Semi-quantitative comparison of the differentiation markers and sodium iodide symporter messenger ribonucleic acids in papillary thyroid carcinomas using RT-PCR

OBJECTIVE: To investigate the levels of expression of the sodium iodide symporter (NIS) and three differentiation markers (thyroglobulin (Tg), thyroid peroxidase (TPO) and thyrotrophin receptor (TSH-R)) in 35 patients with primary (n=31) or recurrent (n=4) papillary thyroid carcinoma, and to compare the findings with clinical data. METHODS: We performed a multiplex semi-quantitative RT-PCR to analyse the relative levels of expression of Tg, TPO and TSH-R mRNAs, and a separate semi-quantitative RT-PCR for NIS mRNA. RESULTS: Tg, TPO and TSH-R mRNAs were expressed in all the patients, whereas NIS mRNA was expressed in all but eight. Analysis of the expression of the differentiation markers in all patients showed a significant correlation among Tg, TPO and NIS. With regard to the relationship between the expression of each gene and the MACIS score, there was significant correlation only for the Tg gene (P<0.05). CONCLUSIONS: The levels of expression of NIS mRNA correlated significantly with those of Tg and TPO mRNAs, but not with those of TSH-R mRNA. The relationship with clinical stage and prognostic score, however, varied among these differentiation markers.     

Correlation between the loss of thyroglobulin iodination and the expression of thyroid-specific proteins involved in iodine metabolism in thyroid carcinomas

Progress in biotechnology has provided useful tools for tracing proteins involved in thyroid hormone synthesis in vivo. Mono- or polyclonal antibodies are now available to detect on histological sections the Na(+)/I(-) symporter (NIS) at the basolateral pole of the cell, the putative iodide channel (pendrin) at the apical plasma membrane, thyroperoxidase (TPO), and members of the NADPH-oxidase family, thyroid oxidase 1 and 2 (ThOXs), part of the H(2)O(2)-generating system. The aim of this study was to correlate thyroglobulin (Tg) iodination with the presence of these proteins. Tg, T(4)-containing Tg, NIS, pendrin, TPO, ThOXs, and TSH receptor (TSHr) were detected by immunohistochemistry on tissue sections of normal thyroids and various benign and malignant thyroid disorders. Tg was present in all cases. T(4)-containing Tg was found in the adenomas, except in Hurthle cell adenomas. It was never detected in carcinomas. NIS was reduced in all types of carcinomas, whereas it was detected in noncancerous tissues. Pendrin was not expressed in carcinomas, except in follicular carcinomas, where weak staining persisted. TPO expression was present in insular, follicular carcinomas and in follicular variants of papillary carcinomas, but in a reduced percentage of cells. It was below the level of detection in papillary carcinomas. The H(2)O(2)-generating system, ThOXs, was found in all carcinomas and was even increased in papillary carcinomas. Its staining was apical in normal thyroids, whereas it was cytoplasmic in carcinomas. The TSHr was expressed in all cases, but the intensity of the staining was decreased in insular carcinomas. In conclusion, our work shows that all types of carcinomas lose the capacity to synthesize T(4)-rich, iodinated Tg. In follicular carcinomas, this might be due to a defect in iodide transport at the basolateral pole of the cell. In papillary carcinomas, this defect seems to be coupled to an altered apical transport of iodide and probably TPO activity. The TSHr persists in virtually all cases.     

Functional characterization of human thyroid tissue with immunohistochemistry.

Immunohistochemistry provides insights in the expression of functional proteins and of their localization in normal thyroid tissue and in thyroid diseases. In hyperfunctional thyroid tissues, staining for sodium/iodide symporter (NIS), pendrin, thyroid peroxidase (TPO), and thyroglobulin (Tg) is increased. In hypofunctioning thyroid tissues, NIS staining is markedly decreased; in benign hypofunctioning adenomas, the expression of the other functional proteins is unmodified or slightly decreased, whereas their expression is profoundly decreased or absent in differentiated thyroid carcinoma.     

促甲状腺激素受体,甲状腺过氧化物酶及甲状腺球蛋白基因表达…

为探讨促甲状腺激素受体。甲状腺过氧化物酶及甲状腺球蛋白ｍＲＮＡ在自身免疫甲状腺疾病（ＡＩＴＤ）病人甲状腺组织的表达情况及其相互关系，应用Ｎｏｒｔｈｅｒｎ印记杂交技术观察了ＴＳＨ受体、ＴＰＯ以及ＴＧ ｍＲＮＡ在７例Ｇｒａｖｅｓ病（ＧＤ），２例桥本甲状腺素炎（ＨＴ）病人甲状腺组织中的表达。研究显示：ＴＳＨ受体ｍＲＮＡ与ＴＰＯ和ＴＧ ｍＲＮＡ在ＧＤ组和ＨＴ组表达不一致，ＧＤ组ＴＳＨ受体ｍＲＮＡ与ＴＰＯ和     

Type I interferons modulate the expression of thyroid peroxidase, sodium/iodide symporter, and thyroglobulin genes in primary human thyrocyte cultures

We evaluated in primary human thyrocyte cultures the effect of interferon (IFN)-alpha and -beta on the expression of thyroid peroxidase (TPO), sodium/iodide symporter (NIS), and thyroglobulin (Tg) as well as T(4) release. Human thyrocyte cultures were carried out with fresh normal thyroid tissue. Gene and protein expression of Tg, TPO, and NIS were assessed by RT-PCR and Western blot analysis after 24, 48, and 72 h of treatment with TSH alone (10 mIU/ml) and in combination with IFN alpha or -beta (10(4) U/ml). IFN inhibited the TSH-stimulated gene expression of Tg, TPO, and NIS in a time-dependent manner without significant differences between IFN alpha and -beta. Moreover, the addition of both type I IFNs clearly reduced the TSH-stimulated protein expression of Tg, TPO, and NIS after 72 h of exposure. Finally, this down-regulation was associated with a reduction of T(4) release by almost 50%. In conclusion, our study shows that both IFN alpha and -beta down-regulate the TSH-stimulated expression of Tg, TPO, and NIS as well as T(4) release. Indeed, the development of hypothyroidism during type I IFN therapy may be related, at least in part, to an abnormal expression and function of key proteins involved in iodine uptake and organification.     

Homologies of the thyroid sodium-iodide symporter with bacterial and viral proteins.

We have demonstrated that Na+/I- symporter (NIS), a novel thyroid autoantigen, has local amino acid sequence homologies with the other thyroid autoantigens: Thyroglobulin (Tg), thyroid peroxidase (TPO) and thyrotropin receptor (TSH-R). These homologies concern the 4th, 5th, 6th extracellular loop and the beginning of the intracellular tail. We have expanded our studies and found that there are significant local homologies with other 11 proteins, most of them of bacterial or viral origin (e.g., Streptococcus or Herpes). These homologies concern the 2nd and 4th extracellular loop, and both the beginning and the end of the intracellular tail. These 11 homologies were retrieved by a computer-assisted search and extracted out of a database containing almost 300,000 amino acid sequences. These homologies were of magnitude greater than those concerning the three thyroid autoantigens [identities=51.1+/-7.3% vs 25.3+/-7.8% (mean+/-SD), p<0.001; similarities=70.6+/-10.7% vs 43.3+/-8.5%; p<0.001]. In addition, extensive, not local, homology was found with a number of unknown proteins from invertebrates (Drosophila melanogaster and Caenorhabditis elegans) and bacteria such as Bacillus subtilis and Xanthobacter. Previously, we had found that NIS has no extensive homology with Tg or TPO or TSH-R. This is the first demonstration of both extensive and local homologies between one thyroid autoantigen (NIS) and microbiological proteins. Taken together with data of the literature on the homologies between other thyroid antigens (Tg, TPO, TSH-R) and bacteria, the homologies we have now found reinforce the view that both bacterial and viral infections may trigger autoimmune thyroid diseases.     

Expression of pendrin and the Pendred syndrome (PDS) gene in human thyroid tissues

The gene recently cloned that is responsible for the Pendred syndrome (PDS), an autosomal recessive disease characterized by goiter and congenital sensorineural deafness, is mainly expressed in the thyroid gland. Its product, designated pendrin, was shown to transport chloride and iodide. To investigate whether the PDS gene is altered during thyroid tumorigenesis, PDS gene expression and pendrin expression were studied using real-time kinetic quantitative PCR and antipeptide antibodies, respectively, in normal, benign, and malignant human thyroid tissues. The results were then compared to those observed for sodium/iodide symporter (NIS) expression. In normal tissue, pendrin is localized at the apical pole of thyrocytes, and this in contrast to the basolateral location of NIS. Immunostaining for pendrin was heterogeneous both inside and among follicles. In hyperfunctioning adenomas, the PDS messenger ribonucleic acid level was in the normal range, although immunohistochemical analysis showed strong staining in the majority of follicular cells. In hypofunctioning adenomas, mean PDS gene expression was similar to that detected in normal thyroid tissues, but pendrin immunostaining was highly variable. In thyroid carcinomas, PDS gene expression was dramatically decreased, and pendrin immunostaining was low and was positive only in rare tumor cells. This expression profile was similar to that observed for the NIS gene and its protein product. In conclusion, our study demonstrates that pendrin is located at the apical membrane of thyrocytes and that PDS gene expression is decreased in thyroid carcinomas.     

Immunohistochemical profile of the sodium/iodide symporter in thyroid,breast, and other carcinomas using high density tissue microarrays and conventional sections

Extrathyroidal cancers could potentially be targeted with (131)I, if the Na(+)/I(-) symporter (NIS) were functional. Using immunohistochemical methods we probed 1278 human samples with anti-NIS antibody, including 253 thyroid and 169 breast conventional whole tissue sections (CWTS). Four high density tissue microarrays containing a wide variety of breast lesions, normal tissues, and carcinoma cores were tested. The results of the normal microarray were corroborated in 50 CWTS. Nineteen of 34 normal tissues, including bladder, colon, endometrium, kidney, prostate, and pancreas, expressed NIS. Nineteen of 25 carcinomas demonstrated NIS immunopositivity; 55.7% of 479 carcinoma microarray cores expressed NIS, including prostate (74%), ovary (73%), lung (65%), colon (62.6%), and endometrium (56%). NIS protein was present in 75% benign thyroid lesions, 73% thyroid cancers, 30% normal-appearing, peritumoral breasts, 88% ductal carcinomas in situ, and 76% invasive breast carcinoma CWTS. Comparatively, breast microarray cores had lower immunoreactivity. Plasma membrane immunopositivity was confirmed in thyrocytes, salivary ductal, gastric mucosa, and lactating mammary cells. In other tissues, immunoreactivity was predominantly intracellular, particularly in malignant lesions. Thus, NIS is present in many normal epithelial tissues and is predominantly expressed intracellularly in many carcinomas. Elucidating the regulatory mechanisms that render NIS functional in extrathyroidal carcinomas may make (131)I therapy feasible.     

Relationship of sodium/iodide symporter expression with I131 whole body scan uptake between primary and metastatic lymph node papillary thyroid carcinomas

The aim of the present study was to evaluate total and membranous Na+/I- symporter (NIS) expressions in papillary thyroid carcinoma (PTC) tissue, correlation of NIS expression between primary and metastatic lymph node (LN) PTC tissues, and relationship of NIS expression with I131 whole body scan (WBS) uptake between primary and metastatic LN PTC tissues by analyzing 17 pairs of primary and metastatic LN PTC tissues. Staining positivity was calculated, and staining intensity was graded as negative (0), weak (1+), moderate (2+) and strong (3+). In primary PTC tissues, positivities and intensities of normal cells were higher than those of carcinoma cells but had no correlation with those in matched metastatic LN PTC tissues. In classic type, positivities, intensities and membranous intensities (mIS) were correlated between primary and matched metastatic LN PTC tissues. In patients aged younger than 45 yr, positivities and intensities in primary PTC tissues had correlation with those in matched metastatic LN PTC tissues. Positivities, intensities, mIS and pathological subtype of carcinoma cells in primary PTC tissues were not correlated with age, tumor size, TNM stage, MACIS score and thyroglobulin (Tg) levels at the time of I131 WBS. Sensitivity, specificity, as well as positive and negative predicted values of mIS in patients with I131 WBS uptake were 69.2, 75, 90 and 42.9% in primary PTC tissues, and 92.3, 100, 100 and 80% in metastatic LN PTC tissues. The results of mIS taken either as positive or negative were correlated with those of I131 WBS after controlling for age. Our results demonstrate that PTC tissues have altered total and membranous NIS expressions, suggesting that NIS expression in primary PTC tissues may predict NIS expression and I131 WBS uptake in matched metastatic LN PTC tissues.