Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells.

The sodium iodide symporter (NIS), first identified in FRTL-5 cells, plays a critical role in iodide transport in the thyroid gland and in the production of the iodine-containing thyroid hormones. The aim of our study was to examine the regulation of NIS RNA steady-state levels and protein expression as well as functional activity in FRTL-5 cells. FRTL-5 cells cycling in media containing thyrotropin (TSH) were incubated for 48 hours with dexamethasone (10(-8)-10(-5) M), triiodothyronine (T3; 10(-9)-10(-6) M), methimazole (100 microM), propylthiouracil (PTU; 100 microM), perchlorate (10 microM) and potassium iodide (40 microM). In other experiments, cells were treated for 48 hours with various cytokines including interleukin-6 (IL-6) (100 U/mL), interferon-gamma (IFN-gamma) (100 U/mL), tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml), IL-1alpha (100 U/mL), and IL-1beta (100 U/mL). Northern blot analysis using a 32P-labeled rat NIS-specific cDNA probe (nucleotides 1397-1937) revealed NIS mRNA as a single species of approximately 3 kb. When normalized for beta-actin mRNA signal intensities, NIS RNA steady-state levels in viable FRTL-5 cells were suppressed by approximately 80% after incubation with dexamethasone and T3 in a concentration-dependent manner. Iodide accumulation was decreased by up to 40% after incubation with dexamethasone and T3, respectively, in a concentration-dependent manner. Using a rabbit polyclonal rNIS-specific antibody, Western blot analysis of FRTL-5 cell membranes revealed a 60% and 70% suppression of NIS protein expression after treatment with T3 (0.1 microM) and dexamethasone (1 microM), respectively. In additon, NIS RNA steady-state levels were decreased by approximately 50% after treatment of monolayers with methimazole, PTU, and potassium iodide, respectively. Incubation with methimazole and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. Treatment of FRTL-5 cells with IL-6 and IL-1beta resulted in a 30% decrease of NIS RNA steady-state levels. IL-6 did not alter NIS functional activity, but IL-1beta suppressed iodide accumulation by approximately 25%. IFN-gamma and perchlorate failed to alter NIS RNA steady-state levels. In contrast to IFN-gamma that had no effect on iodide accumulation, perchlorate almost completely suppressed iodide accumulation. TNF-alpha and IL-1alpha failed to alter NIS RNA steady-state levels in higher passage numbers of FRTL-5 cells, whereas treatment with TNF-alpha and IL-1alpha of early passages of FRTL-5 cells (<20 cell passages) resulted in a 70% and 40% decrease of NIS RNA steady-state levels, respectively, and in a 20% suppression of NIS functional activity. In conclusion, our data suggest that various agents known to affect iodide transport are capable of differentially altering NIS gene expression and function in cultured thyroid cells. Suppression of NIS gene expression and function by certain cytokines may be responsible, at least in part, for the impaired radioiodine uptake by thyroid tissue in certain forms of thyroiditis.     

The mechanism of action of tumour necrosis factor-alpha and interleukin 1 on FRTL-5 rat thyroid cells

Previous work showed that treatment of rats with tumour necrosis factor-alpha produced a model of nonthyroid illness in which there was reduction of circulating thyroid hormones and TSH, reduced thyroid response to TSH, and reduced thyroid iodide uptake. In vitro studies showed that tumour necrosis factor-alpha binds to a specific receptor on FRTL-5 rat thyroid cells, that TSH increases the number of tumour necrosis factor-alpha receptors, and that tumour necrosis factor-alpha inhibits iodide uptake by these cells. In the present study, we obtained additional data on the effects of tumour necrosis factor-alpha on FRTL-5 cells and studied the mechanism of action of tumour necrosis factor-alpha in these cells. Tumour necrosis factor-alpha inhibited both basal and TSH-stimulated [125I]iodide uptake: tumour necrosis factor-alpha slowed the recovery of [125I]iodide trapping after the cells were exposed to TSH and augmented the loss of the [125I]iodide trapping function after the cells were deprived of TSH: tumour necrosis factor-alpha inhibited [125I]iodide trapping in a noncompetitive manner; tumour necrosis factor-alpha did not affect cell growth of FRTL-5 cells. Interleukin-1 (IL-1) also inhibited basal and TSH-stimulated [125I]iodide uptake, but it stimulated cell growth. Tumour necrosis factor-alpha and IL-1 did not affect the generation of cAMP in the presence or absence of TSH; these cytokines blocked the cAMP-induced stimulation of [125I]iodide uptake. Tumour necrosis factor-alpha did not affect [3H]arachidonic acid uptake or release by FRTL-5 cells. The inhibitors of the phospholipase A2-arachidonic acid pathway did not affect the action of tumour necrosis factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lithium on function and growth of thyroid cells in vitro.

Lithium has been reported to alter thyroid function and cause goiter in some patients. To explain the mechanism of lithium action in the thyroid gland, we studied the effect of lithium on thyroid function and cell growth in FRTL-5 rat thyroid cells and on de novo thyroid hormone formation in primary cultures of porcine thyroid follicles. TSH-induced iodide uptake was suppressed at 2 mM lithium in both FRTL-5 cells and porcine follicles. In porcine thyroid follicles, iodide uptake stimulated by 8-bromo-cAMP, iodine organification, and de novo thyroid hormone formation were also reduced by lithium; however, 2 mM lithium did not inhibit TSH-induced cAMP production. In FRTL-5 cells, lithium also inhibited forskolin-stimulated iodide uptake. These results suggested that lithium exerts its effect at a step involving cAMP signal transduction rather than inhibiting cAMP production. In both FRTL-5 thyroid cells and porcine follicles, lithium enhanced cell growth in basal states (lacking TSH) and with TSH treatment. In porcine thyroid cells, the protein kinase C activator, tetradecanoyl phorbol-13-acetate, increased cell growth, and lithium had an additive effect with tetradecanoyl phorbol-13-acetate on cell growth. To examine the possibility that the action of lithium was mediated by the protein kinase C pathway, porcine cells were incubated with lithium and H7, a selective protein kinase C inhibitor. Lithium-induced cell growth was suppressed to the basal level by H7. These results suggest that lithium exerts its growth-promoting effect through the protein kinase C system.     

The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells

The effect of iodide on growth of rat thyroid cells (FRTL-5) was studied. TSH-stimulated cell growth was inhibited by iodide in a concentration-dependent manner, and an effect of iodide was detected at 10(-6) mol/l. KClO4 or 1-methylimidazole-2-thiol blocked the effect of iodide, suggesting that iodide uptake and its organification are required to produce the inhibitory effect of iodide on cell growth. Iodide not only decreased TSH-stimulated cAMP production in FRTL-5 cells but also cell growth induced by cAMP. These observations suggest that iodide inhibits TSH-stimulated growth of the cells by attenuating cAMP production and also by acting on the step(s) distal to cAMP generation. The inhibitory effect of iodide was also seen in growth stimulated by insulin, insulin-like growth factor-I or 12-O-tetradecanoyl phorbol 13-acetate, suggesting multiple sites of action of iodide in the process of growth of FRTL-5 cells.     

Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.     

Combined Cerenkov luminescence and nuclear imaging of radioiodine in the thyroid gland and thyroid cancer cells expressing sodium iodide symporter: initial feasibility study

Radioiodine (RI) such as (131)I or (124)I, can generate luminescent emission and be detected with an optical imaging (OI) device. To evaluate the possibility of a novel Cerenkov luminescence imaging (CLI) for application in thyroid research, we performed feasibility studies of CLI by RI in the thyroid gland and human anaplastic thyroid carcinoma cells expressing sodium iodide symporter gene (ARO-NIS). For in vitro study, FRTL-5 and ARO-NIS were incubated with RI, and the luminometric and CLI intensity was measured with luminometer and OI device. Luminescence intensity was compared with the radioactivity measured with γ-counter. In vivo CLI of the thyroid gland was performed in mice after intravenous injection of RI with and without thyroid blocking. Mice were implanted with ARO-NIS subcutaneously, and CLI was performed with injection of (124)I. Small animal PET or γ-camera imaging was also performed. CLI intensities of thyroid gland and ARO-NIS were quantified, and compared with the radioactivities measured from nuclear images (NI). Luminometric assay and OI confirmed RI uptake in the cells in a dose-dependent manner, and luminescence intensity was well correlated with radioactivity of the cells. CLI clearly demonstrated RI uptake in thyroid gland and xenografted ARO-NIS cells in mice, which was further confirmed by NI. A strong positive correlation was observed between CLI intensity and radioactivity assessed by NI. We successfully demonstrated dual molecular imaging of CLI and NI using RI both in vitro and in vivo. CLI can provide a new OI strategy in preclinical thyroid studies.     

Characterization of complex apurinic/apyrimidinic-site clustering associated with an authentic site-specific radiation-induced DNA double-strand break

Radiation lethality is largely attributed to radiation-induced DNA double-strand breaks (DSBs). A range of structural complexity is predicted for radiation-induced DSBs. However, this lesion has never been analyzed in isolation at the molecular level. To address this problem, we have created authentic site-specific radiation-induced DSBs in plasmid DNA by triplex-forming oligonucleotide-targeted 125I decay. No significant difference in DSB yield was observed after irradiation in the presence or absence of the radical scavenger DMSO, suggesting that DSB formation is a result of the direct effect of the radiation. A restriction fragment terminated by the DSB was isolated and probed with the Escherichia coli DNA repair enzyme endonuclease IV (endo IV), which recognizes apurinic/apyrimidinic (AP) sites. Enzymatic probing demonstrated clustering of AP sites within 10 bases of the 125I-targeted base in the DNA duplex. Our results suggest scavengeable radicals may not play a large role in the generation of AP sites associated with DSB formation, because at least 30% of all fragments have endo IV-sensitive sites, regardless of irradiation conditions. An internal control fragment recovered from the 125I linearized plasmid did not exhibit endo IV sensitivity in excess of that observed for a similar fragment recovered from an undamaged plasmid. Thus, AP site clustering proximal to the DSB resulted from the 125I decays responsible for DSB formation and was not due to untargeted background irradiation.     

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.     

Transforming growth factor-beta blocks protein kinase-A-mediated iodide transport and protein kinase-C-mediated DNA synthesis in FRTL-5 rat thyroid cells.

Recent studies have shown that transforming growth factor-beta (TGF beta) alters DNA synthesis and iodide metabolism in human, porcine, and rat thyroid cells. In the present work we studied the mechanism of TGF beta action in FRTL-5 rat thyroid cells. The cells were treated with TGF beta in the presence of TSH, growth factors, and cellular modulators for various periods of time; then, [3H]thymidine incorporation and DNA content were measured as indicators of DNA synthesis, and [125I]iodide uptake was measured to assess cell function. TGF beta (10 ng/ml) inhibited TSH-induced DNA synthesis and iodide uptake. TGF beta also inhibited DNA synthesis induced by insulin-like growth factor-I, fibroblast growth factor, and endothelial cell growth factor. The protein kinase-A (PKA) activator 8-bromo-cAMP increased both iodide uptake and DNA synthesis; TGF beta inhibited 8-bromo-cAMP-induced [125I]iodide uptake, but not [3H]thymidine incorporation. The protein kinase-C (PKC) activator phorbol 12-myristate 13-acetate increased [3H]thymidine incorporation, and TGF beta inhibited this action of phorbol 12-myristate 13-acetate. The results show that activation of PKA or PKC increases DNA synthesis. TGF beta inhibited PKC-mediated, but not PKA-mediated, DNA synthesis in these cells. The results also show that TGF beta selectively inhibits PKA-mediated iodide uptake, but not PKA-mediated DNA synthesis. These findings suggest that TGF beta is a strong inhibitor of the proliferation and function of thyroid cells.     

Ku70反义寡核苷酸增强甲状腺癌细胞的辐射敏感性

目的 研究DNA修复基因Ku70反义寡核苷酸(ASODN)对甲状腺癌细胞辐射敏感性的影响.方法 设计合成特异性靶向Ku70的ASODN,以不同浓度转染人甲状腺髓样癌细胞(TT),并设脂质体和正义寡核苷酸(SODN)对照组进行比较.采用Western印迹技术检测各组细胞中Ku70蛋白表达水平.利用不同剂量60 Co γ射线照射细胞后,细胞克隆形成实验检测细胞存活分数.CCK-8法、Annexin-V/PI染色法分析ASODN对细胞活力和细胞凋亡的影响.彗星电泳法比较γ射线照射后DNA双链断裂的修复效率.结果 转染ASODN各组细胞Ku70蛋白表达均较脂质体对照组、SODN组明显降低,并呈浓度依赖性;照射后细胞存活率降低(P＜0.01),凋亡率显著增加(P＜0.01),DNA双链断裂的修复效率降低(P＜0.01).结论 ASODN能够特异性地下调甲状腺癌细胞Ku70的表达,抑制γ射线照射后细胞存活和DNA双链断裂修复,提高肿瘤细胞的辐射敏感性.     

Effects of ketoconazole on the iodide uptake by FRTL-5 cells.

Ketoconazole is an imidazole derivative used as an antimycotic agent with reported effects on the endocrine system, but very little is known about its possible actions on thyroid function. Our purpose was to study the influence of this substance on the basal and TSH-stimulated iodide uptake in the rat thyroid cell strain FRTL-5. Ketoconazole (1-50 mumol/l) was shown to slightly increase the basal iodide uptake but, at higher concentrations (75-100 mumol/l), it sharply decreased iodide uptake below the basal levels. When the cells were cultured under bTSH stimulation (30 UI/l), the inhibitory effect of ketoconazole was exerted at concentrations as low as 25 mumol/l. This inhibition was observed even if it was added to the culture medium immediately before the Na125I addition. Forskolin, a stimulator of adenylate cyclase activity, was unable to prevent the iodide uptake inhibition. Low doses of ketoconazole increased cAMP concentrations. In the presence of TSH this effect was more evident in an inverse dose-dependent way. Because of its dual action, it can be assumed that ketoconazole could influence the iodide uptake in the FRTL-5 cells through more than one mechanism.     

Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses

DNA double-strand breaks (DSBs) are generally accepted to be the most biologically significant lesion by which ionizing radiation causes cancer and hereditary disease. However, no information on the induction and processing of DSBs after physiologically relevant radiation doses is available. Many of the methods used to measure DSB repair inadvertently introduce this form of damage as part of the methodology, and hence are limited in their sensitivity. Here we present evidence that foci of gamma-H2AX (a phosphorylated histone), detected by immunofluorescence, are quantitatively the same as DSBs and are capable of quantifying the repair of individual DSBs. This finding allows the investigation of DSB repair after radiation doses as low as 1 mGy, an improvement by several orders of magnitude over current methods. Surprisingly, DSBs induced in cultures of nondividing primary human fibroblasts by very low radiation doses (approximately 1 mGy) remain unrepaired for many days, in strong contrast to efficient DSB repair that is observed at higher doses. However, the level of DSBs in irradiated cultures decreases to that of unirradiated cell cultures if the cells are allowed to proliferate after irradiation, and we present evidence that this effect may be caused by an elimination of the cells carrying unrepaired DSBs. The results presented are in contrast to current models of risk assessment that assume that cellular responses are equally efficient at low and high doses, and provide the opportunity to employ gamma-H2AX foci formation as a direct biomarker for human exposure to low quantities of ionizing radiation.     

Phorbol myristate acetate inhibits alpha 1-adrenergically but not thyrotropin-regulated functions in FRTL-5 rat thyroid cells

The iodination of thyroglobulin and the formation of thyroid hormones are regulated by alpha 1-adrenergic agents as well as TSH in rat FRTL-5 cells. The regulatory effects of the alpha-1-adrenergic agents and TSH on both of these processes are associated with an increase in cytosolic Ca2+ and an increase in that component of iodide efflux that is representative of the movement of iodide from the thyroid cell into the follicular lumen. When FRTL-5 cells are preincubated with phorbol myristate acetate (PMA) for at least 3 min, the norepinephrine-stimulated changes in cytosolic Ca2+ levels and iodide efflux are inhibited. In contrast, PMA pretreatment has no effect on iodide efflux and actually enhances the changes in cytosolic Ca2+ induced by TSH. Phorbol myristate acetate pretreatment has no effect on TSH-stimulated cAMP-mediated iodide uptake in FRTL-5 cells, nor does it affect the binding parameters of the alpha 1-adrenergic receptor antagonist prazosin. These data suggest that protein kinase C is involved in a feedback mechanism regulating alpha 1-adrenergic but not TSH-induced changes associated with the iodination of thyroglobulin and the formation of thyroid hormones; and that this feedback effect occurs after the step of ligand binding but before the increase in cytosolic Ca2+ induced by the alpha 1-adrenergic agents.     

Perchlorate ion enhances mouse thyroid responsiveness to thyrotropin, human chorionic gonadotropin and long acting thyroid stimulator.

Perchlorate treatment of mice increased by 1.5-2-fold the thyroid secretory response to TSH, hCG and LATS, in the McKenzie bioassay. Perchlorate alone did not increase basal plasma radioactivity. Perchlorate augmentation of the secretory response index was roughly proportional to the level of stimulation; it was similar for all three stimulators despite their different time courses of action which were unaltered by perchlorate; it was the same whether perchlorate administration preceded, coincided with or shortly followed injection of the stimulator, a finding in keeping with the slow clearance of this ion. The perchlorate effect was dose-related, although within a narrow range (6.25-12.5 microng/mouse). Near-maximal per chlorate effect was obtained with a dose (12.5 microng) which, when tested in different experimental conditions (MMI-blocked thyroid), discharged 80% of intrathyroidal radioiodide. Perchlorate exerted its augmenting effect by enhancing thyroid secretion: it increased plasma radioiodothyronines and radioiodide concentrations without decreaseing the blood disappearance rates of iodide and iodothyronines. The potentiating effect of perchlorate probably takes place at a step prior to cyclic AMP action since it did not affect dbcAMP-stimulated secretion. The perchlorate effect may be indirect, through mobilization of minute amounts of intrathyroidal iodide.     

Loss of adrenergic regulation of cAMP production in the FRTL-5 cell line

Rat thyroid cells in primary culture augment cAMP production when challenged with beta-adrenergic agonists; at 10(-5) M the potency is isoproterenol greater than norepinephrine greater than epinephrine. In analogy with human thyroid cells, rat thyroid primary cultures display alpha-adrenergic-stimulated cGMP production which inhibits TSH and norepinephrine stimulation of cAMP. Adrenergic regulation of cyclic nucelotide production is lost in the cloned thyroid cell line of rat origin known as FRTL-5. Also the potentiating effect of phentolamine on TSH stimulation of cAMP production in thyroid primary culture becomes an inhibitory one in the FRTL-5 cells. Neither 'soluble factors' nor contamination of other cell populations could account for the different behaviour of the primary culture and the cell line toward adrenergic regulation. The reported activation by norepinephrine of iodide efflux in FRTL-5 cells rules out the loss of specific adrenergic receptors in the FRTL-5 cells. It is proposed that the cloning of FRTL-5 cells from primary cultures causes an 'alteration' in the coupling of adrenergic receptors to the adenylate cyclase system. This alteration does no affect those mechanism of message transduction that do not involve cAMP as the signal.