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F Arturi, D Russo, D Giuffrida, M Schlumberger and S Filetti

OBJECTIVE: To investigate the molecular mechanisms underlying the influence of alteration of iodine trapping on the prognosis of metastatic papillary thyroid carcinomas, focusing on the expression of the Na+/I(-) symporter (NIS). DESIGN: We evaluated the expression of the NIS gene in a series of 11 enlarged neck lymph-node metastases of papillary thyroid carcinomas, including four patients in whom an enlarged lymph node represented the first sign of the tumoral disease. Nine lymph nodes, either reactive or metastatic for non-thyroid tumors, were also investigated. METHODS: Expression of the NIS gene was evaluated by RT-PCR in material obtained by fine-needle aspiration biopsy. RESULTS: The NIS gene was expressed in eight (73%) of 11 differentiated thyroid cancer metastatic lymph nodes examined. Five of these metastatic lymph nodes were positive at the post-treatment total-body iodine-131 scan; in the other three, the total-body scan showed no uptake in the metastatic tissues, indicating an alteration downstream to the NIS mRNA synthesis causing the loss of iodide uptake. As expected, when the NIS mRNA expression was absent, total-body (131)I scan showed no uptake in the metastatic lymph nodes. CONCLUSIONS: Our study demonstrates that NIS gene expression may be absent in metastatic differentiated thyroid carcinomas and that different mechanisms, other than loss of NIS transcription, may also be involved in the loss of iodide uptake in metastatic thyroid cells. Study of NIS gene expression in the metastatic lymph nodes, therefore, may provide useful information in the management of patients with thyroid carcinoma.

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S. Grasso, S. Filetti, D. Mazzone, V. Pezzino, R. Vigo and R. Vigneri

Abstract.

The function of the thyroid pituitary axis was investigated in 8 anencephalic infants with no hypothalamus.

Thyrotrophin (TSH), thyroxine (T4), 3,5,3′-triiodothyronine (T3) and 3,3,′5′-triiodothyronine (reverse T3, rT3) were measured in the cord blood in 5 cases and during the first 4 h of life in 3 cases, TSH response to synthetic thyrotrophin-releasing hormone (TRH) (200 μg iv) was carried out in two cases and thyroid hormone response to bovine TSH (5 IU iv) was evaluated in 3 cases.

The following results were obtained:

1) The pituitary gland was found in all infants and the thyroid was normal both grossly and by microscopic sections.

2) TSH levels at birth were normal but there was no spontaneous post-delivery surge.

3) T4 and T3 values at delivery were within normal range, but no T3 increase was present after birth. rT3 levels at birth were higher than normal in 3 cases.

4) Administration of TRH caused a marked and rapid TSH release.

5) Thyroid hormone response to TSH was normal.

The present findings suggest that in the anencephalic foetus both pituitary TSH-secreting cells and the thyroid gland do develop despite the absence of the hypothalamus and are able to function if adequately stimulated.

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F Trapasso, R Iuliano, E Chiefari, F Arturi, A Stella, S Filetti, A Fusco and D Russo

OBJECTIVE: Decrease or loss of the Na+/I- symporter (NIS) activity profoundly affects the suitability of the use of radioiodine to detect or treat metastatic thyroid tissues. The aim of our study was to verify whether specific oncogene abnormalities were responsible for the alteration in NIS activity in thyroid cells. DESIGN AND METHODS: Expression of the NIS gene was investigated by Northern blot analysis in normal and in some oncogene-transformed cell lines with different degrees of malignancy which had lost the iodide uptake ability. RESULTS: NIS gene expression was up-regulated by TSH in a dose-dependent and time-dependent way in normal PC Cl 3 cells. The same effect was observed by activating the cAMP-dependent pathway by forskolin. Conversely, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA) showed a partial inhibitory effect on NIS gene expression. The oncogene-transformed cell lines PC v-erbA, PC HaMSV, PC v-raf, and PC E1A cells showed reduced NIS mRNA levels compared with the normal PC Cl 3 cells. Conversely, an almost complete absence of NIS gene expression was found in PC RET/PTC, PC KiMSV, PC p53(143ala), and PC PyMLV cell lines. CONCLUSIONS: Our data show that oncogene activation could play a role in affecting the iodide uptake ability in thyroid tumoral cells; different mechanisms are involved in the oncogene-dependent loss of NIS activity in transformed thyroid cells.

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S Filetti, JM Bidart, F Arturi, B Caillou, D Russo and M Schlumberger

The recent cloning of the gene encoding the sodium/iodide symporter (NIS) has enabled better characterization of the molecular mechanisms underlying iodide transport, thus opening the way to clarifying its role in thyroid diseases. Several studies, at both the mRNA and the protein expression levels, have demonstrated that TSH, the primary regulator of iodide uptake, upregulates NIS gene expression and NIS protein abundance, both in vitro and in vivo. However, other factors, including iodide, retinoic acid, transforming growth factor-beta, interleukin-1alpha and tumour necrosis factor alpha, may participate in the regulation of NIS expression. Investigation of NIS mRNA expression in different thyroid tissues has revealed increased levels of expression in Graves' disease and toxic adenomas, whereas a reduction or loss of NIS transcript was detected in differentiated thyroid carcinomas, despite the expression of other specific thyroid markers. NIS mRNA was also detected in non-thyroid tissues able to concentrate radioiodine, including salivary glands, stomach, thymus and breast. The production of specific antibodies against the NIS has facilitated study of the expression of the symporter protein. Despite of the presence of high levels of human (h)NIS mRNA, normal thyroid glands exhibit a heterogeneous expression of NIS protein, limited to the basolateral membrane of the thyrocytes. By immunohistochemistry, staining of hNIS protein was stronger in Graves' and toxic adenomas and reduced in thyroid carcinomas. Measurement of iodide uptake by thyroid cancer cells is the cornerstone of the follow-up and treatment of patients with thyroid cancer. However, radioiodide uptake is found only in about 67% of patients with persistent or recurrent disease. Several studies have demonstrated a decrease in or a loss of NIS expression in primary human thyroid carcinomas, and immunohistochemical studies have confirmed this considerably decreased expression of the NIS protein in thyroid cancer tissues, suggesting that the low expression of NIS may represent an early abnormality in the pathway of thyroid cell transformation, rather than being a consequence of cancer progression. The relationship between radioiodine uptake and NIS expression by thyroid cancer cells require further study. New strategies, based on manipulation of NIS expression, to obtain NIS gene reactivation or for use as NIS gene therapy in the treatment of radiosensitive cancer, are also being investigated.

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F Arturi, D Russo, JM Bidart, D Scarpelli, M Schlumberger and S Filetti

OBJECTIVE: In the present study we analyzed the pattern of pendrin (PDS) and sodium/iodide symporter (NIS) gene expression in some thyroid carcinoma cell lines and a series of thyroid tumoral tissues. METHODS: Total RNA was extracted from all cell lines and from 53 tissues, and gene expression was examined by RT-PCR. Semiquantitative 'multiplex' RT-PCR was used to assess variations in PDS gene expression among various thyroid pathologies. Pendrin expression was determined in the thyroid cell lines by Western blot analysis. RESULTS: PDS mRNA was expressed in all the cells investigated; conversely, NIS mRNA was detectable only in the B-CPAP cells. Pendrin protein was expressed in B-CPAP and WRO cell lines, reduced in FRO and absent in ARO cells. PDS gene expression was not detected in 5 of 25 differentiated thyroid carcinomas (DTC) while NIS gene was not expressed in six carcinomas. A concordance expression of both PDS and NIS transcripts was found in 20 DTC. In contrast, 2 neoplastic thyroid tissues carrying undetectable PDS mRNA maintained NIS transcript, and 3 thyroid carcinomas negative for NIS mRNA retained the expression of PDS gene. A semiquantitative analysis showed that the mean PDS mRNA levels were significantly decreased in DTC tissues. CONCLUSIONS: Our data demonstrate that pendrin expression: (i) is present in the more differentiated thyroid carcinoma cell lines studied; (ii) is reduced or absent in DTC tissues; (iii) may not correlate with the NIS expression. These alterations may contribute to the loss of iodine concentration ability detected in thyroid tumors.

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S. Filetti, B. Rapoport, D. C. Aron, F. C. Greenspan, C. B. Wilson and W. Fraser

Abstract.

Studies were performed on pituitary adenoma tissue obtained from a patient with hyperthyroidism secondary to inappropriate thyrotrophin (TSH) secretion. Cells dispersed enzymatically were established in primary monolayer culture for a period of 39 days. Intact TSH and TSH-subunit release into the culture medium declined over the initial 20 days, followed by a rebound in TSH and β-TSH production until the time of subculture at day 39. In vitro α-TSH production declined more slowly than did TSH and β-TSH, but did not recover in parallel with intact TSH. Production of α-TSH by cultured cells, at different times, was 20–80-fold greater than that of β-TSH. In the pituitary tissue α-TSH content (112 ng/mg tissue) was 8.6-fold greater than the β-TSH concentration (13 ng/mg tissue), but similar to intact TSH content (105 ng/mg tissue). In serum, the α-TSH concentration (6.5 ng/ml) was lower than in most previously reported patients with thyrotrophic adenomata. TSH bioactivity in tumour tissue (approximately 600 ng/mg tissue) was greater than the immunoreactive TSH concentration. LH, FSH and prolactin were undetectable in tumour tissue, and in the culture medium throughout the period of cell culture. Exposure of thyrotroph monolayers to 10−6 m TRH for 4 h led to 32%-– 600% increases in TRH release, depending on the day of culture and the cell flask utilized. The α-TSH response to TRH paralleled that of intact TSH, β-TSH being unmeasureable. Incubation of cell monolayers for 4 h in 5 × 10−7 m dopamine decreased TSH and α-TSH secretion into the medium. In contrast, exposure of cells to T3 at concentrations as high as 10−6 m did not affect TSH secretion. Surprisingly, TRH (10−7 m to 2 × 10−6 m) inhibited cultured thyrotroph cAMP content by about 50%; cGMP levels were unaffected.

This study, the first to examine secretory function of human thyrotrophic tumour cells in vitro, confirms the disproportionate production of α-TSH by these tumours, even though this may not be apparent from the level of the α-subunit in serum. The data indicate that TSH secretion by thyrotroph tumour cells, at least from this particular patient, remains sensitive to TRH stimulation and dopamine inhibition, but insensitive to inhibition by T3. Not all TSH bioactivity is reflected by the immunoassayable TSH concentration, suggesting that the tumour may be producing variants of normal TSH that nevertheless retain biological activity. Finally, the data suggest that, at least in this particular tumour, TRH stimulation of TSH secretion is not mediated by cAMP as a second messenger.

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L Lacroix, C Mian, B Caillou, M Talbot, S Filetti, M Schlumberger and JM Bidart

OBJECTIVE: The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied in a series of various extra-thyroidal human tissues, and especially in those known to concentrate iodide. METHODS: To this end, we used real-time kinetic quantitative PCR to detect NIS and PDS transcripts and immunohistochemistry for the analysis of their protein products. RESULTS: NIS gene and protein expression was detected in most tissues known to concentrate iodine, and particularly in salivary glands and stomach. In contrast, PDS gene expression was restricted to a few tissues, such as kidney and Sertoli cells. Interestingly, in kidney, pendrin immunostaining was detected at the apical pole of epithelial cells of the thick ascending limb of the Henle's loop and of the distal convoluted tubule. CONCLUSION: This study provides new insights on the localization and expression of two genes involved in iodide transport and emphasizes the interest of combining real-time quantitative PCR and immunohistochemistry for the comparison of gene and protein expression in tissues.

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F Arturi, I Presta, D Scarpelli, JM Bidart, M Schlumberger, S Filetti and D Russo

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.

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D Russo, S Bulotta, R Bruno, F Arturi, P Giannasio, M Derwahl, JM Bidart, M Schlumberger and S Filetti

OBJECTIVE: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). METHODS: The levels of NIS and pendrin proteins were analyzed in total protein extracts from nodular and non-nodular tissues by Western blot. RESULTS: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. CONCLUSIONS: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.

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M Torlontano, U Crocetti, L D'Aloiso, N Bonfitto, A Di Giorgio, S Modoni, G Valle, V Frusciante, M Bisceglia, S Filetti, M Schlumberger and V Trischitta

OBJECTIVE: The 'standard' postoperative follow-up of patients with differentiated thyroid cancer (DTC) has been based upon serum thyroglobulin (Tg) measurement and (131)I whole body scan ((131)I-WBS) after thyroid hormone (T(4)) treatment withdrawal. However, (131)I-WBS sensitivity has been reported to be low. Thyroid hormone withdrawal, often associated with hypothyroidism-related side effects, may now be replaced by recombinant human thyroid stimulating hormone (rhTSH). The aim of our study was to evaluate the diagnostic accuracy of (131)I-WBS and serum Tg measurement obtained after rhTSH stimulation and of neck ultrasonography in the first follow-up of DTC patients. DESIGN: Ninety-nine consecutive patients previously treated with total thyroidectomy and (131)I ablation, with no uptake outside the thyroid bed on the post-ablative (131)I-WBS (low-risk patients) were enrolled. METHODS: Measurement of serum Tg and (131)I-WBS after rhTSH stimulation, and ultrasound examination (US) of the neck. RESULTS: rhTSH-stimulated Tg was <or=1 ng/ml in 78 patients (Tg-) and >1 ng/ml (Tg+) in 21 patients, including 6 patients with Tg levels >5 ng/ml. (131)I-WBS was negative for persistent or recurrent disease in all patients (i.e. sensitivity = 0%). US identified lymph-node metastases (confirmed at surgery) in 4/6 (67%) patients with stimulated Tg levels >5 ng/ml, in 2/15 (13%) with Tg>1<5 ng/ml, and in 2/78 (3%) who were Tg-negative. CONCLUSIONS: (i) diagnostic (131)I-WBS performed after rhTSH stimulation is useless in the first follow-up of DTC patients; (ii) US may identify lymph node metastases even in patients with low or undetectable serum Tg levels.