Telomerase Activity in Premalignant and Malignant Lesions of Human Oral Mucosa1

Recent studies have demonstrated a strong association between carcinogenesis and re-activation of telomerase in various human tumors. In the present study, we have analyzed the telomerase activity in 105 oral mucosal samples, including normal mucosa and premalignant and malignant lesions, by using the telomeric repeat amplification protocol assay. The telomerase activity was detected in normal oral squamous epithelium and in 75% of the oral leukoplakias and oral carcinomas. Although the telomerase activity was observed in normal and hyperplastic squamous epithelium, it showed some relationship with certain clinico-pathological factors in malignant lesions. Telomerase activity was found to have a relationship with the grade of tumor differentiation. Of 34 well-differentiated squamous cell carcinomas, only 10 (30%) exhibited high telomerase activity, whereas in moderately or poorly differentiated squamous cell carcinomas, all seven (100%) tumors displayed high activity. In addition, the level of telomerase activity had an inverse correlation with the treatment response in the early-stage tumors, and the activity differed significantly between the tumors in the following intraoral sites: nonkeratinizing mucosa (buccal mucosa, alveolus, and floor of mouth) and tongue. This preliminary result shows that telomerase activity is present in normal oral squamous epithelium, as it is in normal hematopoetic cells and in carcinomas, and that telomerase activity has a relationship with degree of tumor differentiation and treatment response. Thus, assessing the telomerase activity may be a useful prognostic marker in oral squamous cell carcinomas. Introduction Cancer of the oral cavity is the most common neoplasm in India and in most southeast Asian countries (I ). The high incidence of this disease has been attributed to the prevalence of tobacco chewing among the populations of these countries. Cancer data from India show that approximately 30% of all cancers occur in oro-pharyngeal regions and account for about 56,000 deaths annually (2). Recently, the incidence of oral cancer has increased in Western countries, also due to the wide use of smokeless tobacco (3). Oral cancer has a well-defined precancerous lesion, oral leukoplakia, which serves as a good model to 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 fragmentary reports are available regarding the molecular carcinogenic pathway of oral cancer (5-7). These studies showed a difference in genetic aberrations between the oral cancers from Western and southeast Asian countries, which may be due to the differences in the etiology. In Indian oral cancers, a higher incidence of ras mutations and a lower incidence of p53 mutations were reported, compared with Western cases (5). In the process of transformation of a cell from normal state to malignant, one of the key steps is the immortalization of cells. Recent evidence indicates that telomerase plays an important role in cell immortalization (8, 9). Telomerase is a RNA-dependent DNA polymerase that synthesizes telomeric DNA fragments de novo, using its RNA moiety as a template, and compensates for the loss of telomere during the cell division (10-12). The current hypothesis is that, during immortalization of a somatic cell, the telomere length will be maintained by the reexpression of telomerase (I 3). Recently, a simple and highly sensitive PCR-based “single tube” method, called the TRAP3 assay, was developed for telomerase detection (14, 15). Thereafter, a number of studies were reported regarding the telomerase biology of normal, immortalized, and malignant cells. These studies have shown that somatic cells in general are devoid of telomerase, whereas germ-line, immortalized, and malignant 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% of gastric cancers and 95% of colorectal cancers (16), 85% of liver cancers (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 corresponding normal tissues; however, its expression was observed in some preneoplastic 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 August 15, 2017. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from 414 Telomerase Activity in Oral Lesions Table 1 Relationship between telomerase ac tivity and histopath ology in oral cancers Histopathology No. of cases Level 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 in these tumors has been reported by only two groups (14, 22). A recent study has demonstrated telomerase activity in 88% of head and neck squamous cell carcinomas and 39% of oral leukoplakia samples examined (22). In the present study, we have examined the telomerase activity in normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma tissues obtained from Indian patients by using TRAP assay. We have also studied the relation between the telomerase activity and clinico-pathological features of the malignant lesions. Materials and Methods Cell Lines. Six cell lines established from the human oral squamous cell carcinoma, namely, HO-l-u-l (floor of the mouth), HO-i-N-i (buccal mucosa), Ca9-22 (gingiva), HSC-2 (buccal mucosa), HSC-3 (tongue), and HSC-4 (tongue), were examined for telomerase activity. All of these cell lines were obtained from Japanese Cancer Research Resources Bank, Tokyo, Japan, and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mi penicillin, and 100 tg/ml streptomycin sulfate at 37#{176}C with 5% CO2. Confluent cultures were trypsinized and washed with sterile PBS, and the cell pellets were snap frozen in liquid nitrogen and stored at -80#{176}Cuntil use. Before pelleting, small aliquots of culture suspension were taken from each dish, and cell numbers were counted. Tissue Sample Collection. A total of 105 oral mucosal samples, including 52 incision biopsies of oral cancer, 5 wide excised (surgical cases) oral squamous cell carcinoma tissues, 36 punch biopsies of oral leukoplakia, and 2 normal oral mucosae, were examined for the telomerase activity. All of these tissue samples were collected prior to any form of treatment for cancer. From each excised tissue, samples were taken macroscopically from three representative areas: the tumor, the immediately adjacent mucosa, and the uninvolved distal mucosa. All of the tissue materials were briefly rinsed in ice-cold sterile PBS to remove the adherent blood and immediately snap frozen in liquid nitrogen and stored at -80#{176}Cuntil use. Simultaneous with the collection of tissue samples for the TRAP assay, bits from each of the tissue samples were fixed in buffered formalin and processed for routine histopathological examination. Personal data and clinical details of each patients were also collected 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 of tissues were homogenized with TRAP lysis buffer (50-250 jd, depending upon the size of the tissue bit) in Kontes tubes with matching 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 concentrations of tissue extracts were determined using Coomassie brilliant blue assay. TRAP analyses were done in three concentrations 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’-AATCCGTCGAGCAGAGTT-3’). In the second step, the telomerasesynthesized new oligonucleotides were amplified using PCR by including reverse CX primer (5’-CCTTACCClTACCCITACCCTAA-3’) in the presence of [32P}dCTP. Then, the PCRamplified products were resolved on 12% nondenaturing polyacrylamide gel, and the reaction products were finally visualized by autoradiography. All of the gels were autoradiographed for short (14 h) and long (48 h) exposure times. The telomerase activity was graded as strong (dark and prominent ladders in both 14and 48-h exposure autoradiograms), moderate (weak ladder in 14-h exposure and dark ladder in 48-h exposure), weak (detectable only in 48-h exposure), and negative (not detectable in either 14or 48-h exposure). We have also confirmed the specificity of the telomerase activity by RNase treatment of the tissue extract prior to TRAP assay and have run a negative control (instead of tissue extract, lysis buffer 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 intraoral site of the lesions in malignant cases. For this, samples with strong and moderate activity of telomerase were taken together as high-activity group, and samples with negative or weak telomerase activity were taken as a negative group because we observed a weak telomerase activity in samples from normal oral mucosa. f test was applied to validate the significance of the difference between groups.