancerization” concept (24).

Unlike other human tissues, the squamous epithelium hasvery high cell turnover rate, and the constant loss of superficial

cells (desquamation) is replaced by continuous division in thebasal cells (25, 26). Hence, comparatively more stem cells arelikely to be present in the squamous epithelium for the active

self-renewal process (25). As in the case of immortalized cells,

the stems cells of human hematopoietic tissue have been shownto possess telomerase activity (27, 28). Cellular kinetic studieshave also demonstrated a similarity in cell proliferation patternbetween the stem cells of squamous epithelium and hemato-poietic tissues (25). Thus, the telomerase activity in normal oralmucosa may be due to the presence of stem cells. Cell kineticstudies have shown that proliferation rate is comparatively

higher in the hyperplastic epithelia of the oral leukoplakia andcarcinoma (29, 30). Thus, these lesions may have more stem

cells, and the telomerase activity may also be higher. Thedegree of telomerase activity in oral squamous cell carcinomawas related to the histological differentiation. It has been re-

ported that WDSCCs are slow-growing, compared with mod-

erately and poorly differentiated squamous cell carcinomas,which are also more aggressive in nature (29, 31). It is likely

that less differentiated oral squamous cell carcinomas mightcontain more immortal cells than the former histological type;hence, telomerase activity would also be high. Studies onmouse squamous cell carcinoma have found a similar pattern of

Table 4 Relations hip between telomerase a ctivity and clinical type in oral leukoplakia

Clinical type ofleukoplakta

Telomerase activity

Strong Moderate Weak Negative Total

Homogenous

Speckled

Erosive

Verrucous

Total

0

I (17%)

I (13%)

0

2(5’�/)

3(15%)

2(33%)

3(37%)

0

8)22%)

10(50%)

2(33%

3(37%)

2 ( 100%)

17(48%)

7(35%)

1 (17%)

1 (13%)

0

9(25%)

20(56%)

6(17%)

8(22%)

2 (5%)

36



‘�f . . . . ‘

. �‘.:,C. � �

.�_

Carcinoma Leukoplakiaab c deab c de

Fig. 5. Telomerase activity in oral cancer (7). adjacent normal mucosa (A), and

distal normal mucosa (D) of the same patients. Numbers, case numbers.

4

Fig. 6. Differences in telomerase activity between WDSCC on the right side of

the tongue (left panel) and oral leukoplakia on the left side of the tongue (rig/it

panel) of the same patient. Lanes a. 10 jsg of tissue extract: Lanes b. 6 �ig of tissue

extract with RNase treatment: Lanes c, 6 �sg of tissue extract without RNasetreatment: LWi(�S (I. 0.6 �sg of tissue extract: Lanes e. 0.06 p.g of tissue extract.

4/8 Telomerase Activity in Oral Lesions

32 38 41 45 36

DATDATDATAT DAT

telomerase activity in relation to the grade of tumor differen-tiation (32).

An earlier study on the squamous cell carcinomas of thehead and neck have demonstrated telomerase activity in 88% of

the cancer tissues and in 38% (6 of 1 6) of the adjacent mucosaeof these tumors (14). Recently, Taylor et al. (33) have demon-strated the telomerase activity in sun-protected and exposednormal human skin and precancerous and cancerous skin le-sions. Studies on mouse skin tumors also showed a high te-lomerase activity in premalignant papillomas and demonstrated

the correlation between tumor progression and telomerase ac-tivity (34). All these results thus indicate that telomerase ac-tivity is present in normal squamous epithelium, as observed in

normal hematopoietic progenitor cells, and its activity shows acorrelation with tumor progression in some instances. Also, thepresence of high telomerase activity in adjacent or distal normalmucosa of the malignant lesions may be because of “con-demned mucosa,” which are due to the field cancerization

concept, and sometimes the activity may be related to theincidence of second primary tumors or recurrences. Further

study is needed to establish this relationship. However, a recentstudy on oral lesions only showed telomerase activity in 1 (5%)

of the 18 adjacent normal mucosa samples examined (22), butactivity was detected in 88% of carcinomas and 39% of leu-koplakia biopsies. In leukoplakia, a correlation between telo-merase activity and grade of dysplasia was found. It is difficult

to explain the difference in telomerase activity in normaladjacent oral mucosa between that study and the present study,but the variation may be due to racial differences.

With our present results, we have failed to detect telo-

merase activity in 25% of the both leukoplakias and malignantlesions. The reason for this cannot be explained at present.

However, one of the reasons that may be considered is theinactivation of telomerase enzyme due to improper tissue col-lection and storage or the presence of Taq polymerase inhibitors

in tissue. We took the utmost care during the collection oftissues, and all of the samples were collected in a uniformmanner. Also, we have analyzed tissue extracts in 10- and100-fold dilutions to nullify the effect of tissue inhibitors.Hence, inactivation or the presence of tissue inhibitors couldnot be a reason for telomerase negativity. Moreover, the pres-

‘w��

�.

� 10.

ence of strong telomerase activity in leukoplakia and its ab-sence in malignant tissue of the same patient (samples werecollected in an identical manner) also do not support thisreason. Hence, some biological differences may exist for thisreason, or there may have more than one pathway in oralcarcinogenesis, a telomerase-involved and an uninvolved

mechanism. Further detailed analyses are required to elucidate

the reason for this difference. A recent study on cellular im-mortality in human oral squamous cell carcinomas showed that

cultures established from some of the late-stage carcinomaswere senescent ones (35). Because telomerase is closely asso-ciated with immortality, the low or absent telomerase activity insome of the tumor samples might account for many mortalcancer cells. A similar observation was made in lung cancers

(19).Because telomerase activity was reported in normal he-

matopoietic cells, we also looked for any differences in inflam-

matory cell infiltration in these telomerase-negative and

-positive lesions with routine histopathological examination.We could not find any significant differences between thesegroups with regard to inflammatory cell infiltration, because the

telomerase-negative lesions also have marked inflammatorycell infiltration, as do telomerase-positive lesions (data notshown). In vitro studies have demonstrated a pivotal role forviral proteins in the activation of telomerase and immortaliza-tion of various cell types (8, 13). Recently, E6 proteins of HPV



Cancer Epidemiology, Biomarkers & Prevention 419

type 16 were shown to activate telomerase in early passage

human keratinocytes and mammary epithelial cells of precrisis

state (36). As in cervical neoplasia, a role for HPV has also

been implicated in oral carcinogenesis, due to the more frequent

occurrence of HPV infection in oral precancerous and cancer-ous tissues relative to normal oral tissues in some studies (6). A

recent study on the HPV prevalence in oral premalignant and

malignant tissues from the patients of same demographic areas

of the present study showed a high prevalence, 74% HPV

infection (37). Thus, there is a possibility that the HPV infec-

tion status could bring about the telomerase activity of the

present samples. However, detailed studies are required to

attribute an in vivo role for HPV proteins in the activation of

telomerase.

Studies on telomerase activity in cancers such as gastric,

liver, breast, and prostate indicated the usefulness of telomerase

activity as a prognostic marker and also proposed that thepresence of telomerase activity could be used as an early

detection marker to distinguish malignant and benign tissues

(16, 17, 20, 21). Although telomerase activity was observed in

normal and hyperplastic oral mucosae, the telomerase activity

in malignant lesions showed some significant associations with

certain clinico-pathological factors. The present results showed

a significant relationship between the level of telomerase ac-

tivity and treatment response in oral squamous cell carcinomas,

especially in early lesions. However, the present results need to

be reconfirmed in a study with larger sample size because the

sample size is limited in the present study, and the results might

therefore have occurred by chance. We also demonstrated a

marked difference in telomerase activity between tongue and

buccal cancers. One of the reasons for the poor response of

radiotherapy in the majority of tongue cancers may be the

superior blood and lymphatic supply, and the response may not

be related to telomerase status. These variations also indicate a

possible difference in carcinogenic and telomerase-involved

pathways in these tissues. The consistent high telomerase ac-

tivity in oral cancer cell lines obtained from different intraoral

sites might be due to the selection of cells in cell lines with

strong telomerase activity, due to their growth advantage under

in vitro conditions. We have also performed an analysis to see

whether any relationship was existed between personal details,

including sex, age, and oral habits like smoking and betel quid

chewing, and the telomerase activity in their tumor tissues. We

could not find any significant relationship between these factors

and telomerase activity (data not shown).

In conclusion, the results of the present study showed that

telomerase activity was present in normal oral squamous epi-thelium and maintained its expression in premalignant and

malignant lesions of the oral mucosa in varying degrees. In oral

malignant lesions, the degree of telomerase activity varied,depending upon the grade of tumor differentiation. The re-sponse of radiotherapy also correlated well with the level of

telomerase activity, especially in the early stages of disease that

develops in nonkeratinizing sites of oral mucosa. Thus, telo-

merase activity can be an additional tool as a biomarker for

assessing the prognosis or biological grade of the oral carcino-mas. Because telomerase activity is present in normal and

hyperplastic mucosa, it cannot be used as a marker for the early

detection of oral cancer, as proposed in other solid tumors.

However, further study on large number of samples is neces-

sary to confirm the prognostic significance of telomerase ac-

tivity in oral squamous cell carcinomas, including the rigorous

follow-up of the patients.

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1997;6:413-420. Cancer Epidemiol Biomarkers Prev S Kannan, H Tahara, H Yokozaki, et al. human oral mucosa.Telomerase activity in premalignant and malignant lesions of

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