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L Lacroix, C Mian, T Barrier, M Talbot, B Caillou, M Schlumberger and JM Bidart

OBJECTIVE: Genetic alterations involving the thyroid transcription factor PAX8 and the peroxisome proliferator-activated receptor gamma 1 (PPARgamma1) genes have been described in thyroid neoplasms. We investigated in a series of thyroid samples, including 14 normal, 13 hyperfunctioning tissues, 26 follicular adenomas, 21 follicular and 41 papillary carcinomas, both the frequency of the PAX8-PPARgamma1 rearrangement and the expression of the PAX8 and PPARgamma transcripts. METHODS: Using RT-PCR followed by sequencing PCR products, PAX8-PPARgamma1 translocation was not detected in benign tissues nor in papillary carcinomas and was detected in 4 (19%) of 21 follicular carcinomas and in one (4%) of 26 follicular adenomas. RESULTS: Specific real-time quantitative RT-PCR (Q RT-PCR) methods detected high levels of PPARgamma transcripts in follicular carcinomas presenting the rearrangement. Interestingly, the level of PPARgamma transcripts was significantly decreased in papillary carcinomas in comparison with those found in benign adenomas and follicular carcinomas. Finally, PAX8 gene expression was decreased in both papillary and follicular thyroid carcinomas, and in these tumors to the same extent in the presence or absence of the rearrangement. These alterations in both PPARgamma and PAX8 gene expression may explain the poorly differentiated histotype of follicular carcinomas harboring the translocation.Immunohistochemistry showed that nuclear PPARgamma staining was weak in normal tissues, adenomas, papillary carcinomas and in some follicular carcinomas, and strong in the follicular carcinomas positive for the PAX8-PPARgamma1 translocation, but also in some follicular tumors in which no translocation could be evidenced. CONCLUSION: These observations confirm that the PAX8-PPARgamma1 translocation characterizes a subset of thyroid follicular carcinomas but is not a specific marker of carcinoma and that its frequency is lower than that initially reported. Finally, immunohistochemistry is not a reliable method for the specific detection of the translocation, that can be specifically evidenced by Q RT-PCR.

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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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L Vayre, JC Sabourin, B Caillou, M Ducreux, M Schlumberger and JM Bidart

131Iodine concentration has been described in several extra-thyroidal tissues. Recent evidence has shown that iodine uptake is achieved by the recently cloned human Na(+)/I(-) symporter (hNIS) gene. However, conflicting results were observed in the expression of hNIS transcripts in extra-thyroidal tissues. In order to document further the distribution of hNIS, we investigated its expression using an immunohistochemical method, based on a polyclonal antibody raised against a synthetic peptide. Various extra-thyroidal tissues were examined, particularly from the digestive tract. Our results confirm that the salivary glands and the stomach express hNIS protein significantly. In contrast, hNIS was undetectable in the colon but the rectal mucosa, which has never been examined, exhibited positive immunohistochemical staining. Other digestive tissues, including the oesophagus, small intestine and appendix, were negative. Weak staining was observed in the mammary gland, indicating that hNIS is expressed in this tissue. The pancreas, skin, ovaries, spleen and kidney showed no positive immunostaining.

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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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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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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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P Chanson, N De Roux, J Young, JM Bidart, P Jacquet, M Misrahi, E Milgrom and G Schaison

The monoclonal origin of gonadotropin-secreting pituitary adenomas has been well demonstrated but only few molecular abnormalities have so far been recognized in these tumors. For many years, several authors have suggested a role for GnRH and/or GnRH receptors (GnRH-R) in the development of these pituitary adenomas. To test the hypothesis that mutant genes encoding a constitutively activated GnRH-R might be involved in the pathogenesis of these tumors, the sequence of the GnRH-R gene was analyzed in tumoral pituitary tissue obtained from ten patients (six female, four male). The pituitary gonadotropin-secreting adenoma was associated with in vivo hypersecretion of FSH, LH and/or free alpha-subunit (n = 7) or was clinically silent (normal plasma levels of gonadotropins or free alpha-subunit, n = 3). In all cases, immunocytochemical studies of the removed adenoma confirmed their gonadotroph nature by revealing positivity for FSH, LH and/or alpha-subunit. Genomic DNA was extracted from the pathological tissue obtained at neurosurgery. Eight sequencing primers were used to amplify the three exons of the GnRH-R gene from tumoral DNA. The entire coding sequence of the GnRH-R gene was sequenced in the ten adenomas. No mutation was found in any of the tumor specimens examined. In conclusion, mutations in the GnRH receptor coding sequence occur infrequently if at all in gonadotropin-secreting pituitary adenomas.

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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.