Oral Oncology 36 (2000) 347±352
Telomerase activity in oral and maxillofacial tumors J. Liao *, T. Mitsuyasu, K. Yamane, M. Ohishi First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan Received 30 December 1999; accepted 20 January 2000
Abstract Telomerase activity was measured in biopsy specimens as well as surgically resected tissues of 39 oral squamous cell carcinomas (SCCs), 22 oral leukoplakias, 13 normal oral mucosas, 12 malignant salivary gland tumors, 10 benign salivary tumors and ®ve normal salivary gland tissues adjacent to tumors using a polymerase chain reaction (PCR)-based telomerase assay. Telomerase activity was detectable in 38.5% (5/13) of normal oral mucosa samples, 54.5% (12/22) of leukoplakia samples, 82.1% (32/39) of oral SCC samples, 83.3% (10/12) of malignant salivary gland tumor samples and 0% (0/10, 0/5) of benign salivary gland tumor and normal salivary gland samples. High-level enzyme activities were seen in 20% (3/15) of mild dysplasia specimens, 50% (2/4) of moderate±severe dysplasia specimens, 48.7% (19/39) of oral SCC tissue specimens, and no high activity was seen in the normal mucosa and hyperkeratosis specimens (P for trend, <0.001). We also analyzed the proliferative activity of dysplastic leukoplakia, oral SCC, and salivary gland tumor specimens using Ki-67 immunohistochemistry. The Ki-67 labeling indices (LI) were signi®cantly higher in dysplastic leukoplakia and oral SCC with high telomerase activity than in dysplastic leukoplakias and oral SCC with low and negative telomerase activity (P<0.01 and P<0.05). These results indicate that telomerase activity has some correlation with the progression of multistep oral carcinogenesis with the cellular proliferation, and also indicate that telomerase may be a speci®c marker used to distinguish malignant salivary gland tumors from their benign counterparts. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Telomerase; Oral leukoplakia; Oral cancer; Salivary gland tumors; Ki-67
1. Introduction Human telomeres consist of tandem hexametric (TTAGGG)n repeat sequences at the ends of chromosomes . Possible functions of telomere sequences include prevention of chromosome degradation, end-toend fusions, rearrangements, and chromosome loss . The length of telomeres in normal somatic cells progressively shortens with each cell division and somatic loss of telomeric sequences causes cellular senescence . Telomerase is a ribonucleoprotein DNA polymerase containing an RNA component that directs the synthesis of telomeric DNA repeats and compensates for the loss of telomeres during cell division [4,5]. Telomerase is expressed in a broad range of human malignant neoplasms, many of the immortalized cell lines and embryonic tissues, such as the germline, testis, and
* Corresponding author. Tel.: +81-092-642-6447; fax: +81-092642-6383. E-mail address: [email protected]
ovary . Telomerase has been thought to be undetectable in the mortal dierentiated somatic cells. However, recent evidence suggests that the telomerase expression is not only in transformed cells and tumors, but also in a variety of normal human tissues and that it is growth regulated. Skin samples have a very low level of telomerase activity, in which a relatively high level of telomerase activity is found in the proliferative basal cell layer, whereas the quiescent dermis is telomerase negative . Consistent with this ®nding, isolated normal epithelial cells and epithelial cell culture are telomerase positive . Those reports indicated that telomerase activity is present in normal human tissues and is correlated with proliferation. Up to now, although a number of studies have demonstrated telomerase activity in a variety of human tumors and normal tissues, the reports on telomerase activity in oral and maxillofacial tumors, especially on salivary gland tumors are limited [9±11]. In this study, we performed a non-radioisotopic telomeric repeat ampli®cation protocol (Non RI TRAP) assay in normal oral mucosa, oral leukoplakia, oral cancer, and benign
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J. Liao et al. / Oral Oncology 36 (2000) 347±352
and malignant salivary gland tumor samples. We also studied the relation between the telomerase activity of oral cancer and clinico-pathological features and the level of proliferation, assessed by Ki-67 immunohistochemistry.
2.2. Cell line
2. Materials and methods
SOUU B cell line derived from tongue carcinoma  was maintained in Dulbecco's modi®ed Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics. The cell extract was used as a positive control for the set of TRAP assays.
2.3. Telomerase assay
One-hundred and one tissue samples were obtained by biopsy or surgical resection from the First Department of Oral and Maxillofacial Surgery, Kyushu University (Fukuoka, Japan). All of the patients from whom tissue samples were obtained, were not treated with radiotherapy, chemotherapy, or immunotherapy, prior to biopsy or surgical resection. The samples included 13 normal oral mucosal tissues, 22 oral leukoplakias, 39 oral squamous cell carcinomas (SCCs), 12 malignant salivary gland tumors, 10 benign salivary gland tumors, and ®ve salivary gland tissues adjacent to tumors. The clinical data for oral cancer samples are shown in Table 1. Each of the samples was divided into two parts. One part was processed to routine histopathological examination, and the other was snap frozen immediately and stored at ÿ80 C until it was evaluated by the telomeric repeat ampli®cation protocol (TRAP) assay. The SCC and salivary gland tumor tissues were classi®ed according to the World Health Organization (WHO) classi®cation and diagnosis of the epithelial dysplasia (ED) was made based on the presence of one or more of the 12 histological ®ndings symptomatic of oral ED, as de®ned by the WHO Collaborating Center .
TRAP assay was performed as described by Kim et al.  with a minor modi®cation. For telomerase extraction, 20±200 mg of frozen or fresh samples were washed three times in ice-cold phosphate-buered saline (PBS). Then the pellets were homogenized in 20±200 ml ice-cold lysis buer [10 mM Tris±HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 5 mM b-mercaptoethanol, 0.5% 3-[(3-cholamidopropyl)-dimethylamino]1-propanesulfonate (CHAPS) and 10% glycerol]. After 30 min of incubation on ice, the lysate was centrifuged at 15,000 rpm for 30 min at 4 C, and the supernatant was frozen and stored at ÿ80 C or immediately assayed. The protein concentration of the extract was measured by BioRad Protein Assay. The TRAP assay was performed on all of the tissue samples, except the cell line, in three dierent concentrations (2, 0.2, and 0.02 mg protein/ assay). An equivalent of 1000 cells was used as a positive control for the cell line extract. The lysis buer was used for the negative control. The reaction mixture for each telomerase assay (50 ml) consisted of 20 mM Tris± HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA), 50 mM deoxyribonucleotide triphosphates (dNTP), 0.1 mg TS primer (5-AATCCGTCGAGCAGAGTT-3), 0.45 mM T4 gene 32 protein, 2 units Taq DNA polymerase. An aliquot (2 mg of protein) of extract was used for each standard TRAP assay. A 150-bp internal standard was used as an internal control. All extracts showing 6-bp ladders were tested for sensitivity to RNase pretreatment, 5 mg of extract was incubated with 1 ml of RNase (1 mg/ml) for 30 min at 37 C. The reaction was started in a thermal cycler at 30 C for 30 min for extension of the TS primer by telomerase, then at 90 C to inactivate the telomerase, followed by the addition of 0.1 mg biotion-labeled CX primer (5-biotinCCCTTACCCTTACCCTTACCCTAA3) and 31 polymerase chain reaction (PCR) cycles of 94 C for 45 s, 50 C for 45 s,72 C for 60 s and ®nally at 72 C for 3 min. The PCR product was electrophoresed on a 12% polyacrylamide gel and blotted onto Hybond N+ membranes. Blots were recorded by chemiluminescence using Non Radio Isotopic Detection Kit, Imaging with high Chemilumi (Toyobo, Osaka, Japan), and exposures were made using Kodak X-OMATAR ®lm. For estimation of telomerase activity, extracts with telomerase activity were re-examined at 10-fold (0.2 mg of
Table 1 Relation between telomerase expression and clinicopathological variables in 39 squamous cell carcinomas (SCCs)a Telomerase activity Variable
T1 T2 T3 T4
10 19 5 5
2 4 0 1
8 15 5 4
WD, well dierentiated; MD, moderately dierentiated.
J. Liao et al. / Oral Oncology 36 (2000) 347±352
protein) and 100-fold (0.02 mg of protein) dilution. Telomerase activity detected in the 0.02 mg was de®ned as `strong activity', the activity limited to the 2 and 0.2 mg assay was de®ned as `moderate activity', and the activity limited to the 2 mg assay was de®ned as `low activity' . Otherwise, moderate to strong activity was designated as high telomerase level, while weak or undetectable activity was designated as low telomerase level. 2.4. Immunohistochemical analysis Paran-embedded blocks adjacent to the tissue harvested for the TRAP assay were used for immunohistochemistry to detect Ki-67. The sections were pretreated in an autoclave to enhance immunoreactivities according to the method of Shin et al.  with some modi®cations. After deparanization in xylene and dehydration in ethanol, 4-mm sections were autoclaved at 121 C for 15 min in 10 mM citrate buer at a pH of 6.0. The MIB-1 antibody (1:100; Immunotech S.A., Marseille, France) was used as the primary antibody for Ki-67 immunohistochemistry. Subsequent reactions were carried out by the streptavidin±biotin method using an LSAB kit (DAKO, Tokyo, Japan). The nuclei were counterstained with hematoxylin. The positive results were represented by brown nuclear staining. In each case approximately 500 randomly selected nuclei were assessed. The percentage of Ki-67positive cells was calculated. 2.5. Statistical analysis Fisher's exact test or the chi-square test was used to compare categorical data between two groups. The Cochran±Armitage test was used to test for linear association. Statistical correlations between telomerase activity and Ki-67 labeling indices were analyzed by Mann±Whitney U test.
(Table 1). In 22 oral leukoplakia tissues, we uni®ed three leukoplakias with moderate dysplasia and one leukoplakia with moderate to severe dysplasia as a moderate±severe dysplasia group. Twelve samples (54.5%) were positive for telomerase activity. In those samples, we found that both the intensity and frequency of detectable telomerase activity increased with the degree of dysplasia. Three (75%) of four moderate± severe dysplasia leukoplakias had detectable telomerase activity, eight (53.3%) of 15 mild dysplasia leukoplakias had detectable telomerase activity, and one (33.3%) of three hyperkeratosis leukoplakias showed detectable telomerase activity. Furthermore, none of the hyperkeratosis leukoplakias showed high-level telomerase activity, three (20%) of 15 mild dysplasia and two (50%) of four moderate±severe dysplasia had high-level telomerase activity. None of the normal mucosa samples showed strong or moderate telomerase activity and only ®ve of 13 samples showed weak telomerase activity (Table 2). 3.2. Telomerase activity in benign and malignant salivary tumors Ten of the 12 malignant salivary gland tumor samples were shown to be telomerase positive in the TRAP assay (Table 4). The positive samples comprised three mucoepidermoid carcinomas, two adenoid cystic carcinomas (ACC), one adenocarcinoma, one SCC, one acinic cell carcinoma, one malignant pleomorphic adenoma and one papillary adenocarcinoma. One case of ACC and one case of muco-epideriod carcinoma showed no telomerase activity. All 10 cases of benign salivary tumor (eight pleomorphic adenoma and two cases of adenolymphoma) and normal salivary gland showed no telomerase activity (Table 4).
3. Results The results of the modi®ed TRAP assay of the SCC cell line and the fresh specimen are shown in Fig. 1. 3.1. Telomerase activity in oral cancer, oral leukoplakias and normal oral mucosa Of the 39 evaluated oral cancer tissues, 33 cases were well-dierentiated SCC and six cases were moderately dierentiated SCC, and none of the samples was poorly dierentiated SCC, in which 32 (82.1%) were positive for telomerase activity and high telomerase activity was detected in 19 (48.7%) tissue specimens. However, telomerase activity had no speci®c association with age, sex, and other clinicopathological conditions of the cases
Fig. 1. The amount of protein (2, 0.2, 0.02 mg) used in each assay is shown. Protein extracts of SQUU cell line (S) were used as positive control, RNase (R) and lysis buer (L) as a negative control. Telomerase activity detected in the 100-fold dilution was de®ned as `strong activity'; the activity limited to 2 and 0.2 mg assay was de®ned as `moderate activity', and that limited to 2 mg assay was de®ned as `low activity'. In this ®gure, sample 1 (A 2 mg, A1 0.2 mg, A2 0.02 mg) showed weak activity, sample 2 (B 2 mg, B1 0.2 mg, B2 0.02 mg) showed moderate activity.
J. Liao et al. / Oral Oncology 36 (2000) 347±352
Table 2 Telomerase activity in oral squamous cell carcinoma (SCC) and leukoplakia and normal mucosaa Condition
Telomerase activity Strong Moderate Weak Negative Total
SCC WD SCC MD SCC
Leukoplakia Hyperkeratosis Mild dysplasia Moderate±severe dysplasia
0 2 1
0 1 1
1 5 1
2 7 1
3 15 4
Table 3 Correlation of telomerase expression with oral histopathology Tissue histology
Normal mucosa Hyperkeratosis Mild dysplasia Moderate±severe dysplasia SCC Fisher's exact test Test for trend
5/13 (38.5%) 1/3 (33.3%) 8/15 (53.3%) 3/4 (75%) 32/39 (82.1%) P=0.011 P=0.001
0/13 (0%) 0/3 (0%) 3/15 (20%) 2/4 (50%) 19/39 (48.7%) P=0.002 P<0.001
WD SCC, well-dierentiated squamous cell carcinoma; MD SCC, moderately dierentiated squamous cell carcinoma.
3.3. Ki-67 LI in normal oral mucosa, leukoplakias, oral cancers and salivary gland tumors In normal oral mucosa and leukoplakia samples, Ki67-positive cells were predominantly found in the basal and parabasal layers (Fig. 2A). In the super®cial layer of moderate±severe dysplasia sample, the Ki-67-positive cells were also observed (Fig. 2B). In SCC, the Ki-67positive cells were located in the periphery of the tumor nests (Fig. 2C). The relationship between the telomerase activity and Ki-67 LI in dysplastic leukoplakias, oral SCC and salivary gland tumors is shown in Tables 4 and 5. Dysplastic leukoplakia and oral SCC samples with high telomerase activity exhibited signi®cantly higher Ki-67 LI than the dysplastic leukoplakias and oral SCC samples with low and no telomerase activity (P<0.01 and P<0.05)
Histologically, cancerization is expressed as progression from normal epithelium to hyperplastic lesions, dysplastic lesions, and invasive carcinoma. In order to investigate and compare telomerase activity in the process of oral tumor progression, we used modi®ed semiquantitative Non-RI TRAP to observe the frequency and intensity of telomerase activity in oral mucosa, oral leukoplakias and oral cancers. In our study, we observed that telomerase activity was detectable in 38.5% (5/13) of normal oral mucosa, 33.3% (1/3) of hyperkeratosis, 53.3% (8/15) of mild dysplasia, 75% (3/ 4) of moderate±severe dysplasia, and 82.1% (32/39) of oral SCC (P for trend, 0.001). Moreover, high-level enzyme activities were seen in 20% (3/15) of mild dysplasia samples, in 50% (2/4) of moderate±severe dysplasia samples, in 48.7% (19/39) of oral SCC samples, and no high activity was seen in the normal mucosa and hyperkeratosis samples (P for trend, <0.001) (Table 3). The signi®cant linear increase of telomerase activity along with the progression of oral SCC suggests an intimate relationship of telomerase activity with oral tumor development.
Fig. 2. Immunohistochemical detection of Li-67 labeling indices (LI) in (A) normal oral epithelia, (B) dysplastic leukoplakia, and (C) squamous cell carcinoma (SCC). In (A) and (B), Ki-67-positive cells were seen in the basal and parabasal layers in the epithelia of the mucosa. In (C), the positive cells were seen in the periphery of the tumor nests. The scale bar corresponds to 50 mm.
J. Liao et al. / Oral Oncology 36 (2000) 347±352 Table 4 Telomerase activity and Ki-67 labeling indices (LI) in salivary gland tumors Samples
Malignant Papillary adenocarcinoma Adenoid cystic carcinoma Mucoepidermoid carcinoma Acinic cell carcinoma Adenocarcinoma Squamous cell carcinoma Malignant pleomorphic adenoma Total Benign Pleomorphic adenoma Adenolymphoma Total Normal salivary gland a
1 3 4 1 1 1 1
21.5 20.33.76 25.24.25 19.8 39.2 31.1 NDa
12 8 2
1 2 3 1 1 1 1 10 (83.3%)
Positive telomerase activity
0 0 0 (0%)
ND, not done.
Previous studies have shown con¯icting results with respect to whether the activation of telomerase is an early or late event during carcinogenesis . Recent reports suggest that telomerase activity be involved at an early stage in human tumor development . Mutirangura et al.  reported that telomerase activity was positive in 10 of 23 cases with leukoplakia, including cases with hyperplasia, mild dysplasia and moderate dysplasia. Kannan et al.  reported 27 of 36 leukoplakias expressing telomerase activity and 17 of these 27 cases expressed weak telomerase activity and 10 cases showed strong and moderate activity. Another report showed that telomerase activity was seen in moderate or severe dysplastic tissue, but hyperkeratosis and mild dysplasia did not have telomerase activity . Our present study is consistent with those results. The telomerase activity was positive in 12 (54.5%) of 22 oral leukoplakias. Among them, high-level enzyme activities were seen in 20% (3/15) of mild dysplasia leukoplakia and 50% (2/4) of moderate±severe dysplasia leukoplakias. The presence of telomerase activity in a high proportion of leukoplakia indicates the increased potential for cellular transformation. In the previous studies on normal oral mucosa, telomerase was either absent or detected in only a few samples [19,20]. In this study, weak enzyme activity was expressed in ®ve of the 13 (38.5%) normal oral mucosa samples. Some reports showed that telomerase activity was detectable in many actively proliferating somatic cells and supported the hypothesis that all continuously proliferating epithelia were telomerase-positive [7,20, 22,23]. The telomerase activity in normal oral mucosa may be due to the presence of stem cells . However,
Table 5 Comparison of Ki-67 labeling indices (LI) between high telomerase and low, negative telomerase activity of dysplastic leukoplakia and oral cancer Variable
DLa Ki-67 LI SCC Ki-67 LI a
Telomerase activity High
Low and negative
30.53.41 (n=5) 39.711.1 (n=14)
22.74.65 (n=14) 32.19.50 (n=21)
DL, dysplastic leukoplakia.
Bickenbach et al.  reported that epidermal stem cells were not the major source of telomerase activity and demonstrated that proliferating transit amplifying cells exhibited more active telomerase. It has also been demonstrated that a normal hematopoietic cell lineage has weak telomerase activity  and that the telomere shortening occurred in the hematopoietic cell lineage which is subject to aging, implying that the level of telomerase activity in those cells may be insucient for the prevention of telomere erosion. The present study also demonstrated that telomerase activity was signi®cantly associated with Ki-67 expression in dysplastic leukoplakia oral cancer, suggesting an association with cellular proliferation. Our ®ndings are supported by some reports [25,26]. A study done by Ogoshi et al.  showed that the expression of Ki-67 was seen only in a subset of cells expressing telomerase RNA. This result also supported the evidence that telomerase activity is associated with cell proliferation. Considering that leukoplakia is often associated with in¯ammation in the submucosa, it is possible that telomerase activity of normal lymphocytes shows low positive signals for telomerase activity and may confound the interpretation of the results. Hiyama et al.  found no activity in normal lymph nodes or splenic tissue while activity has been found in some early hemopoietic precursor stem cells. In several of our examined leukoplakia samples, despite the lymphocytic in®ltration, telomerase activity could not be measured. There are few reports which studied telomerase activity of salivary gland tumors . In this study, we found telomerase activity in 83.3% (10/12) of malignant salivary gland tumors. Among these positive cases, one adenocarcinoma and one SCC showed high telomerase activity. On the other hand, one ACC and one mucoepideriod carcinoma (well dierentiated) showed no telomerase activity. We could not detect telomerase activity in all cases of benign tumors (eight pleomorphic adenomas and two adenolymphomas). Although the TRAP assay at present should not be used as a stand alone diagnostic tool, it has great potential value for the judgment of malignancy of salivary gland tumors. In conclusion, our ®ndings suggest that the oral cancer progression is associated with the intensity of telomerase
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