Presence of TSH receptor in thyroid neoplasms.

The nature of the TSH receptor in adenoma and carcinoma of the thyroid gland was studied using a radioreceptor assay technique. A membrane fraction of tissue homogenate was obtained by discontinuous sucrose gradient ultracentrifugation, and 125I-TSH, labelled by a lactoperoxidase method, was purified with a receptor adsorption method. Both the capacities and the association constants of high affinity receptors (4 x 10(9) M-1) and of low affinity receptors (0.073 x 10(9) M-1) observed in the normal thyroid were almost identical to those of the thyroid of Graves' disease and those of thyroid adenoma. Although the two papillary carcinomas examined were found to have two kinds of TSH receptors, one of the carcinomas showed decreased association constants for both high affinity and low affinity receptors.     

Expression of the thyroid hormone receptor, the oncogenes c-myc and H-ras, and the 90 kD heat shock protein in normal, hyperplastic, and neoplastic human thyroid tissue.

Expression of the thyroid hormone receptor (TR beta), the 90 KD heat shock protein (HSP 90), and the oncogenes H-ras and c-myc mRNA in normal hyperplastic, and neoplastic human thyroid tissue was investigated by Northern blot and slot blot analyses. The TR beta mRNA was present in all normal and neoplastic thyroid tissue samples. The levels were significantly higher in normal and hyperplastic tissues (7.91 +/- 0.48 and 7.60 +/- 0.68 arbitrary units, respectively) than in neoplastic tissues (3.82 +/- 0.67) (p < 0.001). H-ras and c-myc mRNA were also detected in glandular tissue specimens, but no significant difference was observed in their expression levels. Furthermore, there was a tendency to a negative correlation between the level of TR beta and c-myc mRNA (p = 0.06). In normal thyroid tissue, HSP 90 mRNA levels were significantly higher than in hyperplastic and papillary carcinoma tissue specimens (p < 0.001). These results indicate that mRNA of TR beta and HSP 90 (the latter only in papillary thyroid carcinoma) are expressed in relation to the degree of cellular differentiation. Furthermore, the presence of TR beta in normal thyroid tissue implies that T3 and T4 may be involved in the regulation of their own production via TR beta activated feedback mechanism(s).     

Positive regulation of the guinea pig thyrotropin receptor

Subcutaneous administration of bovine (b) TSH (up to 10 IU) to 8-week-old male guinea pigs was followed by a transient elevation in serum thyroid hormone levels (T4 and T3) and an increase in thyroid weight (approximately 25-40%), which returned to control levels by 3 days. Total thyroid TSH receptor content was assessed by the binding of receptor-purified [125I]bTSH to 15,000 X g fractions of thyroid homogenate. The TSH receptor content paralleled the increase in thyroid weight, with no detectable change in the TSH-binding capacity per mg tissue. Intraperitoneal minipump infusions of bTSH (1 IU/day) over 6 days produced marked and persistent increases in thyroid hormone levels and thyroid weight (greater than 300%) and a similar increase in TSH receptor content. There was no change in the single site equilibrium association constant for bTSH [1.1 X 10(9) M-1 +/- (SE) 9.6 X 10(7) M-1] and no alteration in the binding capacity per mg tissue (67.2 +/- 6.4 pg/mg). Investigation of the in vitro adenylate cyclase response to bTSH (10 mU/ml) and the production of immunoassayable cAMP showed no difference between thyroid tissue obtained from bTSH-treated animals and that obtained from untreated control animals. These observations demonstrated that TSH exerted a positive regulatory effect on its receptor and, under the in vivo conditions used, failed to induce TSH receptor loss or physiologically important desensitization. Such data may explain how TSH receptor antibodies are able to act as TSH agonists and maintain increased thyroid hormone output in human disease.     

Mechanisms of increased sensitivity for detection of thyroid stimulating antibodies by use of hypotonic medium

We have attempted to clarify the mechanism whereby the sensitivity to detect thyroid stimulating antibodies by use of cultured thyroid cells is enhanced when sodium chloride is removed from isotonic medium. Preexposure of cultured porcine thyroid cells to 10(-4) mol/l cycloheximide increased the subsequent cAMP response to TSab stimulation in isotonic NaCl medium, but not in hypotonic NaCl-free medium. Crude membrane fractions from porcine thyroid tissues were incubated with 6 Graves' IgGs in NaCl or NaCl-free medium at 37 degrees C for 1 h, centrifuged, washed, added [125I]TSH, and incubated for another hour. The result of the 'residual TBII assay' indicated that all 6 IgGs exhibited greater inhibition of [125I]TSH binding to the membranes when pre-incubated under hypotonic than isotonic conditions. When cultured porcine thyroid cells were incubated with 7 TSab samples in NaCl or NaCl-free medium depleted with 3-isobutyl-1-methylxanthine at 37 degrees C for 1 h, washed, added the NaCl-free medium containing 3-isobutyl-1-methylxanthine, and incubated for another 2 h, the use of NaCl-free medium during the pre-incubation resulted in greater increase in cAMP elicited by all 7 samples. In summary, 1) an inhibitory mechanism upon adenylate cyclase seems to be involved under isotonic conditions and may require new protein synthesis; 2) the binding of TSH receptor antibodies to the TSH receptor increases when NaCl is removed from isotonic medium, and 3) these factors are considered responsible for the enhanced sensitivity under hypotonic conditions.     

Antibodies to membrane antigens in autoimmune thyroid disease

The possibility that sera from patients with autoimmune thyroid diseases contain autoantibodies to thyroid membrane proteins distinct from microsomal antigen and the TSH receptor has been investigated using (a) solid phase assay system based on human thyroid membranes and 125I-labelled protein A and (b) immunoprecipitation of detergent solubilized 125I-labelled thyroid membranes followed by gel electrophoresis and autoradiography. In the solid phase assay binding to membranes showed a highly significant correlation with binding to microsomes (r = 0.82; P less than 0.001; N = 82) indicating that the interaction between the serum and the membranes was due principally to microsomal antibody binding to microsomal antigen contaminating the membrane preparations. However, there were some discrepancies suggesting that an additional antigen-antibody system was involved. This possibility was then investigated using immunoprecipitation of 125I-labelled thyroid membranes. A labelled protein with mol wt 54 K was specifically immunoprecipitated (relative to normal pool serum) by 3 out of 4 sera from patients with Graves' disease who showed high binding to thyroid membranes. A further 4 sera from such patients with low membrane binding affinity failed to immunoprecipitate the 54 K protein. Sera from some patients with Hashimoto's disease and some patients with rheumatoid arthritis and one patient with Addison's disease also immunoprecipitated the 54 K protein from solubilized thyroid membranes. These studies suggested that antibodies interacting with the 54 K protein contributed to the discrepancies between thyroid membrane and microsome binding. However, the 54 K protein was also immunoprecipitated from detergent solubilized membranes prepared from human placenta, skeletal muscle and adrenal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotropin receptor-adenylate cyclase system in plasma membranes from normal and diseased human thyroid glands.

Thyrotropin binding characteristics and adenylate cyclase (AC) activity of thyroid plasma membranes were studied in 52 tissues from normal and diseased human thyroids. Data from normal glands, Graves' goiters, non toxic multinodular goiters and nodular and perinodular tissue of toxic nodular goiters show the same basal, TSH- and NaF- stimulated adenylate cyclase activities (no. = 45; 34.1 +/- 3.2 (m +/- SE), 378 +/- 43, 298 +/- 48 pmol cAMP x min-1 x mg membrane protein-1), the same stimulability of AC by TSH (11.3 +/- 1.4--fold over basal level) and by NaF (8.1 +/- 1.8-fold), the same apparent TSH binding equilibrium constants (5.6 +/- 0.7 and 406 +/- 57 nM) and the same TSH binding site concentrations (2.2 +/- 0.4, 27.8 +/- 5.9 pmol x mg membrane protein-1). Alterations of the TSH receptor and of the AC were detected in membranes from tumoral and metastatic lymph node tissues from thyroid papillary carcinoma and in the thyroid tissue from post-radioiodide therapy thyroiditis. These observations suggest that: (i) hyperthyroidism in Graves' disease or toxic nodular goiter does not result in and is not a consequence of an alteration in the TSH receptor-adenylate cyclase system; (ii) there is no evidence supporting a relationship between the studied membrane properties and clinical or histological status; (iii) membrane abnormalities detected in thyroid carcinoma vary widely; (iv) studies of these membrane alterations might be of interest in the therapeutic management of thyroid carcinoma and may lead to a better understanding of the receptor-adenylate system.     

Increased thyroid epithelial cell proliferation in toxic thyroid nodules.

Most toxic thyroid nodules (TTN) result from clonal expansion of a single cell caused by a somatic mutation in the thyrotropin (TSH) receptor, the Gsalpha protein, or yet unknown proteins. Expanding a single cell into a TTN with thousands of cells suggests a prolonged increase in proliferation compared to nonaffected surrounding cells. To test this hypothesis, we evaluated cell proliferation in TTN. Tissue from 20 TTN and their surrounding normal thyroid tissue was studied for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 epitope as markers for cell proliferation. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. Nineteen samples were evaluated for PCNA immunohistochemistry. In 16 TTN, a significant (p< or =0.05%) up to 3-fold increase in the labeling index for PCNA was detectable. In only 3 toxic nodules (2 without a detectable TSH receptor or Gsalpha protein mutation), we found no significant difference in the labeling index compared to the surrounding tissue. Because labeling for KI-67 was much lower, only 16 toxic thyroid nodules were quantified. Twelve of these showed significantly (p< or =0.05%) increased labeling indices. The increase of the labeling index for both markers was similar for histologically defined adenoma versus adenomatous nodule or nodules with or without TSH receptor mutation or clonal versus polyclonal origin of toxic nodules studied. These findings are evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most TTNs, independent of their histopathological or molecular characteristics. Although increased proliferation in many TTNs is very likely the result of TSH receptor mutations, the cause of increased proliferation in TTN without a mutation is unknown.     

Characterization of tumor necrosis factor-alpha receptors in human and rat thyroid cells and regulation of the receptors by thyrotropin

Administration of recombinant human tumor necrosis factor-alpha (TNF) to rats and mice produces a model of nonthyroid illness in which there is impairment of hypothalamic-pituitary thyroid function, including reduced serum concentrations of T4 and T3, reduced thyroid radioiodine uptake, and reduced response to TSH. In this study, we tested the binding and effects of TNF on FRTL-5 cells and on four human thyroid carcinoma cell lines. The TSH-stimulated [125I]iodide uptake by FRTL-5 cells was inhibited by TNF in a dose-dependent manner. The four human thyroid carcinoma cell lines (NPA, MRO, ARO, WRO) have TSH receptors but did not respond to TSH in regard to iodide uptake and thymidine incorporation. Both human thyroid carcinoma cells and FRTL-5 cells contain specific receptors for TNF. Scatchard analysis showed that the receptor numbers and dissociation constants in human thyroid carcinoma cells and FRTL-5 cells were, respectively; 2.4 x 10(4), 5.4 nM (WRO); 8 x 10(3), 3.4 nM (MRO); 4 x 10(3), 1 nM (ARO); 7 x 10(3), 1 nM (NPA); 3 x 10(3), 1 nM (FRTL-5), and 9 x 10(3), 1 nM (FRTL-5 cells treated with TSH). The results indicate that TNF affects thyroid cell function through binding to the TNF receptor and that the number of TNF receptors is regulated by TSH.     

Telomerase activity in thyroid malignancy.

Telomerase activity seems to play a role in the development and pathogenesis of thyroid carcinoma. Its incidence of expression and its application as a tumor marker remain to be elucidated. Thyroid tissues obtained during thyroidectomy from 1996-1998 were rapidly frozen and stored at -80 degrees C until processed. Telomerase activity was determined using telomeric repeat amplification protocol (TRAP). Histology of the tissue examined (67 benign and 59 malignant) was reviewed. Telomerase activity was detected in 15 of 52 papillary carcinomas (29%); 1 of 1 thyroid lymphoma (100%); 1 of 2 anaplastic carcinomas (50%); and 2 of 16 lymphocytic thyroiditis specimens (13%). Telomerase activity was not detectable in 35 normal thyroid, 9 follicular adenoma, 7 nodular hyperplasia, 2 follicular carcinoma, and 2 medullary carcinoma. Lymphocytic thyroiditis was detected in 8 of 37 (22%) apparently normal thyroid tissues adjacent to papillary thyroid carcinoma and telomerase activity was present in 2 of these 8 specimens (25%). In conclusion, telomerase does not appear to be frequently activated in papillary thyroid carcinoma. The association of lymphocytic thyroiditis with papillary thyroid carcinoma may limit its clinical usefulness as a tumor marker.     

Lack of desensitization of 3',5'-cyclic AMP response to thyrotropin stimulation in differentiated thyroid carcinoma

The course of the cAMP response to thyrotropin (TSH) in 23 specimens of differentiated thyroid carcinoma and the adjacent normal tissue was studied. In the carcinomatous tissue, the cAMP concentration in slices incubated with TSH was significantly greater after 2 h than after 15 min of incubation; the level was almost the same at both times in normal tissue. The effects of an initial exposure of thyroid slices to TSH (50 U/l) on the subsequent cAMP responsiveness to the hormone were investigated in seven more differentiated thyroid carcinomas. In normal tissue, the cAMP level in slices exposed to TSH, washed, and exposed again was lower after the third incubation than in slices exposed to TSH only in the third. In contrast, the cAMP level tended to be greater after the second of 2 TSH incubations in 5 out of the 7 specimens of carcinomatous tissue. The data suggest that differentiated thyroid carcinoma lacks desensitization of the cAMP response to TSH.     

Thyrotropin receptors in normal and pathological human thyroid tissues.

The properties of TSH receptors in normal and pathological human thyroid tissues were studied. Highly purified bovine TSH after lactoperoxidase iodination retained full biological activity, as assessed by radioreceptor assay. Binding of bovine [125I]TSH to 1000 x g pellets of human thyroid homogenate was specific with respect to hormone and tissue. Total binding amounted to 50-60% of total radioactivity using 10 mg (wet weight) normal thyroid tissue. Nonspecific binding was only 6% of total radioactivity. Normal thyroid tissue contained two orders of binding sites, which were shown to be independent of each other by Hill plot analysis. The high affinity sites [equilibrium dissociation constant (Kd), 0.015-0.16 x 10-9 M] were present in concentrations of 1.05-9.30 pmol/mg protein and concentrations of low affinity sites (Kd, 1.2-2.4 x 10-9 M) were 35.9-213 pmol/mg. In all pathological thyroid tissue studied, two orders of binding sites were found with dissociation constants similar to those of normal tissues, but the number of binding sites was markedly reduced. Both orders of binding sites in solitary "cold" adenomas and only the low affinity sites in thyroid tissue from patients with Graves' disease were significantly reduced in number (P less than 0.01). There was a questionable decrease in high affinity sites in the Graves' tissue (P = 0.05). We have found the definite presence but a decreased number of binding sites in both orders of receptors in papillary and follicular carcinomas. There were few or no binding sites in medullary carcinoma.     

Studies of the TSH radioreceptor assay.

We have examined several variables in the reagents and procedures used in the TSH radioreceptor assay, the binding of iodinated TSH to its thyroidal receptor. We found that iodinated bovine TSH (S.A. 30 U/mg) was more effectively bound to receptor than iodinated human TSH (S.A. 7.3 U/mg). Iodination of TSH was the Bolton-Hunter acylation method apparently prevented binding to TSH receptor. Surgically removed human thyroid tissue specifically bound 10.3 +/- 1.0 (mean +/- SEM) of added [125I]TSH, but post-mortem human thyroid bound only 3.9 +/- 0.4% of [125I]TSH (p less than 0.001). Maximal binding of [125I]TSH was found at pH 5.8. Many tissue preparations contained activity, possibly due to proteases, which inactivated TSH, and inclusion of a protease inhibitor, aprotinin, significantly increased specific binding.     

甲状腺滤泡型肿瘤分子标记物的研究进展

良、恶性甲状腺结节的鉴别主要是根据B超、放射性核素扫描以及甲状腺细针抽吸活检(FNAB),FNAB对甲状腺乳头状癌、髓样癌的诊断准确率较高,但对那些包括增生性结节、滤泡型腺瘤、滤泡癌以及乳头状癌滤泡性变等在内的所谓“滤泡型新生物”或“可疑性滤泡型病变”的诊断准确率较低.近年来研究发现,许多蛋白质分子如DNA损伤诱导转录...     

Localization and expression of thyroid hormone receptors normal and neoplastic human thyroid

The aim of this study was to investigate the regional expression of thyroid hormone nuclear receptor forms (TR(alpha) and TR(beta)) and isoform (TR(alpha1) and TR(beta2)) mRNAs in normal and neoplastic (benignant and malignant) human thyroid tissue. Tumor specimens from patients with thyroid carcinomas (papillary: 5 cases; follicular: 5 cases; anaplastic: 2 cases), thyroid follicular adenomas (7 cases) and tissue from normal thyroid glands (12 cases) were analyzed by in situ hybridization and semiquantitative RT-PCR for the expression of TR(alpha1) and beta, as well as for the isoform alpha2 that does not bind the hormone. In normal tissues, TR(alpha2) was expressed at lower levels compared to TR(alpha1) (alpha1/alpha2 = 4.3). In papillary and follicular carcinomas, the expression of TR(alpha1) and TR(beta) did not change as compared with normal thyroid tissue and adenomas (0.87 +/- 0.15 SD vs 0.89 +/- 0.17 densitometric units, DU, and 0.15 +/- 0.02 vs 0.14 +/- 0.03 DU, respectively). However, the expression of TR(alpha2) was significantly higher in differentiated carcinomas compared to normal thyroid tissue and adenomas (0.47 +/- 0.05 vs 0.20 +/- 0.05 DU, p < 0.05) with alpha1/alpha2 = 1.4. In anaplastic carcinoma all TRs were absent. We concluded that both normal and pathological thyroid tissues, with the exception of anaplastic carcinoma, express all TRs in thyreocites and that differentiated thyroid carcinomas are associated in enhancing the expression of TR(alpha2) mRNA.     

Coexistence of Thyrotropin-Producing Pituitary Adenoma with Papillary Adenocarcinoma of the Thyroid—A Case Report and Surgical Strategy

We report a very rare case of thyrotropin (thyroxin stimulating hormone, TSH)-producing pituitary adenoma coexisting with a papillary adenocarcinoma of the thyroid. A 45-year-old woman presented with hyperhidrosis and a nodule in the left thyroid that was first noticed one year earlier. An endocrinological examination showed elevated serum levels of free triiodothyronine (T3) and free throxin (T4) without inhibition of TSH, suggesting the presence of syndromes of inappropriate secretion of TSH. A specimen obtained by needle aspiration of the thyroid nodule revealed the presence of papillary adenocarcinoma. Magnetic resonance images demonstrated a pituitary macroadenoma.The patient was diagnosed as having a TSH-producing pituitary adenoma coexisting with a papillary adenocarcinoma of the thyroid. The patient underwent a total thyroidectomy with resection of the neighboring lymph nodes. Two weeks after this surgery, the pituitary adenoma was totally removed via a pterional approach. Histological and immunohistochemical examinations of the surgical specimens confirmed the lesion as a papillary adenocarcinoma of the thyroid and a TSH-producing pituitary adenoma. Serum TSH levels decreased to undetectable levels immediately after the surgery for the pituitary adenoma.Prolonged stimulation of the thyroid gland by TSH may be involved in the growth of thyroid carcinoma. In cases with a TSH-producing pituitary adenoma, the possible coexistence of thyroid carcinoma should be carefully ruled out. In such cases, a total thyroidectomy followed by TSH level normalization should be performed. Incomplete removal of the thyroid might enable the carcinoma to re-grow if TSH level can not be normalized after the pituitary adenomectomy.     

Clonal origin of non-medullary thyroid tumours assessed by non-random X-chromosome inactivation.

OBJECTIVE: X-chromosome inactivation analysis was performed in order to assess the clonal origin of non-medullary thyroid tumours and to distinguish between multicentricity and multifocality in multiple papillary thyroid carcinoma (PTC). METHODS: One hundred and thirteen tumour samples from 31 patients with isolated PTC, 16 patients with multinodular PTC, 14 patients with follicular thyroid adenoma (FTA) and 15 patients with follicular thyroid carcinoma (FTC) were collected. The corresponding normal thyroid tissues were analysed, and in 14 cases, tumour-surrounding tissue was also studied. Genomic DNA was digested with HpaII and HhaI previous to PCR amplification of the polymorphic CAG repeat, on exon 1 of the human androgen receptor gene (HUMARA). PCR products were analysed by denaturing gel electrophoresis, silver staining and densitometric analysis. PCR products were also used to determine the number of CAG repeats of patients with isolated PTC, FTA, FTC and of 41 healthy volunteers. RESULTS: Heterozygosity for the HUMARA polymorphism was found in 64/76 (84%) cases. Lyonization of the thyroid was observed in 15/76 (20%) cases, which were excluded from clonal analysis. Except for two cases of isolated PTC, all tumour samples studied presented monoclonal X-inactivation patterns, while normal thyroid tissue was polyclonal. Monoclonal patterns were also found in 4/14 tumour-surrounding tissues. No difference was found in the length of CAG alleles between patients and controls. Of eight informative cases of multinodular PTC, three showed evidence of multicentricity and five revealed patterns consistent with multifocality. CONCLUSIONS: Both isolated and multinodular PTC as well as FTA and FTC are of monoclonal origin. Our results also suggest that approximately one-third of multiple PTC have an independent origin for the different nodules (multicentricity). Monoclonality was also found in tissues surrounding some PTC nodules. No association was found between the length of CAG alleles and thyroid malignancies.     

Determination of galectin-3 messenger ribonucleic Acid overexpression in papillary thyroid cancer by quantitative reverse transcription-polymerase chain reaction

Galectin-3, a lectin-family protein that appears to be involved in malignant transformation, has been reported to be an accurate immunohistochemical marker for thyroid cancer. However, immunohistochemistry is a subjective method that can be difficult to apply to cytologic specimens. Therefore, we sought to develop an objective and quantitative assay to measure galectin-3 mRNA in thyroid tissue to enhance potential clinical use of galectin-3 in the molecular analysis of thyroid nodules. In this study, total RNA from 37 snap-frozen thyroid tissue specimens was isolated from eight papillary and nine follicular thyroid cancers, six follicular adenomas, seven adenomatoid nodules, and seven normal thyroid lobes from patients undergoing thyroidectomy. Normalized levels of galectin-3 mRNA, expressed as picograms per nanogram GAPDH mRNA, were higher in papillary carcinomas (3327 pg/ng) and follicular adenomas (1314 pg/ng) than in thyroid normal tissue (426 pg/ng; P = 0.0012 and 0.032, respectively). Galectin-3 mRNA levels were also higher in papillary cancers than in adenomatoid nodules (P = 0.0012). However, galectin-3 mRNA levels were not statistically greater in follicular carcinomas than either normal tissue or follicular adenomas (P = 0.068 and 0.12, respectively). In summary, in comparison to galectin-3 immunohistochemistry, quantitative measurement of galectin-3 mRNA appears useful in the identification of papillary thyroid cancers (PTCs) but does not appear to be useful in distinguishing follicular carcinomas from follicular adenomas.