Rat Thyroid Cell Line Research Articles

The effect of dimethyl sulfoxide on the induction of DNA strand breaks in plasmid DNA and colony formation of PC Cl3 mammalian cells by alpha-, beta-, and Auger electron emitters (223)Ra, (188)Re, and (99m)Tc.

BackgroundDNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by 223Ra compared to 188Re and 99mTc modulated by the radical scavenger dimethyl sulfoxide (DMSO).MethodsRadioactive solutions of 223Ra, 188Re, or 99mTc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay.ResultsExposure to 120 Gy of 223Ra, 188Re, or 99mTc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy 223Ra and 500 Gy 188Re or 99mTc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter 223Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with 223Ra, 188Re, and 99mTc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for 223Ra, 188Re, and 99mTc, respectively.ConclusionsFor 223Ra, as well as for 188Re and 99mTc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0203-x) contains supplementary material, which is available to authorized users.

Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells

Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent l-Leucyl-l-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes.

CFTR and SLC5A8 Associate in Thyroid Epithelial Cells

The cystic fibrosis transmembrane conductance regulator, CFTR, is expressed at the apical membranes of many epithelial cells, where it mediates cAMP‐stimulated anion secretion. In thyroid, CFTR may indirectly and/or directly furnish iodide for hormone synthesis. CFTR functionally interacts with many membrane transporters via PSD‐Discs large homologous‐ZO‐1 (PDZ) domain‐containing scaffolding proteins, thereby forming macromolecular transport complexes. SLC5A8 is an electrogenic, sodium‐coupled monocarboxylate transporter that also is expressed at thyrocyte apical membranes. CFTR and SLC5A8 contain the identical PDZ domain interaction motif; preliminary studies utilizing overexpression systems indicate a role for PDZ adaptor proteins in linking CFTR and SLC5A8. The present studies use the Fisher rat thyroid cell line, FRTL‐5, to test the hypothesis that PDZ proteins facilitate CFTR/SLC5A8 interactions in an endogenously expressing system. Expression of CFTR (~150 kDa), SLC5A8 (~68 kDa) and NHERF‐1 (~50 kDa) in total FRTL‐5 lysates was confirmed by immunoblot analysis using anti‐CFTR (M3A7 and Cystic Fibrosis Foundation Therapeutics (CFFT) 660), anti‐SLC5A8 (Abcam) and anti‐NHERF‐1 (Alomone) antibodies. Anti‐SLC5A8‐immunoprecipitated (IPd) fractions of FRTL‐5 lysates reacted with both CFFT 660 and anti‐NHERF‐1. Fractions IPd using anti‐CFTR (M3A7) were probed with anti‐SLC5A8 and anti‐NHERF‐1. M3A7‐IPd fractions showed positive reactivity with anti‐SLC5A8, but NHERF‐1 was not detected. These data suggest that although CFTR and SLC5A8 associate in FRTL‐5 cells, the participation of NHERF‐1 is not required.
 Support: NIH/NIGMS Award 1R15GM101674‐01A1 (to PF); Office of the Director, NIH Award Number T35OD010979.

Mutations in the TASK‐3 Potassium Channel Pore Modify Its Inhibition by Breathing Stimulant Compounds

BACKGROUND Drugs that inhibit TASK-1 and TASK-3 potassium channel function (e.g., PKTHPP, A1899, and doxapram) cause stimulation of breathing in rodents. To better understand the mechanism(s) of action of these drugs, we undertook studies to identify residues in TASK-3 that mediate this inhibition. HYPOTHESIS: Guided by in silico drug docking, we hypothesized PKTHPP and A1899 bind in the TASK-3 intracellular pore. METHODS: To test our hypothesis, we mutated residues in or near the predicted PKTHPP and A1899 binding site (residues 120 to 128). We quantified each mutation's effect on the TASK-3 concentration-response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in a Fischer rat thyroid cell line; channel function was measured by Ussing chamber. RESULTS: One mutant, TASK-3 L122D, was markedly resistant to block by PKTHPP, A1899, and doxapram (Table 1). Other pore mutations had more modest effects. Three residues when mutated (L120D, M124D, L128D) were without function. Conclusions Our data are consistent with a model in which breathing stimulant drugs PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding within the channel intracellular pore. FUNDING: Massachusetts General Hospital Department of Anesthesia, Critical Care & Pain Medicine; NIH/HL117871; NSF/CHE0809413; Simons Collaboration on the Origins of Life. IC50 Wild-type T121D L122D V123D M125D Q126D S127D PKTHPP (nM) 10 (9-11) 19 (15-24)* >10,000t 5 (4-7)* 31 (21-48)* 160 (96-265)* 575 (522-635)* A1899 (nM) 18 (15-22) nd >10,000t nd nd nd nd Doxapram (uM) 16 (11-21 ) nd 1,300 (1,100-1,700)* nd nd nd nd

DuOx2 Promoter Regulation by Hormones, Transcriptional Factors and the Coactivator TAZ

The production of H₂O₂, which is essential to thyroid hormone synthesis, involves two NADPH oxidases: dual oxidases 1 and 2 (DuOx1 and DuOx2). A functional study with human DuOx genes and their 5′-flanking regions showed that DuOx1 and -2 promoters are different from thyroid-specific gene promoters. Furthermore, their transcriptional activities are not restricted to thyroid cells. While regulation of Tg (thyroglobulin) and TPO (thyroperoxidase) expression have been extensively studied, DuOx2 promoter regulation by hormones and transcriptional factors need to be more explored. Herein we investigated the role of TSH, insulin and insulin-like growth factor 1 (IGF-1), as well as the cAMP effect on DuOx2 promoter (ptx41) activity in transfected rat thyroid cell lines (PCCL3). We also assessed DuOx2 promoter activity in the presence of transcriptional factors crucial to thyroid development such as TTF-1 (thyroid transcription factor 1), PAX8, CREB, DREAM, Nkx2.5 and the coactivator TAZ in HeLa and HEK 293T-transfected cells. Our results show that TSH and forskolin, which increase cAMP in thyroid cells, stimulated DuOx2 promoter activity. IGF-1 led to pronounced stimulation, while insulin induction was not statistically different from DuOx2 promoter basal activity. All transcriptional factors selected for this work and coactivator TAZ, except DREAM, stimulated DuOx2 promoter activity. Moreover, Nkx2.5 and TAZ synergistically increased DuOx2 promoter activity. In conclusion, we show that DuOx2 expression is regulated by hormones and transcription factors involved in thyroid organogenesis and carcinogenesis, reinforcing the importance of the control of H₂O₂ generation in the thyroid.

Mitochondrial Respiratory Chain Inhibitors Involved in ROS Production Induced by Acute High Concentrations of Iodide and the Effects of SOD as a Protective Factor.

A major source of reactive oxygen species (ROS) generation is the mitochondria. By using flow cytometry of the mitochondrial fluorescent probe, MitoSOX Red, western blot of mitochondrial ROS scavenger Peroxiredoxin (Prx) 3 and fluorescence immunostaining, ELISA of cleaved caspases 3 and 9, and TUNEL staining, we demonstrated that exposure to 100 μM KI for 2 hours significantly increased mitochondrial superoxide production and Prx 3 protein expression with increased expressions of cleaved caspases 3 and 9. Besides, we indicated that superoxide dismutase (SOD) at 1000 unit/mL attenuated the increase in mitochondrial superoxide production, Prx 3 protein expression, and lactate dehydrogenase (LDH) release and improved the relative cell viability at 100 μM KI exposure. However, SOD inhibitor diethyldithiocarbamic acid (DETC) (2 mM), Rotenone (0.5 μM), a mitochondrial complex I inhibitor, and Antimycin A (10 μM), a complex III inhibitor, caused an increase in mitochondrial superoxide production, Prx 3 protein expression, and LDH release and decreased the relative cell viability. We conclude that the inhibitors of mitochondrial respiratory chain complex I or III may be involved in oxidative stress caused by elevated concentrations of iodide, and SOD demonstrates its protective effect on the Fischer rat thyroid cell line (FRTL) cells.

Expression analysis of stemness genes in a rat thyroid cell line FRTL5.

Detection and analysis of a small subpopulation of cells such as stem cells or cancer stem cells are recognized to be a key technique in a recent regeneration and cancer science. However, in the thyroid, no marker that identifies stem cells has been established yet. We previously established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS), named mRNA quantification after FACS (FACS-mQ). By using this method, the biological characteristics of the sorted cells can be determined by analyzing their gene expression profile. In this study, we analyzed the expression of stemness genes in a rat thyroid cell lines FRTL5 using FACS-mQ. 3 stemness genes, NANOG, ABCG2 and GATA4, were expressed in FRTL5. In FRTL5 cells, varied expression of thyroglobulin (TG) among cells was observed by flow cytometry. Cell populations with high or low TG expression were analyzed by FACS-mQ. The cell population with low TG expression showed increased expression of the stemness genes. Furthermore, Ki67-positive cells showed increased expression of TG, which suggested that cells with high TG proliferated rapidly. These results indicated that FRTL5 contains a cell population with high stemness gene expression and less differentiated features, resembling stem cells. These cells might regulate proliferation in FRTL5.

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte.

Iodide is captured by thyrocytes through the Na(+)/I(-) symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca(2+)-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

Differential Regulation of the Production of Reactive Oxygen Species in Th1 Cytokine–Treated Thyroid Cells

Th1 cytokines exert pleiotropic effects in Hashimoto's thyroiditis. Previous studies reported a downregulation of thyroperoxidase and dual oxidase (DUOX) protein and mRNA expression in thyroid cells treated with Th1 cytokines. Although this effect is partially mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species, the nature and the source of the ROS involved are currently unknown. The aim of this study was to examine further the nature and source of the ROS produced in response to Th1 cytokines. Two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes were incubated with Th1 cytokines (interleukin [IL]-1α and interferon-γ) in the presence or absence of the Th2 cytokine IL-4, the nitric oxide synthase inhibitor N-nitroso-L-arginine methyl ester (L-NAME), or the synthetic antioxidant N-acetylcysteine. The nature and source of the intracellular and extracellular ROS produced were determined. A rapid increase in intracellular ROS was observed in cells incubated with Th1 cytokines. This increase was not caused by extracellular hydrogen peroxide (H2O2) produced by DUOX because both DUOX expression and extracellular H2O2 synthesis were decreased by Th1 cytokines. Confocal colocalization experiments showed that the Th1 cytokine-triggered ROS were not produced from mitochondria. Electron paramagnetic resonance investigations of PCCL3 cells indicated that the highly reactive hydroxyl radical was not involved in the response to Th1 cytokines. NOX2 mRNA expression was significantly increased in PCCL3 cells incubated with Th1 cytokines, as was the expression of the protein in the thyroid of Hashimoto's thyroiditis patients. NOX4 expression was by contrast unaffected. These results suggest that at least superoxide could be produced after exposure of thyroid cells to Th1 cytokines. The effects of L-NAME and IL-4, both of which partially or totally reverse Th1 cytokine-induced effects, on ROS release were also analyzed. L-NAME and IL-4 significantly reduced the Th1 cytokine-induced surge of intracellular ROS in PCCL3 and human thyroid cells. The data presented here reinforce the idea that ROS, other than extracellular H2O2 produced by DUOX, are released from NOX2 after exposure of thyroid cells to Th1 cytokines. ROS/reactive nitrogen species act as important, but as further explained, not exclusive intracellular mediators of Th1 cytokine-induced effects in thyroid cells.

Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter

NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2.− and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog 99mTcO4− nor of the K+ cation mimic 86Rb+ in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

The thyroid cancer PAX8–PPARG fusion protein activates Wnt/TCF-responsive cells that have a transformed phenotype

A chromosomal translocation results in the production of a paired box 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARG) fusion protein (PPFP) in ∼35% of follicular thyroid carcinomas. To examine the role of PPFP in thyroid oncogenesis, the fusion protein was stably expressed in the non-transformed rat thyroid cell line PCCL3. PPFP conferred on PCCL3 cells the ability to invade through Matrigel and to form colonies in anchorage-independent conditions. PPFP also increased the fraction of cells with Wnt/TCF-responsive green fluorescent protein reporter gene expression. This Wnt/TCF-activated population was enriched for colony-forming and invading cells. These actions of PPFP required a functional PPARG DNA binding domain (DBD) within PPFP and were further stimulated by PPARG agonists. These data indicate that PPFP, through its PPARG DBD, induces Wnt/TCF pathway activation in a subpopulation of cells, and these cells have properties of cellular transformation including increased invasiveness and anchorage-independent growth.

Abstract 87: Apigenin further enhances Akti1/2-induced radioactive iodine uptake (RAIU) and rescues TGF-β1-reduced RAIU in PCCl3 rat thyroid cells.

Abstract Background: Na+/I− Symporter (NIS) is a glycoprotein expressed on the basolateral membrane of thyroid follicular cells and it facilitates iodide uptake from circulating blood. In thyroid cancer, NIS-mediated RAIU serves as the basis for therapeutic ablation of post-thyroidectomy remnants. However, patients with advanced thyroid cancer do not benefit from radioiodine therapy due to reduced or absence of NIS expression/function. Thus, it is of clinical importance to investigate the underlying mechanisms of NIS modulation such that strategies to selectively upregulate NIS expression/ function can be devised. Approach: Clinical and experimental evidences suggest an inverse correlation between Epithelial-to-mesenchymal transition (EMT) and NIS-mediated RAIU in thyroid cancer. To investigate if there is a functional role for EMT in decreasing RAIU, we determined the expression levels of 83 EMT-associated genes in PCCl3 rat thyroid cell lines treated with DMSO (control), Akti1/2 (small molecule inhibitor of pAkt1/2 known to increase RAIU) or TGF-β1 (cytokine known to decrease RAIU) using SABiosciences RT2 profiler EMT PCR array and subjected the results to Ingenuity Pathway Analysis (IPA). To mimic clinical conditions, cells were subjected to ≈5-day Thyroid Stimulating Hormone (TSH) withdrawal followed by acute stimulation by TSH for 24h. Cells were then treated for 24h with reagents in the presence of TSH followed by RNA extraction or RAIU assay or protein isolation. Results: From IPA analysis, we predicted apigenin, a plant derived flavonoid could potentially increase radioactive iodide uptake. Indeed, in-vitro RAIU assays and western blots revealed that apigenin (20μM) further enhances Akti1/2 (10μM)-induced RAIU up to 2-fold by increasing iodide influx and not by increasing NIS protein levels or decreasing iodide efflux in thyroid cells. pAkt inhibition was permissive for apigenin to increase RAIU. Apigenin (20μM) also rescued TGF-β1 (10ng/ml)-reduced RAIU up to 85%. Apigenin is known to affect many kinases and the molecular mechanism of action of apigenin in increasing RAIU is currently under investigation using candidate approach. Conclusion: Apigenin is the only compound reported so far that consistently further increases Akti1/2-induced RAIU. Apigenin promises to reveal novel molecular mechanisms of NIS modulation in thyroid. Apigenin also serves as a potential candidate for dietary supplement in combination with other drugs for future efficacy studies related to radioiodide therapy of thyroid cancer. Citation Format: Aparna Lakshmanan, Sissy Meihua Jhiang. Apigenin further enhances Akti1/2-induced radioactive iodine uptake (RAIU) and rescues TGF-β1-reduced RAIU in PCCl3 rat thyroid cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 87. doi:10.1158/1538-7445.AM2013-87

Notch Pathway Is Activated by MAPK Signaling and Influences Papillary Thyroid Cancer Proliferation

Mutually exclusive genetic alterations in the RET, RAS, or BRAF genes, which result in constitutively active mitogen-activated protein kinase (MAPK) signaling, are present in about 70% of papillary thyroid carcinomas (PTCs). However, the effect of MAPK activation on other signaling pathways involved in oncogenic transformation, such as Notch, remains unclear. In this study, we tested the hypothesis that the MAPK pathway regulates Notch signaling and that Notch signaling plays a role in PTC cell proliferation. Conditional induction of MAPK signaling oncogenes RET/PTC3 or BRAFT1799A in normal rat thyroid cell line mediated activation of Notch signaling, upregulating Notch1 receptor and Hes1, the downstream effector of Notch pathway. Conversely, pharmacological inhibition of MAPK reduced Notch signaling in PTC cell. Thyroid tumor samples from transgenic mice expressing BRAFT1799A and primary human PTC samples showed high levels of Notch1 expression. Down-regulation of Notch signaling by γ-secretase inhibitor (GSI) or NOTCH1 RNA interference reduces PTC cell proliferation. Moreover, the combination of GSI with a MAPK inhibitor enhanced the growth suppression in PTC cells. This study revealed that RET/PTC and BRAFT1799A activate Notch signaling and promote tumor growth in thyroid follicular cell. Taken together, these data suggest that Notch signaling may be explored as an adjuvant therapy for thyroid papillary cancer.

Functional coupling of TRPC2 cation channels and the calcium‐activated anion channels in rat thyroid cells: Implications for iodide homeostasis

The initial step in a synthesis of thyroid hormones is the uptake of iodide from the circulation. Iodide (I(-)) is transported into thyroid cells via a Na(+)/I(-) symporter (NIS), which is electrogenic and thus sensitive to alterations in membrane potential (V(m)). I(-) is then released to the lumen of thyroid follicles where the hormones are synthesised and stored. The mechanisms of I(-) release to follicle lumen are poorly characterised. Our whole-cell voltage clamp recordings revealed the presence of a Ca(2+) activated Cl(-) current (CaCC) in Fisher rat thyroid cell line 5 (FRTL-5). Transcripts of anoctamin 1 (ANO1) and anoctamin 10 (ANO10), putative molecular constituents of CaCC, were detected. The anion channels underlying CaCC are highly permeable to I(-). Both niflumic acid (NFA) and 2-aminoethyl diphenylborinate (2-APB), antagonists of CaCC and transient receptor potential channels, respectively, inhibited CaCC. Canonical transient receptor potential channel 2 (TRPC2) is the only TRPC member present in FRTL-5 cells. The activation rate of CaCC was markedly slower in shTRPC2 knock-down cells, indicating that Ca(2+) entry via TRPC2 contributes to CaCC activation. The uptake of iodide was enhanced and the resting V(m) was more depolarised in TRPC2 knock-down cells. We suggest that the interplay between TRPC2 and ANO1 may have dual effects on iodide transport, modulating I(-) release via ANO channels and I(-) uptake via the V(m) sensitive NIS.

Synthesis and Evaluation of 3,4‐Dihydropyrimidin‐2(1H)‐ones as Sodium Iodide Symporter Inhibitors

The sodium iodide symporter (NIS) is responsible for the accumulation of iodide in the thyroid gland. This transport process is involved in numerous thyroid dysfunctions and is the basis for human contamination in the case of exposure to radioactive iodine species. 4-Aryl-3,4-dihydropyrimidin-2(1H)-ones were recently discovered by high-throughput screening as the first NIS inhibitors. Described herein are the synthesis and evaluation of 115 derivatives with structural modifications at five key positions on the pyrimidone core. This study provides extensive structure-activity relationships for this new class of inhibitors that will serve as a basis for further development of compounds with in vivo efficacy and adequate pharmacokinetic properties. In addition, the SAR investigation provided a more potent compound, which exhibits an IC(50) value of 3.2 nM in a rat thyroid cell line (FRTL5).

FUNCTIONAL EXPRESSION OF TSH RECEPTOR IN THE VENTRICLE OF RATS AND MICE: INVOLVEMENT A REGULATION OF HMGCR BY TSH VIA THE CAMP/PKA/PCREB SIGNALLING PATHWAY

ObjectivesElevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in subclinical hypothyroid, which can lead to and aggravate heart disease if it gets serious. However, there is no...

Let-7a Down-Regulation Plays a Role in Thyroid Neoplasias of Follicular Histotype Affecting Cell Adhesion and Migration through Its Ability to Target theFXYD5(Dysadherin) Gene

Thyroid neoplasias of the follicular histotype include the benign follicular adenomas and the malignant follicular carcinomas. Although several genetic lesions have already been described in human thyroid follicular neoplasias, the mechanisms underlying their development are still far from being completely elucidated. MicroRNAs (miRs or miRNAs) have recently emerged as important regulators of gene expression, also playing a key role in the process of carcinogenesis. The aim of our work has been to identify the miRNAs differentially expressed in human thyroid follicular neoplasias and define their role in thyroid carcinogenesis. The miRNA expression profile of 10 human thyroid follicular adenomas was compared to that of 10 normal thyroid tissues. The miRNA expression profiles revealed the down-regulation of let-7a in thyroid follicular adenomas compared to normal thyroid. Then, quantitative RT-PCR analyses validated the microarray data and showed a significantly higher decrease in let-7a expression in follicular carcinomas. Enforced let-7a expression in the follicular thyroid carcinoma cell line WRO induces an epithelial-like phenotype, increases cell adhesion, and decreases cell migration. Conversely, silencing of let-7a in the normal rat thyroid cell line PC Cl 3 has opposite effects. We identified dysadherin (FXYD5), a cell membrane glycoprotein, correlated with tumor progression and invasiveness, as a target of let-7a. Consistently, an inverse correlation between dysadherin and let-7a expression levels was found in human thyroid follicular adenomas and carcinomas. These results suggest a role of let-7a down-regulation in the development of thyroid neoplasias of the follicular histotype, likely regulating dysadherin protein expression levels.

Abstract 3348: Pax8-PPARδ fusion protein activates β-catenin/TCF pathways

Abstract Pax8-PPARδ Fusion Protein (PPFP) occurs in ∼35% of follicular thyroid carcinomas. Expression of PPFP in the non-transformed rat thyroid cell line PCCL3 conferred on the cells the ability to invade through matrigel and to form colonies in anchorage independent conditions. PPFP also increased the percentage of cells that have activated β-catenin/TCF. We transfected control and PPFP expressing PCCL3 thyroid cell lines with two different β-catenin/TCF-GFP reporter systems. We then sorted a population of GFP positive cells and expanded them to obtain 100% transfected cell lines. These GFP positive cells gave rise to a new heterogenous population with a stable equilibrium between GFP positive, β-catenin/TCF active cells and GFP negative, β-catenin/TCF inactive cells. PPFP expressing cell lines contained more than twice the percentage of GFP positive cells compared to control cell lines. GFP positive PPFP expressing thyroid cells were enriched for cells exhibiting the transformed phenotypes previously observed in the unsorted, untransfected cell lines. Sorted β-catenin/TCF active single cells gave rise to heterogenous clonal populations containing both β-catenin/TCF active and negative cells. Clonal populations derived from β-catenin/TCF inactive cells remained purely negative. Clonal populations derived from GFP positive cells were twice as enriched for anchorage independent colony forming cells than GFP negative clones. More strikingly, GFP positive clones were 5-10 times more invasive than negative clones, which exhibited the same invasive potential as control cells. Treatment with PPARδ full or partial agonists further increased the β-catenin/TCF population in PPFP expressing cells. Again, the effects were confined to populations containing β-catenin/TCF active cells. Co-treatment with PPARδ antagonists reversed the effects of PPARδ agonists. These data suggest that β-catenin/TCF active cells have properties of cancer stem cells and that PPFP acts on this population to expand the stem cell like fraction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3348. doi:1538-7445.AM2012-3348

Effect of early acute high concentrations of iodide exposure on mitochondrial superoxide production in FRTL cells

Excessive oxidative stress has been suggested as one of the underlying mechanisms in the development of thyroid cytotoxicity. Although the involvement of mitochondria has been hypothesized, the effect of early acute high concentrations of iodide on mitochondrial superoxide production remains largely unknown, especially within a 24h time frame. By using a novel fluorescent probe, MitoSOX Red, we demonstrated the concentration response and time-course response of KI-induced mitochondrial superoxide production in the Fischer rat thyroid cell line (FRTL). A strong increase of MitoSOX Red fluorescence intensity in FRTL cells can be seen at 2h following high concentrations of iodide exposure. Besides, we indicated that 6-propyl-2-thiouracil (PTU, 300μM), thyroid-stimulating hormone (TSH, 10mU/ml), and perchlorate (KClO4, 30μM) can inhibit excessive iodide-induced strong mitochondrial superoxide production; however, diethyldithiocarbamic acid (DETC, 2mM) can further increase excessive iodide-induced mitochondrial superoxide production. By using transmission electron microscopy (TEM), we noted accumulated myelinoid bodies with lipid droplets and numerous apoptotic nuclear bodies at 24h in FRTL cells. In addition, we demonstrated a significant decrease in cytochrome c (cyt c) content in the mitochondria by enzyme linked immunosorbent assay (ELISA), and DNA fragments and significant increases in lactate dehydrogenase (LDH) activity were detected. We propose a sequence of events mediated by a strong mitochondrial superoxide production at 2h, followed by lipid peroxidation, cell membrane damage with significant cyt c release, culminating in DNA fragmentation and apoptotic nuclear formation at 24h, which may partly contribute to the underlying mechanisms of early acute iodide excess.

Rethinking the Role of Oncogenes in Papillary Thyroid Cancer Initiation

Rethinking the Role of Oncogenes in Papillary Thyroid Cancer Initiation