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Original Studies |
Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles, Campus Erasme, 1070 Bruxelles, Belgium
Address all correspondence and requests for reprints to: F. Miot, IRIBHN, Université Libre de Bruxelles, Campus Erasme, Bat C, 808, route de Lennik, 1070 Bruxelles, Belgium. E-mail: fmiot{at}ulb.ac.be
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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It has been shown that some somatic mutations of the TSH receptor
confer a constitutive activity to these receptors, resulting (even in
the absence of TSH) in the stimulation of cAMP accumulation. Such
mutations are responsible for ±70% of the autonomous adenomas (2).
Mutations impairing the GTPase activity of the G protein Gs
account for less than 10% (3). The homogenous biochemistry of the
tumor (4), the existence of a single mutation, and its absence in the
rest of the tissue suggested the clonality of the defect, which was
recently confirmed (5). The gain in growth of these nodules has been
poorly studied. Somatic cells have a limited capacity of division
ascribed, at least in part, to the shortening of the telomeres at the
end of their chromosomes (6). Telomeres are protein-DNA complexes
required for protecting and maintaining chromosome ends (7).
Progressive shortening of telomeres in cultured somatic cells with
population doubling and aging suggests that this process also occurs
in vivo (8). Telomerase, a ribonucleoprotein DNA polymerase
that synthesizes telomeric DNA repeats at the 3' ends of eukaryotic
chromosomes, can compensate for such shortening. Telomerase activity
has been reported in germline cells and embryonic tissues (9), in
somatic cells with a high proliferation capacity (10, 11), in immortal
cell lines (12), and in a large number of human malignant tumors
(13, 14, 15). In this study, we have characterized the telomere length and
telomerase activity in autonomous thyroid adenomas and in their
surrounding tissues (Table 1
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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The nodules were identified clinically as being autonomous or cold, by 131I radioiodide or 99Tc pertechnetate scanning. To check the identity of autonomous and quiescent tissue, iodide trapping was measured in vitro in slices, as previously described (4). The thyroid tissues were collected after surgical resection from patients undergoing a partial or total thyroidectomy for an autonomous or hypofunctioning adenoma. The tissue was immediately kept at 4 C, carefully separated in autonomous and quiescent extranodular tissue before freezing in liquid nitrogen, and stored at -80 C. Samples of each nodule were systematically submitted to an anatomopathological analysis.
Determination of terminal restriction fragment (TRF) length
The genomic DNA was extracted from ±100 mg frozen thyroid by
grinding the tissue under liquid nitrogen. The powder was resuspended
in 700 µL of the lysis buffer [50 mmol/L Tris-HCl (pH8), 100 mmol/L
NaCl, 100 mmol/L EDTA, 1% SDS, 800 µg proteinase K (Merck,
Darmstadt, Germany)] and incubated for 20 h at 56 C. Ten
micrograms of DNA from extranodular tissues or 20 µg DNA from the
nodules were digested with HinfI and RsaI [10
U/µg (Gibco BRL, Life Technologies, Brussels, Belgium)] at 37 C for
3 h. DNA fragments were separated on a 0.8% agarose gel during
20 h at 70 V. The gel was dried and hybridized at 37 C for 8
h in 5x Denhardt, 5x saline-sodium citrate, 0.5% SDS, 1 mg/mL
BSA, 50 µg/mL denatured salmon sperm DNA with a 5'
(TTAGGG)3 telomeric probe end labeled with
32P ATP using T4 polynucleotide
kinase (Gibco BRL). Washes were performed in 1x saline-sodium
citrate/0.1% SDS at 42 C. The gel was dried at 65 C for 1 h and
autoradiographed for 3 days at -80 C or exposed on a phosphorimager
plate (Molecular Dynamics, Inc., Sunnyvale, CA) for
1 day. The lengths of the fragments were determined by use of molecular
weight DNA markers: 1-kilobase (1-kb) DNA ladder (Gibco BRL), Raoul
(Appligene, Pleasanton, CA) High Molecular Weight (Gibco
BRL).
Telomerase assay
HL-60 and Molt-4 cells were cultivated in a 10% FCS 1640 RPMI with L-glutamine medium. Then, 106 cells or 100 mg of tissue, ground under liquid nitrogen, were lyzed in 200 µL buffer containing 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L benzamidine (ICN Biomedicals, Inc., Cosa Mesa, CA), 5 mmol/L ß-mercaptoethanol, 0.5% CHAPS (Serva, Heidelberg, Germany), 10% glycerol at 4 C for 30 min. The lysates were centrifuged at 12,000 x g for 20 min at 4 C. The supernatants were used to determine the telomerase activity. Their protein content was determined using the Bradford assay (16).
Telomerase activity was determined using the Oncor TRAPEZE
detection kit (Oncor Inc., Gaithersburg, MD), following the
manufacturer’s protocol, based on the original method described by Kim
et al. (17). Briefly, a TS primer (5'-AATCCGTCGAGCAGAGTT-3')
from the kit was radiolabeled using T4 polynucleotide
kinase with 32P ATP. TS was elongated during 30 min at
37 C using 1–2 µg of protein extract. PCR amplification was
performed at 94 C for 30 sec and 55 C for 30 sec with 30 cycles, ended
by a 3-min step at 72 C with an internal standard of 36 base pairs
(bp). An 80-C heated sample was systematically used as negative
control. The efficiency of this treatment had been demonstrated on
telomerase positive cancer samples. The absence of primer-dimers and
PCR contamination was checked in an assay without any protein extract.
The PCR products were resolved by electrophoresis in a nondenaturing
12.5% polyacrylamide gel at 400 V for 4 h. The gel was then
exposed for autoradiography for 3 days at -80 C.
Fingerprint
DNA fragments, obtained after digestion of 5 µg DNA with HinfI, were separated on a 0.8% agarose gel during 16 h at 100 V. After blotting on nitrocellulose membrane, the DNA fragments were hybridized with a minisatellite probe, as previously described (18). The membrane was autoradiographed for 1 week at -80 C.
Data analysis
The mean length of the TRF was estimated using the ImageQuaNT program (Molecular Dynamics, Inc.), which creates a curve of signal intensity according to the migration length. The mean TRF length of each sample is defined as the median of this curve caused by the asymmetry of its shape. The mean TRF length values obtained from normal and pathological tissues were analyzed using a statistical paired t test.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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The analysis of telomere restriction fragment (TRF) lengths in 12
autonomous nodules revealed a significant shortening, when compared
with the TRF lengths of their corresponding collateral tissue. The mean
value of TRF lengths in the normal surrounding tissue of the nodule was
12.4 ± 1.6 kb. This was comparable with the TRF length measured
from control DNA extracted from leukocytes (lane C in Fig. 1A). In the nodules, the TRF lengths were
reduced to 8.6 ± 1.1 kb (Fig. 1, A and B). The shortening of
3.8 ± 1.0 kb was significant, with a P < 0.0001
with the paired t test. The same kind of decrease was also
observed in 16 hypofunctioning adenomas classified as microfollicular
(12 of 16), macrofollicular (2 of 16), and multinodular with cystic
degenerescences. The TRF mean size in the 16 surrounding tissues was
12.3 ± 1.7 kb. The size decreased to 8.8 ± 1.6 kb in the
hypofunctioning adenomas, demonstrating a shortening of 3.5 ± 1.2
kb (P < 0.0001). There exists in all the tissues a
broad distribution of the TRF sizes, which could be caused by both
interchromosomal and intercellular heterogeneity. In the adenomas, a
more widespread distribution of the smaller sizes of TRF was
systematically observed (Fig. 1A). Indeed, the ImageQuaNT curves from
all nodules were asymmetric (not shown). Therefore, the distribution of
the logarithmic migration distance was analyzed by calculating the
SD. Using the paired t test, the
SDs, calculated for each curve obtained from normal and
nodule tissues, were found to be statistically different
(P < 0.0001). The quality of DNA was evaluated for
each sample by fingerprint experiments (Fig. 1C). Degradation of DNA
was not observed in the normal tissues or in the adenomas, except for
sample N12, which was not taken into account in this study. The
fingerprints showed that DNA digestion by HinfI was
complete. Each adenoma and its respective surrounding tissue showed an
identical pattern of bands.
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. Ten of the 12 quiescent surrounding
tissues also did not show telomerase activity, whereas 2, infiltrated
with lymphocytes, presented the 6-base repeat ladder characteristic of
telomerase activity (Fig. 2). This activity seemed weak, compared with
the one measured in HL-60 immortal cell line or in a cancer tissue
prepared exactly in the same way as the thyroid samples. The absence of
telomerase activity in the majority of the samples was not a false
negative result. Indeed, the PCR reaction used to amplify the DNA
fragment elongated by the telomerase was not inhibited by any tissue
extract. This was proven by the presence of the internal 36-bp
amplified PCR control for each sample. On the other hand, telomerase
activity did not seem to be inhibited in our assay. As we can see in
Fig. 3, the mixing of 0.7 µg HL-60
extract, showing a high telomerase activity, with 2 µg of thyroid
nodule extracts, did not result in a significant inhibition of HL-60
telomerase activity.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Like all normal eukaryotic somatic cells, thyrocytes have a limited life span. It is known that each division leads to an erosion of telomeric sequences at the end of the chromosomes. The telomere hypothesis, which is well documented in human fibroblasts (28, 29), suggests that, in the absence of telomerase, the telomere shortening constitutes the mitotic clock for replicative senescence in normal somatic cells. At a critical telomere length, some cells escape from senescence by reactivating the telomerase and stabilizing their telomeres, bypass the crisis, and acquire unlimited replicative capacity. This occurs in the majority of malignant tumors and immortal cell lines (17).
The functional hyperactivity in the thyroid autonomous adenomas occurs with an increase of cell proliferation, as has been measured by an immunohistochemical staining procedure using monoclonal antibody MIB-1 (Deleu, unpublished). Proliferation seems to be more important at the periphery of the lesion than in the middle, whereas the quiescent tissue has a normal low proliferation index.
The data presented in this work showed a significant decrease (3.8
± 1.0 kb) of the TRF lengths in the adenomas vs. the
surrounding tissue. This decrease is not characteristic of autonomous
nodules, because it has been also observed in 16 hypofunctional
adenomas (decrease of 3.5 ± 1.2 kb). Assuming a loss of 100 bp
per population doubling, it would represent about 30 divisions,
i.e. the minimal number of cell divisions required for
growth from 1 cell (1 ng) to 109 cells (1 g), the average
size of such adenomas when they are surgically removed. The fingerprint
experiments were used to check the equal quality and digestion of the
DNAs prepared from the normal and pathological tissues. The same
pattern of bands obtained for both normal and pathological DNA samples
indicated that: 1) the samples came from the same patient; and 2) no
major chromosome instability (at least in the conditions described in
Fig. 1C
) was detected in the nodules, as has been described in some
tumors or cells at the end of their life span (30, 31).
The TRF length distribution is systematically more widespread in the autonomous adenomas than in the normal collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule. The proliferation data obtained by MIB-1 immunolabeling and the data on telomere length presented in this work for autonomous adenomas could suggest that the telomeres of cells in the nodule center have reached the short critical size and they stop dividing; whereas cells in the periphery, with longer telomeres, still proliferate. The idea that the cells reach the end of their life span is supported by the fact that hyperfunctioning adenomas never or rarely lead to carcinoma or invasion. Moreover, it has been shown that the expression of immediate early protooncogenes, like c-myc, c-jun, and c-fos, is decreased in these nodules, compared with the collateral tissue (Deleu, unpublished).
The well-known rarity of malignant degeneration of autonomous adenomas is in agreement with the absence of telomerase activity in these tumors. We were unable to detect any telomerase activity in the 12 analyzed autonomous adenomas, whereas the positive control included in each assay showed the 6-base repeat ladder. The presence of the internal amplified PCR control proved also that the results were not false negatives. Two of the quiescent surrounding tissues and also 1 collateral tissue of a hypofunctioning adenoma were positive for telomerase activity. The anatomopathological analysis revealed the presence of important lymphocytic infiltration in these 3 tissues. This could explain the weak telomerase activity, which has already been described in several thyroid studies (32, 33, 34, 35). Telomerase activity was not present in the cold nodules tested, except in one microfollicular adenoma lacking morphological characteristics of follicular carcinoma. Almost 100% of such carcinomas show telomerase activity (32). This unexpected result has already been described in a previous report (36), and it raised the question as to whether such an adenoma may contain a subpopulation of malignant cells. This patient is now carefully followed.
The hyperfunctioning adenomas are constituted of highly differentiated cells, which may explain the absence of telomerase activity. It has been shown in several telomerase-positive cell lines that differentiation is able to down-regulate the telomerase activity (37, 38). Thus, the high proliferation capacity in the adenomas results only in a decrease in telomere length. This decrease of TRF lengths, the wide-spread distribution of these lengths, and the absence of telomerase activity in 12 thyroid autonomous adenomas suggest that the cells have undergone a larger number of divisions than the surrounding normal tissue; some of them have reached the end of their life span, whereas others still proliferate without possessing malignant potential.
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Acknowledgments |
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Received May 5, 1998.
Revised July 2, 1998.
Accepted September 4, 1998.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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protein gene in 31 toxic thyroid
nodules. J Clin Endocrinol Metab. 82:3885–3891.This article has been cited by other articles:
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  A. Hebrant, J. Van Sande, P. P. Roger, M. Patey, M. Klein, C. Bournaud, F. Savagner, J. Leclere, J. E. Dumont, W. C. G. van Staveren, et al. Thyroid Gene Expression in Familial Nonautoimmune Hyperthyroidism Shows Common Characteristics with Hyperfunctioning Autonomous Adenomas J. Clin. Endocrinol. Metab., July 1, 2009; 94(7): 2602 - 2609. [Abstract] [Full Text] [PDF]
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