Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles

Isolated porcine thyroid cells were cultured in agarose-coated dishes and allowed to reform follicles with normal polarity. The thyroid cells reaggregated into follicles were compared with cells cultured in monolayer for cAMP responsiveness to TSH and thyroid-stimulating antibody (TSAb). The cells in follicles were sensitive to TSH stimulation and responded to the hormone at concentrations as low as 3.3-10 microU/ml with an increase in cAMP production. In contrast, 10-50 microU/ml TSH were required to elicit a cAMP response in cells cultured in monolayer using identical conditions. cAMP responsiveness to TSAb also was greater in the cells organized into follicles. TSAb was detected in serum from 89.4% of 66 untreated patients with hyperthyroid Graves' disease using thyroid follicles, but TSAb was detected in serum from only 60% of the patients when assayed using cells in monolayer. The assay using thyroid follicles was used to measure TSAb in 27 euthyroid patients who were euthyroid while receiving thionamide therapy and compared with 20-min thyroid 131I uptake after T3 suppression. TSAb was present in 11 of 12 nonsuppressible patients and in 5 of 15 suppressible patients. TSAb was positively correlated with 20-min 131I thyroid uptake. We conclude that thyroid cells cultured in follicles are suitable for measuring TSAb.     

Thyroid stimulating immunoglobulin bioassay using cultured normal human thyroid cells

It is currently believed that the thyroid stimulating immunoglobulin (TSI) of Graves' disease is involved in the pathogenesis of hyperthyroidism through the stimulation of the adenylate cyclase-cyclic AMP system. To evaluate this mechanism, TSI in the serum of patients with Graves' disease was determined by its ability to generate cyclic AMP (cAMP) in monolayer cells prepared from a normal thyroid gland. The thyroid tissue was digested with collagenase, and the liberated follicles were collected from the supernatant and cultured for 7 days. One gram of thyroid tissue yielded more than 1 X 10(7) monolayer cells which were stored in aliquots at -80C. Cells (1 approximately 2 X 10(4)/0.28 cm2 microtiter well) were incubated for 4 hours in 0.2 ml Hanks solution poor in NaCl, with various amounts of bovine TSH (bTSH) or 1.5 mg/ml Graves' serum IgG extracted by polyethylene glycol. cAMP accumulated in medium and cells was measured by RIA. Total cAMP (both medium and cells) was about 4 times higher when NaCl was deleted from Hanks solution. Moreover, as more than 90% of the cAMP was released into the medium, it was possible to omit the measurement of cellular cAMP, which requires extraction. The increase in medium cAMP concentration was dependent upon the number of cells, incubation time, and dose of bTSH. Time course and dose response curves in medium cAMP stimulated by IgG from 3 Graves' patients paralleled those of bTSH equivalent units. Accordingly, TSI activity could be expressed in bTSH equivalent units (bTSH microUeq). The assay could detect 1.0 or 3.3 microU/ml of bTSH and was highly reproducible. TSI activity in all of 16 IgGs from normal subjects was under 3.3 bTSH microUeq/ml, while it was greater than 3.3 bTSH microUeq/ml in IgGs from 33 of 37 (89%) untreated patients with Graves disease. Of the 13 patients followed for 2 to 7 months while on antithyroid drugs, 12 had greater than 3.3 bTSH microUeq/ml and, with the exception of one, all showed a decrease in their TSI activity. Moreover, 5 of 12 patients treated continuously for more than 1 year were TSI negative (less than 3.3 bTSH microUeq/ml), and except for one case, all had TSI values below 8 bTSH microUeq/ml (a value found in only 25% of untreated patients). This in vitro bioassay for TSI is simple and sensitive. It detects the presence of TSI in virtually 90% of untreated patients with Graves' disease. TSI activity showed a clear decrease during the course of antithyroid drug therapy.     

Clinical experience with a human thyroid cell bioassay for thyroid-stimulating immunoglobulin

A sensitive, specific, and practical bioassay for thyroid-stimulating immunoglobulin (TSI) is now available for clinical use. Fifty-seven of 61 patients with untreated hyperthyroid Graves' disease were TSI positive (sensitivity, 93%). TSI was undetectable in all normal subjects and in patients with Hashimoto's thyroiditis (without concurrent Graves' ophthalmopathy), nontoxic goiter, and toxic nodular goiter (specificity, 100%). The prevalence of TSI in serum declined after therapy, particularly during methimazole or propylthiouracil treatment. TSI correlated well with relapse or remission after antithyroid drug therapy. All 12 patients who were TSI negative at the time of discontinuing antithyroid drug therapy remained in remission (average follow-up of 10 months). TSI values in Graves' disease correlated better with thyroid dysfunction than with ophthalmopathy. Prenatal TSI activity tended to be higher in mothers of infants who developed neonatal Graves' disease than in at-risk mothers who delivered normal infants. However, there was considerable overlap between the two groups.     

The specificity of inhibin bioassay using cultured pituitary cells

It has been established that the inhibition of the cellular content of FSH in cultured pituitary cells can be used as a sensitive and precise bioassay for inhibin. During studies on the inhibin content of human plasma, FSH suppression was noted to occur together with LH suppression. Further studies revealed that where combined FSH and LH suppression occurred, cytological changes in the pituitary cells suggestive of toxicity were found, indicating non-specific effects of these substances. Charcoal treatment or gel filtration of seminal plasma removed or separated the toxic substances from the inhibin activity, the latter characterized by FSH suppression parallel to the standard preparation, no LH suppression and no light-microscopic changes in pituitary cells. It is recommended that careful evaluation of all inhibin bioassay systems should be undertaken to detect substances producing non-specific effects and additional guidelines for the assessment of specificity are suggested.     

Blocking anti-thyrotropin receptor antibodies desensitize cultured human thyroid cells

Stimulating anti-TSH receptor antibodies (TSAb) mimic TSH in the induction of refractoriness in cultured thyroid cells; TSAb and TSH desensitize one another. We investigated whether blocking anti-TSH receptor antibodies (TBkAb) have the same desensitizing effects in cultured human thyroid cells. Prolonged exposure of cells (20 h) to TBkAb followed by antigen-antibody dissociation by an acid wash step was required to induce refractoriness to subsequent stimulation of cAMP accumulation with TSH and TSAb. Cycloheximide prevented this desensitization effect. The cAMP response to forskolin was not reduced in cells pretreated by TBkAb and was increased in cells desensitized by TSH or TSAb. The pattern of the TSH dose-response curves suggested that desensitization by TSH or TSAb involved only a postreceptor mechanism but both receptor and postreceptor phenomena in the case of TBkAb. In conclusion, like TSH or TSAb, TBkAb may induce a homologous desensitization in human thyroid cells which is not mediated by cAMP.     

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 μM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.     

Triiodothyronine and 3',5'-cyclic AMP secretion by cultured human thyroid cells in response to thyrotropin and thyroid-stimulating immunoglobulin

We have established a relatively simple and sensitive system for measuring T3 as well as cAMP secretion using cryopreserved human thyroid cells in culture. We defined optimal culture conditions and characterized the system. T3 secretion from human thyrocytes (only 1 x 10(5) cells/well) could be stimulated in a time- and dose-dependent fashion by both TSH (doses as low as 10 mU/l) and thyroid-stimulating immunoglobulin to levels 5- to 10-fold above baseline. The response to the thyroid stimulating agents was preserved for at least 3 weeks. Experiments with inhibitors of iodothyronine synthesis (propylthiouracil and methimazole) indicated that the bulk of the TSH-stimulated T3 secretion measured apparently derives from de novo iodothyronine biosynthesis rather than preformed T3. We utilized the system to investigate some aspects in the regulation of human thyrocyte T3 and cAMP secretion. Maximum stimulation of the thyroid hormone was achieved at TSH doses capable of evoking a further rise in levels of cAMP. A rise in cAMP accumulation was observed as early as 15 min following exposure to TSH, whereas it took 1-4 days to detect a significant increase in T3 secretion. Within 6 h of incubation, the bulk of TSH-stimulated intracellular cAMP was found released into the medium. 1-methyl-3-isobutylxanthine (MIX) caused a dose-related decrease (beyond 0.1 mmol/l MIX) in TSH-stimulated T3 secretion which contrasted with a concomitant expected increase in cAMP accumulation. Hence, as also observed in adrenal and testicular tissue, xanthines at high concentration seem to exhibit a dual action: potentiation of cAMP accumulation by inhibiting phosphodiesterase activity and a concomitant reduction of hormone formation.     

体外培养的人甲状腺细胞对TSH反应的实验研究

目的 研究促甲状腺激素（TSH）对甲状腺细胞分泌细胞的影响，探讨TSH的细胞内信号传导通路。方法 用含10%小牛血清的RPMI-1640培养液体外培养人甲状腺细胞，对照组48h后加入TSH，实验组24h内加入TSH（2U/L)，观察细胞培养1，3，5，7d的T3，T4分泌量及培养细胞内的cAMP含量和TPO活性。结果 对照组甲状腺细胞呈上皮样贴壁生长，实验组细胞则呈假滤泡样立体生长。实验组细胞T3、T4分泌量及细胞内cAMP含量均较同期对照组明显增加，但实验组TPO活性仅于3d之内较对照组增加，3d之后显著下降，至培养第5d2组细胞TPO活性均消失。结论 TSH作为一种生长刺激因子至少有一部分是以cAMP为第二信使起作用的；TSH对细胞增殖及分化的作用与细胞周期相关，可通过同一信号分子调节细胞生长，是细胞增值与分化的关键因素。     

Simultaneous measurement of TSH-binding inhibitor immunoglobulin and thyroid stimulating autoantibody activities using cultured FRTL-5 cells in patients with untreated Graves' disease

A new and practical assay was developed using cultured FRTL-5 cells for simultaneous assessment of TS-ab and TSH-binding inhibitor immunoglobulin, allowing direct comparison of these two activities under the same conditions. Subsequent to the TS-ab assay in which extracellular cAMP concentration in Hanks' medium without NaCl was determined, [125I]b TSH in this medium was added to observe the ability of serum Ig to inhibit the binding of [125I]b TSH to FRTL-5 cells. We found a much higher specific binding of [125I]b TSH to FRTL-5 cells and a much greater inhibition of [125I]bTSH binding to the cells exposed to Graves' Ig in hypotonic NaCl-free than in NaCl containing Hanks' medium, indicating that the binding of both TSH and Graves' Ig to the TSH receptor was salt-sensitive. The inhibitory activity of [125I]bTSH binding to the cells was 0.2 +/- 4.6% (mean +/- SD) in 45 normals. Inter-assay coefficients of variation in two positive controls with the mean values of 18.0 and 65.8% were 15.8 and 16.5%, respectively. Among 46 patients with untreated hyperthyroidism owing to Graves' disease, 45 (97.8%) were positive for TS-ab; 35 (76.1%) and 40 (87.0%) were positive for TSH-binding inhibitor in Ig assays using FRTL-5 cells and solubilized porcine thyroid membranes, respectively. TS-ab activities correlated less closely with TSH-binding inhibitory activities determined using FRTL-5 cells (r = 0.576, p less than 0.001) than with those determined using porcine thyroid membranes (r = 0.745, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of thyrotropin and thyroid stimulating immunoglobulins on gangliosides labelling of human thyroid cultured pathological cells

Gangliosides are known to play an important role in different cell processes. We have investigated the effect of TSH on [14C] glucosamine incorporation into gangliosides of cultured pathological cells isolated from colloid nodular goiter, follicular adenoma, and toxic adenoma. The effect of thyroid stimulating immunoglobulins (TSI) has also been similarly studied on primary cultures from colloid nodular goiter and from porcine thyroid cells. Immunoglobulins were purified from sera of Graves' disease patients. Cells were cultured with or without either stimulator and labelled with [14C] glucosamine during 24 h. Total lipid extracts were fractionated on DEAE Sephadex A 25. Eluted gangliosides were analysed by thin layer chromatography, detected by autoradiography and scraped off for radioactivity measurement. Radioactivity was related to the free cholesterol content. The ganglioside pattern of human thyroid cells in primary cultures is found to vary and to closely reflect the ganglioside pattern of the corresponding gland. No ganglioside pattern could be related to a specific thyroid disorder. When cells are treated with TSH, the amount of [14C] glucosamine incorporated in total gangliosides varies according to diseases. The radioactivity incorporated in gangliosides from colloid nodular goiter cells is not similarly affected by TSH and TSI. In porcine thyroid cells, TSH and TSI have the same effect on glucosamine incorporation into gangliosides. Thus it appears that TSH and TSI may differently affect the gangliosides labelling according to the origin of the thyroid cells.     

2-methoxyestradiol induces apoptosis in cultured human anaplastic thyroid carcinoma cells.

Anaplastic thyroid carcinoma (ATC) is one of the most malignant tumors in humans, and currently there is no effective treatment. In the present study we investigated the effect of an endogenous estrogen metabolite, 2-methoxyestradiol (2-ME), on the growth of human ATC cells. 2-ME treatment had a strong growth inhibitory effect on five human ATC cell lines (HTh7, HTh 74, HTh83, C643, and SW1736), but showed no effect on one cell line (KAT-4). Cell cycle analysis of the growth-inhibited cells showed that 2-ME induced a G2/M-arrest, followed by an increased fraction of cells in sub-G1. Analysis of internucleosomal DNA laddering as well as DNA fragmentation in a terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay demonstrated a high number of cells undergoing apoptosis after 2-ME treatment. An increased activation of caspase-3 and caspase-8 by 2-ME was observed, and inhibition of caspase-3 decreased the apoptotic effect. Addition of 2-ME increased activity of p38 mitogen-activated protein kinase (MAPK) in the sensitive HTh7 as well as the refractory KAT-4 cells, however, activation of stress-activated protein kinase/c-jun aminoterminal kinase (SAPK/JNK) was seen only in the HTh7 cells. Inhibitors of p38 MAPK and SAPK/JNK significantly attenuated the 2-ME effect. Taken together, our data demonstrate an antiproliferative and apoptotic effect of 2-ME on ATC cells involving activation of MAPKs.     

Electrical responses of cultured thyroid cells to serotonin

Cultured porcine thyroid cells, maintained in the differentiated state by dibutyryl cyclic AMP, responded to serotonin (5-HT; 10 nmol/l to 1 mumol/l) with a depolarization of the membrane potential, but did not respond to histamine (100 mumol/l) or dopamine (1 mumol/l). The resting membrane potential of these cells was about -71 mV, maximal concentrations of 5-HT (1 mumol/l) inducing a depolarization to approximately -53 mV. Methysergide or phenoxybenzamine, but not propranolol, abolished the response to 5-HT. Sensitivity to 5-HT was reduced by previous exposure of cultures to TSH, the beta-adrenoceptor agonist salbutamol or 5-HT itself.     

Control of growth in cultured rat thyroid cells

The purpose of this study was to determine the role of cAMP in the growth of FRTL and FRTL5 cells, 2 continuous cultured thyroid lines. TSH, at concentrations similar to those reported to induce growth in primary dog thyroid cultures, played an essential role for growth. Stimulators of adenylate cyclase, cholera toxin and forskolin, and cAMP analogues, dibutyryl cAMP and 8-bromo cAMP, mimicked the effect of TSH in both groups of cultured cells. The present data confirm the role of TSH in controlling growth of both cell lines and suggest that cAMP is an essential intracellular mediator of TSH action.     

Nuclear thyroid hormone receptors in cultured bone cells

Thyroid hormones influence bone metabolism, but a direct interaction of triiodothyronine with nuclear T3 receptors in bone cells has not yet been reported. We investigated 125I-T3 binding to nuclei isolated from the cloned osteoblastlike rat osteosarcoma cells ROS 17/2.8. At 37 degrees C, saturable 125I-T3 binding to isolated nuclei reached equilibrium by 30 minutes and was completely displaced upon the addition of 500 nmol/L unlabeled T3. Nonsaturable binding represented about 0.5% of the radioactivity added (20% of the total binding). Thyroxine and 3,3',5'triiodothyronine competed with 125I-T3 with a 20-fold and 400-fold lower affinity than T3, respectively. Analysis of equilibrium competition experiments revealed the presence of a single class of homogeneous binding sites with an association constant of 5.0 +/- 0.3 X 10(9) mol/L-1 and a maximum nuclear binding capacity of 0.13 +/- 0.02 ng/mg DNA. A twofold increase of bone Gla protein (BGP) secretion was observed with T3 treatment suggesting that these T3 nuclear receptors are coupled with a biological response.     

A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase

A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.