 |
|
Quantitative Assay of Telomerase Activity in Head and Neck Squamous Cell Carcinoma and Other Tissues
Songzhi Zhang, MM;
Mingmin Dong, MD;
Xuejing Teng, MB;
Tiehe Chen, MM
Arch Otolaryngol Head Neck Surg. 2001;127:581-585.
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.
INTRODUCTION
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).1, 2, 3, 4, 5
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:
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.
|
|
|
|
Table 1. The Relationship Between Telomerase Content and Its Activity
(HeLa Cell Lines)
|
|
|
|
|
|
|
Table 2. The Testing Results of Reproductiveness (HeLa Cell Lines)
|
|
|
|
|
|
|
Table 3. Clinical Data and Telomerase Activity in Samples*
|
|
|
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).
|
|
|
|
Table 4. Levels of Telomerase Activity in Specimens of Malignant Neoplasms,
Normal Tissue, Head and Neck Squamous Cell Carcinomas (HNSCC), and Other Tumors*
|
|
|
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,7, 8, 9 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.9, 10, 11, 12, 13
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.
AUTHOR INFORMATION
Accepted for publication June 16, 2000.
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.
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 NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells
and cancer. Science. 1994;266:2011-2015.
FREE FULL TEXT
2. Mutirangura A, Supiyaphun P, Trirekapan S, et al. Telomerase activity in oral leukoplakia and head and neck squamous
cell carcinoma. Cancer Res. 1996;56:3530-3533.
FREE FULL TEXT
3. Mao L, El-Naggar AK, Fan YH, et al. Telomerase activity in head and neck squamous cell carcinoma and adjacent
tissues. Cancer Res. 1996;56:5600-5604.
FREE FULL TEXT
4. Takubo K, Nakamura K, Izumiyama N, et al. Telomerase activity in esophageal carcinoma. J Surg Oncol. 1997;66:88-92.
FULL TEXT
|
ISI
| PUBMED
5. Cheng RY, Yuen PW, Nicholls JM, et al. Telomerase activation in nasopharyngeal carcinomas. Br J Cancer. 1998;77:456-460.
ISI
| PUBMED
6. Califano J, Ahrendt SA, Meininger G, Westra WH, Koch WM, Sidransky D. Detection of telomerase activity in oral rinses from head and neck
squamous cell carcinoma patients. Cancer Res. 1996;56:5720-5722.
FREE FULL TEXT
7. Haugen BR, Nawaz S, Markham N, et al. Telomerase activity in benign and malignant thyroid tumors. Thyroid. 1997;7:337-342.
ISI
| PUBMED
8. Umbricht CB, Saji M, Westra WH, Udelsman R, Zeiger MA, Sukumar S. Telomerase activity: a marker to distinguish follicular thyroid adenoma
from carcinoma. Cancer Res. 1997;57:2144-2147.
FREE FULL TEXT
9. Aldous WK, Grabill NR. A fluorescent method for detection of telomerase activity. Diagn Mol Pathol. 1997;6:102-110.
FULL TEXT
|
ISI
| PUBMED
10. Ohyashiki JH, Ohyashiki K, Sano T, Toyama K. Non-radioisotopic and semi-quantitative procedure for terminal repeat
amplification protocol. Jpn J Cancer Res. 1996;87:329-331.
FULL TEXT
|
ISI
| PUBMED
11. Ohyashiki JH, Ohyashiki K, Toyama K, Shay JW. A nonradioactive, fluorescence-based telomeric repeat amplification
protocol to detect and quantify telomerase activity. Trends Genet. 1996;12:395-396.
FULL TEXT
|
ISI
| PUBMED
12. Hiyama E, Yokoyama T, Tatsumoto N, et al. Telomerase activity in gastric cancer. Cancer Res. 1995;55:3258-3262.
FREE FULL TEXT
13. Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non–small-cell lung cancers. J Natl Cancer Inst. 1995;87:895-902.
FREE FULL TEXT
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter
What's this?
RELATED ARTICLE
Archives of Otolaryngology–Head & Neck Surgery Reader's Choice: Continuing Medical Education
Arch Otolaryngol Head Neck Surg. 2001;127(5):606-608.
FULL TEXT
|
