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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 12 4368-4372
Copyright © 1998 by The Endocrine Society

Original Studies

Decrease of Telomere Length in Thyroid Adenomas without Telomerase Activity

X. De Deken, C. Vilain, J. Van Sande, J. E. Dumont and F. Miot

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


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8 ± 1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6 ± 1.1 kb), compared with the TRF length of their surrounding tissues (12.4 ± 1.6 kb). The same kind of decrease, 3.5 ± 1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3 ± 1.7 kb in surrounding tissue and 8.8 ± 1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
TOXIC, or autonomous, thyroid adenomas are benign, well-delimited, and encapsulated tumors characterized by TSH-independent function and growth (1). Autonomous thyroid hormone synthesis and secretion suppress normal TSH secretion, leading to the quiescence of surrounding tissue. In nuclear medicine, the tumors are called hot nodules, because they concentrate radioiodide or 99Tc pertechnetate, whereas the collateral tissue takes up less isotope. On the contrary, hypofunctioning adenomas, poorly metabolizing iodide, are called cold nodules.

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{alpha} 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 1Go).


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  		Table 1. Telomerase activity and TRF lengths in thyroid adenomas included in this study

 

   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Tissue collection

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 {gamma}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 {gamma}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.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
The tissues used in this study were characterized as autonomous nodules, by scintigraphy in vivo and by in vitro T/M values higher than in quiescent surrounding tissue (T/M = tissue-to-medium ratio of radioiodide content; range of T/M values in nodules = 2.4–128, and range of T/M values in adjacent tissue = 1.1–26).

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. 1AGo). In the nodules, the TRF lengths were reduced to 8.6 ± 1.1 kb (Fig. 1Go, 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. 1AGo). 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. 1CGo). 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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  		Figure 1. Panel A, Lengths of TRF in thyroid tissues of 12 patients with autonomous adenomas. Genomic DNA was extracted from each nodule (HN) and its surrounding quiescent tissue (N), submitted to electrophoresis, and hybridized with a 32P(TTAGGG)3 probe. The length was determined with radioactive 1-kb ladder (1st lane), Raoul ladder, and high weight molecular ladder (staining not shown). Lane C, TRF in normal leukocytes as DNA digestion control. Panel B, Histogram representation of the TRF lengths shown in panel A. Panel C, Fingerprint of autonomous adenoma (HN) and surrounding tissue (N) from 7 of the 12 patients studied. Lane 1, K562 cell line DNA used as digestion control. Last lane, Raoul standard ladder.

 
None of the 12 autonomous nodules presented detectable telomerase activity; the results of 5 are shown in Fig. 2Go. 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. 2Go). 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. 3Go, 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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  		Figure 2. Telomerase activity in five representative cases of autonomous nodules and their quiescent collateral tissue. The activity was measured by the telomeric repeat-amplification protocol (TRAP) assay using 1 µg protein extract. Each sample was heated at 80 C to inactivate the telomerase activity [only shown for patient 10 (N10*, HN10*) and positive controls (C+*)]. The internal PCR control appears at 36 bp (arrow). B0, Blank of PCR, all reagents except the protein extract. HL-60 and a brain cancer tissue were used as positive controls for the TRAP assay.

 


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  		Figure 3. Telomerase activity in HL-60 cells in the presence of protein extract from telomerase negative samples from autonomous nodules. HL-60 cell extract (0.7 µg) was mixed with 2 µg protein extract of hot nodule before the TRAP assay. C+, Telomerase activity from HL-60 cell line alone; C+*, HL-60 extract heated at 80 C; B0, PCR blank as in Fig. 2Go.

 
No telomerase activity was measured in the majority of the hypofunctioning adenomas and their surrounding tissues. A collateral tissue, infiltrated by lymphocytes and a cold nodule issued from another patient, showed a weak telomerase activity. This last case, CN7, and a representative negative one are shown in Fig. 4Go.



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  		Figure 4. Telomerase activity in 2 of the 16 hypofunctioning adenomas (CN) and their respective collateral tissue (N). The activity was measured by the TRAP assay using 1 µg protein extract. Each sample was heated at 80 C (*). Internal PCR control at 36 bp; B0, blank of PCR; C, positive control for telomerase activity.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Autonomous thyroid adenomas have been characterized in vivo as hyperfunctioning tumors secreting thyroid hormones independently of the plasma TSH. The patients suffer from hyperthyroidism when the size of the lesion and the intake of iodide are sufficient (19). The hyperactivity of the nodule contrasts with the quiescence of the surrounding tissue itself because of low levels of plasma TSH. Seventy percent of the autonomous adenomas are caused by constitutive stimulation of the cAMP cascade by mutations in the gene of the TSH receptor (2, 3, 20, 21, 22, 23). cAMP induces the expression of thyrocyte differentiation (expression of thyroid-specific genes like thyroglobulin, thyroperoxidase, and Na+/I- symporter) and stimulates the functional activity of the gland (iodide uptake and hormone secretion) and its growth (24, 25, 26, 27).

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. 1CGo) 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.


   Acknowledgments
 
We thank Professor P. Rocmans (Thoracic Surgery Department) and Dr. I. Salmon (Department of Anatomopathology, Erasme University Hospital) for their collaboration in providing us the tissues. We are grateful to Dr. C. Streydio for helping us in the fingerprint experiments and Dr. S. Swillens for valuable discussion.

Received May 5, 1998.

Revised July 2, 1998.

Accepted September 4, 1998.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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