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Table 1 
The Relationship Between Telomerase Content and Its Activity (HeLa Cell Lines)
The Relationship Between Telomerase Content and Its Activity (HeLa Cell Lines)
Table 2 
The Testing Results of Reproductiveness (HeLa Cell Lines)
The Testing Results of Reproductiveness (HeLa Cell Lines)
Table 3 
Clinical Data and Telomerase Activity in Samples*
Clinical Data and Telomerase Activity in Samples*
Table 4 
Levels of Telomerase Activity in Specimens of Malignant Neoplasms, Normal Tissue, Head and Neck Squamous Cell Carcinomas (HNSCC), and Other Tumors*
Levels of Telomerase Activity in Specimens of Malignant Neoplasms, Normal Tissue, Head and Neck Squamous Cell Carcinomas (HNSCC), and Other Tumors*
1.
Kim  NWPiatyszek  MAProwse  KR  et al Specific association of human telomerase activity with immortal cells and cancer. Science.1994;266:2011-2015.
2.
Mutirangura  ASupiyaphun  PTrirekapan  S  et al Telomerase activity in oral leukoplakia and head and neck squamous cell carcinoma. Cancer Res.1996;56:3530-3533.
3.
Mao  LEl-Naggar  AKFan  YH  et al Telomerase activity in head and neck squamous cell carcinoma and adjacent tissues. Cancer Res.1996;56:5600-5604.
4.
Takubo  KNakamura  KIzumiyama  N  et al Telomerase activity in esophageal carcinoma. J Surg Oncol.1997;66:88-92.
5.
Cheng  RYYuen  PWNicholls  JM  et al Telomerase activation in nasopharyngeal carcinomas. Br J Cancer.1998;77:456-460.
6.
Califano  JAhrendt  SAMeininger  GWestra  WHKoch  WMSidransky  D Detection of telomerase activity in oral rinses from head and neck squamous cell carcinoma patients. Cancer Res.1996;56:5720-5722.
7.
Haugen  BRNawaz  SMarkham  N  et al Telomerase activity in benign and malignant thyroid tumors. Thyroid.1997;7:337-342.
8.
Umbricht  CBSaji  MWestra  WHUdelsman  RZeiger  MASukumar  S Telomerase activity: a marker to distinguish follicular thyroid adenoma from carcinoma. Cancer Res.1997;57:2144-2147.
9.
Aldous  WKGrabill  NR A fluorescent method for detection of telomerase activity. Diagn Mol Pathol.1997;6:102-110.
10.
Ohyashiki  JHOhyashiki  KSano  TToyama  K Non-radioisotopic and semi-quantitative procedure for terminal repeat amplification protocol. Jpn J Cancer Res.1996;87:329-331.
11.
Ohyashiki  JHOhyashiki  KToyama  KShay  JW A nonradioactive, fluorescence-based telomeric repeat amplification protocol to detect and quantify telomerase activity. Trends Genet.1996;12:395-396.
12.
Hiyama  EYokoyama  TTatsumoto  N  et al Telomerase activity in gastric cancer. Cancer Res.1995;55:3258-3262.
13.
Hiyama  KHiyama  EIshioka  S  et al Telomerase activity in small-cell and non–small-cell lung cancers. J Natl Cancer Inst.1995;87:895-902.
Original Article
May 2001

Quantitative Assay of Telomerase Activity in Head and Neck Squamous Cell Carcinoma and Other Tissues

Author Affiliations

From the Department of Otolaryngology–Head and Neck Surgery, Third Teaching Hospital, Xinxiang Medical College, Xinxiang (Dr Zhang and Mr Teng), Department of Otolaryngology, the First Hospital of Henan Medical University, Zhengzhou (Dr Dong), and the Shanghai Naval Medical Institute, Shanghai (Dr Chen), People's Republic of China.

Arch Otolaryngol Head Neck Surg. 2001;127(5):581-585. doi:10.1001/archotol.127.5.581
Abstract

Objectives  To confirm the applicability and use of a new technique to detect and quantify telomerase activity of specimens from head and neck malignant neoplasms and to explore whether the levels of telomerase activity can be a useful marker for cancer risk assessment in head and neck malignant neoplasms.

Design  Ninety-six specimens from 39 patients with head and neck malignant neoplasms were obtained. The specimens included 39 from patients with primary tumors (25 with head and neck squamous cell carcinoma and 14 with others), 10 from patients with neck metastases, 10 from patients with dysplasias, and 37 from patients with normal tissue. HeLa cell lines were used as positive control samples.

Main Outcome Measure  The levels of telomerase activity were determined using a liquid scintillation counter.

Results  The new method has a high rate of outcome reproducibility. The intrabatch and extrabatch variations were 15.6% and 16.4%, respectively. The linear relationship was good between the telomerase activity and the value within 700 radioactive cpm (rcpm) to approximately 7000 rcpm. The levels of telomerase activity determined by radioactive count were more than 1000 rcpm in 42 of the 49 malignant specimens and much more than that in the normal tissues, with the exception of 3 specimens. The levels of telomerase activity in normal tissues were less than 1000 rcpm in every sample and less than that in the malignant neoplasm samples, with the exception of 1 specimen (P<.000). Higher levels of telomerase activity in 2 of 10 tissues from patients who had dysplasias were detected (2 specimens from patients who had severe dysplasia). The differences in the levels of telomerase activity between the head and neck squamous cell carcinomas and the other tumors were not statistically significant (P>.05).

Conclusions  Detection of telomerase activity in head and neck malignant neoplasms can be a useful marker for the assessment of cancer. Telomerase reactivation may play an important role in tumorigenesis in head and neck squamous cell carcinoma. The quantification of telomerase activity may have clinical diagnostic value for head and neck malignant neoplasms.

TELOMERES are specialized structures at the ends of eukaryotic chromosomes that consist of repeated TTAGGG hexamers that can prevent chromosomes from degrading and fusing with other chromosomes. The length of telomeres in somatic cells is shortened during each cell replication cycle. However, the length of telomeres is maintained in germline cells. Telomerase, an RNA-dependent DNA polymerase, is a ribonucleoprotein that maintains telomere length. The primary function of telomerase is the synthesis of telomeric DNA, the reactivation of which is associated with escape from cellular senescence and cell immortalization.

Recently, telomerase activity has been detected in tissue samples from many human cancers but not in most normal tissue samples,1 which suggests that telomere stabilization and telomerase activation may play a role in tumorigenesis. However, there are few reports about telomerase reactivation in head and neck malignant neoplasms, especially in neck metastases, and the samples obtained were mostly from subjects who had head and neck squamous cell carcinoma (HNSCC).15 The question does activation of telomerase occur frequently in the pathogenesis of head and neck malignant neoplasms cannot be answered. Moreover, the original telomerase rapid amplification protocol (TRAP) assay is limited by its being a time-consuming procedure and that makes quantifying the enzyme activity difficult. Therefore, it is necessary to find a quantitative method to detect telomerase activity.

To confirm the applicability of a new technique and then to use it to quantify telomerase activity in tissue samples from head and neck malignant neoplasms and to explore whether levels of telomerase activity can be a useful marker for cancer risk assessment in head and neck malignant neoplasms, 96 specimens from 39 patients with head and neck malignant neoplasms and HeLa cell lines were detected by use of a liquid scintillation counter (LSC). The basic principle of this method is that a guanosine-rich oligonucleotide strand of telomeric sequence is used as primer, tritium-labeled deoxythymidine triphosphates (3H-dTTPs) are incorporated into the products while telomerase elongates the primers, and then free 3H-dTTPs are removed by rinsing. Finally, the radioactive counts per minute (rcpm) of products is detected, and the levels of telomerase activity can be evaluated according to the radioactive counts per minute.

MATERIALS AND METHODS
TISSUES AND CELLS

Ninety-six fresh specimens were obtained from 39 patients with head and neck malignant neoplasms, including 39 primary tumors (25 specimens from patients with HNSCCs and 14 specimens from patients with other carcinomas), 10 specimens from patients with neck metastases, 10 specimens from patients with dysplasia, and 37 specimens from patients with normal tissue. In 10 patients, both primary and neck metastatic tumors were obtained. All the specimens were confirmed by histological examination. Specimens were stored immediately after surgical excision or biopsy at −80°C until analysis. The weight of each specimen was between 50 and 150 mg. The specimens were obtained from the nose, pharynx, larynx, thyroid gland, and neck segment of the esophagus. For control samples, HeLa cell lines were tested.

TELOMERASE ASSAY

After removing the fat and connective tissues, each frozen specimen was first washed in buffer consisting of a combination of 10-mmol/L TRIS buffer and choloride (pH 7.4), 1.5-mmol/L magnesium chloride, 10-mmol/L potassium chloride, and 2-mmol/L 2-dithiothreitol, then the sample was dried with sterile filter paper. The sample was weighed, divided into 1- to 2-mm pieces, and lysed in microcentrifuge tubes containing cold lysate buffer that consisted of a combination of 20-mmol/L TRIS buffer and chloride (pH 8.3), 3.0-mmol/L magnesium chloride, 5-mmol/L 2-dithiothreitol, 100-mmol/L potassium chloride, 100-mmol/L sodium chloride, 1.0% polyglycol ether (nonionic) surfactant 40, 0.2-mmol/L phenyl methylsulfomyl fluoride, 10 U/mL RNasin, 10% glycerol, and 2.0-mmol/L spermidine. This was then homogenized using a manual homogenizer. After 30 minutes of incubation on ice, the lysate was centrifuged at 3000g for 5 minutes at 4°C. The supernatant was aliquoted and its protein concentration was determined using a combination of 10 mg of Coomassie brilliant blue G-250, 10 mL of 100% alcohol, 20 mL of 85% phosphoric acid, and 70 mL of water. The protein concentration of the extract was regulated at 1 mg/mL. The supernatant was aliquoted, flash frozen in liquid nitrogen, and stored at −20°C. When the cell line was tested, the sample was washed once or twice, then the lysate buffer was added directly, without homogenization. The next steps were the same as those used for tissue specimen analysis.

Two plastic tubes were used as an assay tube and a control tube, 10 µL of supernatant was added to the 2 tubes. Then 2.0 µL of the reaction-ending mixture, consisting of a combination of 100-mmol/L EDTA, 10-mmol/L TRIS buffer and chloride (pH 7.0), 0.1 mg/mL of RNase A, and 1.0 mg of plasmid DNA, was added to the control tube. After reacting in a thermostat for 10 minutes at 30°C, 10 µL of the reaction mixture, consisting of a combination of 2.0-mmol/L deoxyadenosine triphosphate, 2.0-mmol/L deoxyguanosine triphosphates, 5.0-µmol/L 3H-dTTP (1 Bq/mmol), and 2.0-µmol/L deoxyoligonucleotide (TTAGGGTTAGGGTTAGGG), was added to the 2 tubes; then the reaction was performed in a thermostat for 60 minutes at 35°C. When the reaction was ended, 2.0 µL of the reaction-ending mixture was added to the assay tube. The mixture was moved to the center of a No. 49 glassine filter 1.0 cm in diameter, and the glassine filter was placed in the drying oven at 80°C for 80 minutes. The glassine filter was placed on the suction tube and was washed with 5.0 mL of precipitate washing mixture (consisting of 2.25-mol/L ammonium acetate and 66% alcohol) to remove free 3H-dTTP; next, then the glassine filter was again placed in the drying oven at 80°C for 40 minutes. Finally, the glassine filter was placed in the scintillation bottle with 3.0 mL of scintillation liquid (consisting of 0.03% 1,4-di[2-(5-phenyloxazolyl)]-benzene, 0.5% 2,5-diphenyloxazole, 66% alcohol, and 100% xylene); the levels of telomerase activity were determined with an LSC. The radioactive counts per minute of products were detected.

The assay functions in determining telomerase activity are as follows:

Image description not available.
RESULTS

This new method has a high rate of reproducibility. The intrabatch and extrabatch variations were 15.6% and 16.4%, respectively. The linear relationship was good between the telomerase activity and the value within 700 rcpm to approximately 7000 rcpm (Table 1 and Table 2). The levels of telomerase activity and characterization of samples are listed in Table 3.

The levels of telomerase activity determined by radioactive counts per minute were more than 1000 rcpm in 42 (85.7%) of 49 malignant specimens and much more than that in the normal tissue samples, with the exception of 3 samples. The levels of telomerase activity in the normal tissue samples were less than 1000 rcpm for every sample, and less than that in the malignant neoplasms, with the exception of 1 specimen (P<.000) (Table 4). The differences in the levels of telomerase activity between the HNSCCs and the other tumors were not statistically significant (P>.05) (Table 4). Higher levels of telomerase activity were detected in 2 of 10 tissue specimens from patients with dysplasia (2 specimens were from patients who had severe dysplasia).

COMMENT
QUANTITATIVE ASSAY OF TELOMERASE ACTIVITY

Detection of telomerase activity using the TRAP assay was first reported in 19941 and the method has been widely used to examine the possible role of telomerase in the development of neoplasia. However, the original TRAP is limited in its time-consuming nature and the difficulty in quantifying the enzyme activity using TRAP. Since recent studies have demonstrated low, but distinct, levels of telomerase activity in peripheral blood cells from normal individuals, at least semiquantitative determination of telomerase activity in samples is needed. To overcome these problems, this study developed a method that uses telomeric repeated liquid scintillation counting (LSC). The levels of telomerase activity were detected with a liquid scintillation counter and determined by the value of radioactive counts per minute. The new method has a high rate of reproductibility. The intrabatch and extrabatch variations were 15.6% and 16.4%, respectively. The linear relationship was good between the telomerase activity and the value within 700 rcpm to approximately 7000 rcpm. To compare our data with the data obtained by TRAP assay, the levels of telomerase activity greater than 1000 rcpm in this study were defined as positive for telomerase. In our study, the levels of telomerase activity determined by the radioactive counts per minute were more than 1000 rcpm in 42 (85.7%) of 49 malignant specimens and much more than that in the normal tissue samples, with the exception of 3 specimens. The levels of telomerase activity in the normal tissue samples were less than 1000 rcpm in every sample, and less than that in the malignant specimens, with the exception of 1 specimen. These results were similar to the reports about telomerase activity in HNSCCs from Kim et al1 (87.5%), Mutirangura et al2 (87.5%), Mao et al3 (90%), and Califano et al6 (80%). It is suggested that this quantitative assay can replace the TRAP assay and be used to detect the telomerase activity of clinical samples.

THE ADVANTAGES AND DISADVANTAGES OF THE LSC METHOD

The TRAP assay was designed to increase the detection of telomerase activity over the original telomerase assay by using a polymerase chain reaction amplification step. The advantages of the TRAP assay were increased sensitivity and rapidity. The disadvantages of the TRAP assay were the use of radioisotopes for the detection process, the time involved, and the poor autoradiographic resolution of telomeric repeats. To improve the TRAP assay, several laboratories modified the TRAP assay. However, those methods, including nonradioactive ones,79 all required polymerase chain reaction amplification; detection of telomerase activity was in an indirect way, and the levels of telomerase activity were shown in a qualified or semiquantitative way, rather than a quantitative way. Moreover, the false-negative results can be obtained when the inhibitors of amplification were present in some tissue extracts. The method developed by our laboratory was called "telomeric repeat LSC protocol." The LSC assay can detect levels of telomerase activity in a direct and quantitative way. The basic principle of the method is that a guanosine-rich oligonucleotide strand of telomeric sequence is used as primer, 3H-dTTPs are incorporated into the products while telomerase elongates the primers, and then free 3H-dTTPs are removed by rinsing, finally, the radioactive counts per minute of products are detected, and the levels of telomerase activity can be evaluated according to the radioactive counts per minute.

Because the products elongated by telomerase were short single-stranded oligonucleotide, the strand was not easy to precipitate and fix. To overcome the problems, the moderate complementary single- or double-stranded telomeric DNA was added to the reaction mixture to form double- or triple-stranded DNA; this innovation improved the separation efficiency and sensitivity of detection. In comparison with the TRAP assay or a modified TRAP assay, the LSC assay features several advantages for detection of telomerase activity, both basic and clinical, eg, ease, rapidity, less expensive, and quantification. However, the problem of isotopic waste remained in the LSC assay.

TELOMERASE REACTIVATION AND TUMORIGENESIS OF HEAD AND NECK MALIGNANT NEOPLASMS

The role of telomerase expression in the process of malignant transformation is undergoing intensive scrutiny. Initial investigations have focused on the association of cellular immortalization with the ability of cells to maintain telomere length during cell division. Concurrent investigations in primary human tumor specimens have demonstrated a unique association among telomerase activity, neoplastic transformation, and cellular immortalization.913 In this study, we found that higher levels of telomerase activity are present in all stages of tumor progression in patients with HNSCC, ranging from early preinvasive dysplasias to fully malignant invasive tumors. Our results are consistent with several reports about detection of telomerase activity in many human cancers, including HNSCC. Moreover, higher levels of telomerase activity were detected in specimens from patients with other types of malignant tumors and neck metastases. To our knowledge, this is the first report about telomerase activity in both primary tumors and corresponding neck metastases; its findings indicated that metastases also have telomerase reactivation. We also found that the levels of telomerase activity between the specimens from patients with HNSCC and those from patients with other tumors, including adenocarcinoma, malignant melanoma, and lymphoma, have no statistically significant differences. It is suggested that telomerase expression may be present in all head and neck malignant neoplasms and telomerase reactivation may play an important role in tumorigenesis in HNSCC, and the detection of telomerase activity in head and neck specimens can be a useful marker for cancer assessment.

In this study, the percentile (P) was used to estimate reference ranges, because these data are not a standard normal distribution. With statistical analysis, the P5 level (radioactive counts per minute) of telomerase activity in malignant neoplasms, also called 95% medical reference ranges, was 890 rcpm; the P95 level of telomerase activity in normal tissue samples, also called 95% medical reference ranges, was 754 rcpm. Two levels can be used to estimate the presence of malignancy in the dysplastic lesions. Two SDs at a lower cut-off were not used to define the presence of malignancy in the dysplastic lesions. In fact, when the level of telomerase activity in a specimen is higher than 890 rcpm, the specimen has the probability of being malignant if telomerase activation is a general phenomenon in malignant neoplasms.

With the aforementioned reference ranges, higher levels of telomerase activity were present in 2 specimens from patients with dysplasias. This may indicate that telomerase mainly expresses in late HNSCC carcinogenesis but prior to a fully developed cancer phenotype. The significant correlation between telomerase activity and late-stage carcinogenesis of HNSCC may be the result of a higher mutation rate involving telomerase repression during this stage. However, the detection of telomerase activity in dysplasias also suggests that reactivation of telomerase may be involved in early tumorigenesis of HNSCC. Further investigations are necessary to determine whether detection of telomerase activity can be used in an early-screening method for asymptomatic high-risk patients.

CONCLUSIONS

This new technique can be used for quantitative analysis of the telomerase activity of tissues or cells. Detection of telomerase activity in head and neck malignant neoplasms can be a useful marker for cancer assessment. Telomerase reactivation may play an important role in tumorigenesis of HNSCC. The quantification of telomerase activity has clinical diagnostic value for head and neck malignant neoplasms.

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Article Information

Accepted for publication June 16, 2000.

Corresponding author: Songzhi Zhang, MM, Department of Otolaryngology–Head and Neck Surgery, Third Teaching Hospital, Xinxiang Medical College, Xinxiang 453003, Henan, People's Republic of China.

References
1.
Kim  NWPiatyszek  MAProwse  KR  et al Specific association of human telomerase activity with immortal cells and cancer. Science.1994;266:2011-2015.
2.
Mutirangura  ASupiyaphun  PTrirekapan  S  et al Telomerase activity in oral leukoplakia and head and neck squamous cell carcinoma. Cancer Res.1996;56:3530-3533.
3.
Mao  LEl-Naggar  AKFan  YH  et al Telomerase activity in head and neck squamous cell carcinoma and adjacent tissues. Cancer Res.1996;56:5600-5604.
4.
Takubo  KNakamura  KIzumiyama  N  et al Telomerase activity in esophageal carcinoma. J Surg Oncol.1997;66:88-92.
5.
Cheng  RYYuen  PWNicholls  JM  et al Telomerase activation in nasopharyngeal carcinomas. Br J Cancer.1998;77:456-460.
6.
Califano  JAhrendt  SAMeininger  GWestra  WHKoch  WMSidransky  D Detection of telomerase activity in oral rinses from head and neck squamous cell carcinoma patients. Cancer Res.1996;56:5720-5722.
7.
Haugen  BRNawaz  SMarkham  N  et al Telomerase activity in benign and malignant thyroid tumors. Thyroid.1997;7:337-342.
8.
Umbricht  CBSaji  MWestra  WHUdelsman  RZeiger  MASukumar  S Telomerase activity: a marker to distinguish follicular thyroid adenoma from carcinoma. Cancer Res.1997;57:2144-2147.
9.
Aldous  WKGrabill  NR A fluorescent method for detection of telomerase activity. Diagn Mol Pathol.1997;6:102-110.
10.
Ohyashiki  JHOhyashiki  KSano  TToyama  K Non-radioisotopic and semi-quantitative procedure for terminal repeat amplification protocol. Jpn J Cancer Res.1996;87:329-331.
11.
Ohyashiki  JHOhyashiki  KToyama  KShay  JW A nonradioactive, fluorescence-based telomeric repeat amplification protocol to detect and quantify telomerase activity. Trends Genet.1996;12:395-396.
12.
Hiyama  EYokoyama  TTatsumoto  N  et al Telomerase activity in gastric cancer. Cancer Res.1995;55:3258-3262.
13.
Hiyama  KHiyama  EIshioka  S  et al Telomerase activity in small-cell and non–small-cell lung cancers. J Natl Cancer Inst.1995;87:895-902.
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