NIS Function Research Articles

Reduced or absent NA+/I- symporter expression in papillary thyroid microcarcinomas

<h3>Background</h3> Radioactive iodine (RAI) is a treatment for patients with thyroid carcinoma after thyroidectomy. The Na+/I- symporter (NIS) plays a role in this ability to concentrate iodide within tumor cells. Recent studies have shown a correlation between immunohistochemical staining of NIS and the ability to concentrate iodide mainly in tumors > 1.0cm. Here only papillary thyroid microcarcinomas (PTMC) with or without LN metastasis (LNM) are evaluated to observe NIS expression. <h3>Design</h3> A retrospective study involved 47 cases of PTMC (24 with and 23 without LNM). Immunostains were performed using two different monoclonal anti-NIS antibodies: clone SPM186 from Abeam and clone FP5A from Millipore. <h3>Results</h3> The findings were very similar using two different monoclonal antibodies. One out of 24 cases with LNM displayed focal expression of NIS while two cases showed several NIS-positive cells. None of 23 cases without LNM and none of metastatic tumors showed NIS expression. Ten benign nodules with Hiirthle cell features exhibited moderate membranous stain. <h3>Conclusion</h3> PTMC with or without LNM show minimal to no expression of NIS. NIS expression and function may not be the same in PTMC vs a non-PTMC and RAI therapy may not be effective in treating PTMC due to lack of NIS.

The Na+/I- symporter (NIS): mechanism and medical impact.

The Na(+)/I(-) symporter (NIS) is the plasma membrane glycoprotein that mediates active I(-) transport in the thyroid and other tissues, such as salivary glands, stomach, lactating breast, and small intestine. In the thyroid, NIS-mediated I(-) uptake plays a key role as the first step in the biosynthesis of the thyroid hormones, of which iodine is an essential constituent. These hormones are crucial for the development of the central nervous system and the lungs in the fetus and the newborn and for intermediary metabolism at all ages. Since the cloning of NIS in 1996, NIS research has become a major field of inquiry, with considerable impact on many basic and translational areas. In this article, we review the most recent findings on NIS, I(-) homeostasis, and related topics and place them in historical context. Among many other issues, we discuss the current outlook on iodide deficiency disorders, the present stage of understanding of the structure/function properties of NIS, information gleaned from the characterization of I(-) transport deficiency-causing NIS mutations, insights derived from the newly reported crystal structures of prokaryotic transporters and 3-dimensional homology modeling, and the novel discovery that NIS transports different substrates with different stoichiometries. A review of NIS regulatory mechanisms is provided, including a newly discovered one involving a K(+) channel that is required for NIS function in the thyroid. We also cover current and potential clinical applications of NIS, such as its central role in the treatment of thyroid cancer, its promising use as a reporter gene in imaging and diagnostic procedures, and the latest studies on NIS gene transfer aimed at extending radioiodide treatment to extrathyroidal cancers, including those involving specially engineered NIS molecules.

Systemic Image-Guided Liver Cancer Radiovirotherapy Using Dendrimer-Coated Adenovirus Encoding the Sodium Iodide Symporter as Theranostic Gene

Currently, major limitations for the clinical application of adenovirus-mediated gene therapy are high prevalence of neutralizing antibodies, widespread expression of the coxsackie-adenovirus receptor (CAR), and adenovirus sequestration by the liver. In the current study, we used the sodium iodide symporter (NIS) as a theranostic gene to investigate whether coating of adenovirus with synthetic dendrimers could be useful to overcome these hurdles in order to develop adenoviral vectors for combination of systemic oncolytic virotherapy and NIS-mediated radiotherapy. We coated replication-deficient (Ad5-CMV/NIS) (CMV is cytomegalovirus) and replication-selective (Ad5-E1/AFP-E3/NIS) adenovirus serotype 5 carrying the hNIS gene with poly(amidoamine) dendrimers generation 5 (PAMAM-G5) in order to investigate transduction efficacy and altered tropism of these coated virus particles by (123)I scintigraphy and to evaluate their therapeutic potential for systemic radiovirotherapy in a liver cancer xenograft mouse model. After dendrimer coating, Ad5-CMV/NIS demonstrated partial protection from neutralizing antibodies and enhanced transduction efficacy in CAR-negative cells in vitro. In vivo (123)I scintigraphy of nude mice revealed significantly reduced levels of hepatic transgene expression after intravenous injection of dendrimer-coated Ad5-CMV/NIS (dcAd5-CMV/NIS). Evasion from liver accumulation resulted in significantly reduced liver toxicity and increased transduction efficiency of dcAd5-CMV/NIS in hepatoma xenografts. After PAMAM-G5 coating of the replication-selective Ad5-E1/AFP-E3/NIS, a significantly enhanced oncolytic effect was observed after intravenous application (virotherapy) that was further increased by additional treatment with a therapeutic dose of (131)I (radiovirotherapy) and was associated with markedly improved survival. These results demonstrate efficient liver detargeting and tumor retargeting of adenoviral vectors after coating with synthetic dendrimers, thereby representing a promising innovative strategy for systemic NIS gene therapy. Moreover, our study-based on the function of NIS as a theranostic gene allowing the noninvasive imaging of NIS expression by (123)I scintigraphy-provides detailed characterization of in vivo vector biodistribution and localization, level, and duration of transgene expression, essential prerequisites for exact planning and monitoring of clinical gene therapy trials that aim to individualize the NIS gene therapy concept.

Histone Deacetylase Inhibition Affects Sodium Iodide Symporter Expression and Induces 131I Cytotoxicity in Anaplastic Thyroid Cancer Cells

Anaplastic thyroid cancers (ATCs) represent only 1%-2% of all thyroid tumors, but they account for up to 50% of the mortality. Treatment of differentiated thyroid carcinomas is well standardized and the use of radioiodine represents an essential step; in contrast, there is no standardized therapeutic approach for anaplastic tumors and their prognosis is poor. The resistance of ATC to radioiodine treatment is principally due to the absence of expression of the sodium iodide symporter (NIS), mainly due to epigenetic silencing. The acetylation status of histones is involved in the epigenetic control of gene expression and is usually disrupted in advanced thyroid cancer. Histone deacetylase inhibitors have been demonstrated as potent anticancer drugs with several different effects on cell viability and differentiation. Stabilized ATC cell lines (BHT-101 and CAL-62) and primary cultures from patients who underwent thyroidectomy for ATC were treated with the histone deacetylase inhibitor LBH589. After treatment, we evaluated the expression and function of NIS. Gene expression was evaluated by real-time polymerase chain reaction (RT-PCR), NIS promoter activity was determined with a luciferase reporter assay, and protein expression was assessed through immunofluorescence. We tested the protein function by (125)I uptake and efflux experiments; finally the cytotoxic effect of (131)I was determined with a clonogenic assay. Our results demonstrate that treatment with LBH589 leads to NIS RNA expression as shown by RT-PCR and luciferase assay, and to protein expression as determined by immunofluorescence in vitro and by immunohistochemistry in xenograft tumors. Moreover, (125)I uptake and efflux experiments show the correct protein function and iodine retention, which translate into cytotoxicity effects, as demonstrated by a clonogenic assay with (131)I. This study supplies a new potential strategy for the treatment of ATC by modifying gene expression with the aim of inducing responsiveness towards radioiodine therapy.

Cooperation of histone deacetylase inhibitors SAHA and valproic acid in promoting sodium/iodide symporter expression and function in rat Leydig testicular carcinoma cells

The presence of the sodium/iodide symporter (NIS) is the prerequisite for the use of the radioiodine in the treatment of thyroid cancer. Thus, stimulators of NIS expression and function are currently investigated in cellular models of various human malignancies, also including extrathyroid cancers. In this study, we analyzed the effects of the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), on NIS expression and function in rat Leydig testicular carcinoma cells (LC540). LC540 cells were exposed to SAHA 3 μM and VPA 3 mM (alone and in combination), and cell viability evaluated by MTT assay and cell counting, NIS mRNA and protein levels by using, respectively, real-time RT-PCR and western blotting. NIS function was evaluated by iodide uptake assay. We found that both HDACi were able to stimulate the transcription of NIS gene, but not its protein expression, while the association of SAHA and VPA increased both NIS transcript and protein levels, resulting in significant sixfold enhancement of radioiodine uptake capacity of LC540 cells. These data demonstrate the presence of an epigenetic control of NIS expression in Leydig tumor cells, suggesting the possibility to use the combination of these two HDACi for a radioiodine-based treatment of these malignancies.

Sodium iodide symporter for nuclear molecular imaging and gene therapy: from bedside to bench and back.

Molecular imaging, defined as the visual representation, characterization and quantification of biological processes at the cellular and subcellular levels within intact living organisms, can be obtained by various imaging technologies, including nuclear imaging methods. Imaging of normal thyroid tissue and differentiated thyroid cancer, and treatment of thyroid cancer with radioiodine rely on the expression of the sodium iodide symporter (NIS) in these cells. NIS is an intrinsic membrane protein with 13 transmembrane domains and it takes up iodide into the cytosol from the extracellular fluid. By transferring NIS function to various cells via gene transfer, the cells can be visualized with gamma or positron emitting radioisotopes such as Tc-99m, I-123, I-131, I-124 and F-18 tetrafluoroborate, which are accumulated by NIS. They can also be treated with beta- or alpha-emitting radionuclides, such as I-131, Re-186, Re-188 and At-211, which are also accumulated by NIS. This article demonstrates the diagnostic and therapeutic applications of NIS as a radionuclide-based reporter gene for trafficking cells and a therapeutic gene for treating cancers.

Stimulation of Cultured H9 Human Embryonic Stem Cells with Thyroid Stimulating Hormone Does Not Lead to Formation of Thyroid-Like Cells

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells, and is responsible for the radioactive iodine accumulation. However, treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression, 125I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells, as was previously done for mouse embryonic stem cells. First, we obtained definitive endoderm from human ESCs. Second, we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors, with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency, endoderm and thyroid markers and 125I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood.

Fifteen years' experience in thyroid surgery.

Thyroid disease is common, thyroid cancer is uncommon. Most goitres are investigated using blood tests, fine needle aspiration cytology together with ultrasound. Surgery usually entails either lobectomy or total thyroidectomy, and for malignancy, patients may need a neck dissection. Recently, significant advances have been made regarding mechanisms involved in both thyroid growth and function (goitrogenesis) and carcinogenesis at a molecular level. In the study cohort, 1113 patients had benign disease and 387 malignancy. For benign disease, 716 patients had lobectomy or isthmusectomy, 44 had near-total thyroidectomy and 318 a total thyroidectomy. For malignancy, patients received initial lobectomy (180) or total thyroidectomy (152). One hundred and eleven had completion surgery. Thirty patients had extensive surgery. Thyroid growth and function was investigated using 500 human thyroid cell primary cultures obtained at surgery, as well as in three animal models. The role of pituitary tumour transforming gene (PTTG), PTTG binding factor (PBF) and sodium iodide symporter (NIS) in thyroid cell function was then evaluated. Temporary and permanent recurrent laryngeal nerve palsy rates were 2.4% and 0.4%. Other complications included temporary (21%) and permanent (3%) hypoparathyroidism, wound infection (1.2%), haematoma (1.2%) and poor scar (0.8%). Six patients have died. Regarding thyroid growth and function, TSH represents (either directly or indirectly) the main factor mediating thyroid follicular cell growth. For carcinogenesis, over-expression of the proto-oncogenes PTTG and PBF induces tumours in nude mice, and PTTG can induce proliferation of human thyroid cells and, in addition, both repress expression and function of NIS.

Retinoic acid modulation of thyroid dual oxidase activity in rats and its impact on thyroid iodine organification

The sodium-iodide symporter (NIS) mediates iodide uptake into the thyrocytes, which is important for the diagnosis and therapy of thyroid disorders. Decreased ability to uptake iodide in thyroid carcinomas reduces the efficacy of radioiodine therapy, and retinoic acid (RA) treatment reinduces iodide uptake. The effectiveness of treatment depends not only on iodide uptake but also on the ability of thyrocytes to organify iodine, which is catalyzed by thyroperoxidase (TPO) in the presence of H(2)O(2). Our goal was to determine the influence of RA on thyroid iodide uptake, iodine organification, and TPO and dual oxidase (DuOx) activities. Normal rats were treated with all-trans-RA or 13-cis-RA (100 or 1500 microg/100 g body weight (b.w.), s.c.) for 14 and 28 days. The 2 h thyroid radioiodine content significantly decreased in rats treated with all-trans-RA (100 microg/100 g b.w.) for 14 days. In this group, NIS function and TPO activity were unchanged, whereas DuOx activity was significantly decreased, which might have contributed to the decrease in iodine organification. Both doses of 13-cis-RA for 28 days increased the 15 min thyroid radioiodine uptake, while the 2 h radioiodide uptake increased only in rats treated with the highest dose of 13-cis-RA. While TPO activity did not change, H(2)O(2) generation was increased in this group, and serum thyroxine levels were normal. Since radioiodine half-life in the thyroid gland is important for treatment efficacy, our results highlight the importance of correctly choosing the RA isomer, the time and the dose of treatment, in order to improve the efficacy of radioiodine therapy.

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake ( 125I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell–Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC 50 values were essentially identical to those determined using Na 125I.

Implications of the molecular characterization of the sodium-iodide symporter (NIS).

The recently cloned sodium-iodide symporter (NIS) represents a key molecule for thyroid function by efficiently accumulating iodide from the circulation into the thyrocyte against an electrochemical gradient. This uptake requires energy, is coupled to the action of Na+/K+-ATPase, and stimulated by TSH, the main hormone regulating thyroid-specific functions. NIS mutations are found in congenital hypothyroidism, and potential defects in the NIS gene, its expression, or function of the NIS protein are currently under investigation in various thyroid diseases. Increased NIS expression has been found in autonomous adenoma and Graves' disease, decreased levels of NIS protein and/or mRNA were observed in Hashimoto's disease, cold nodules, most thyroid cancers and cell lines derived therefrom. Autoantibodies directed against NIS have been identified in autoimmune thyroid disease and blocking antibodies isolated from sera of patients with Hashimoto's disease inhibit NIS function in NIS-transfected CHO cells. NIS mRNA expression can be up-regulated by retinoic acid in human thyroid carcinoma cell lines whereas retinoic acid treatment decreases NIS expression and function in differentiated rat thyroid FRTL-5 cells. Apart from thyrocytes, NIS is also expressed in other tissues known to transiently accumulate radioiodide, albeit at much lower levels, requiring RT-PCR for detection of the transcript. Diagnostic and therapeutic implications of the recent cloning of the human NIS gene such as development of NIS-directed drugs, ligands, antibodies, vaccines, gene therapeutic approaches combining NIS targeting and expression together with the long-established, efficient and safe method of radioiodide therapy are discussed both for application to thyroid related diseases and carcinoma, and non-thyroid benign and malignant diseases. Apart from these therapeutic and diagnostic perspectives the availability of the NIS gene will also open new opportunities to develop sensitive and homologous diagnostic test systems to identify factors involved in autoimmune thyroid disease, evolution of goitre, adenoma and thyroid cancer as well as NIS-directed new drugs. Advanced and sophisticated molecular diagnostic approaches (RT-PCR from fine needle aspirations, screening for mutations, analysis of gene defects) are already developed for NIS and will complement or overcome some established procedures in thyroid diagnostics.

Quantitative Molecular Imaging of Viral Therapy for Pancreatic Cancer Using an Engineered Measles Virus Expressing the Sodium-Iodide Symporter Reporter Gene

Our objectives were to, first, determine the oncolytic potential of an engineered measles virus expressing the sodium-iodide symporter gene (MV-NIS) for intratumoral (i.t.) therapy of pancreatic cancer and, second, evaluate NIS as a reporter gene for in vivo monitoring and quantitation of MV-NIS delivery, viral spread, and gene expression in this tumor model. Cultured human pancreatic cancer cells were infected with MV-NIS. Light microscopy, cell viability, and iodide uptake assays were used to confirm viral infection and NIS gene expression and function in vitro. Human pancreatic tumor xenografts were established in mice and infected via i.t. MV-NIS injections. NIS-mediated i.t. iodide uptake was quantitated by (123)I micro-SPECT/CT. i.t. MV-NIS infection was confirmed by immunohistochemistry of excised pancreatic xenografts. The oncolytic efficacy of MV-NIS was determined by measurement of tumor growth and mouse survival. Infection of human pancreatic cancer cell lines with MV-NIS in vitro resulted in syncytia formation, marked iodide uptake, and ultimately cell death. Tumor xenografts infected with MV-NIS concentrated radioiodine, allowing serial quantitative imaging with (123)I micro-SPECT/CT. i.t. MV-NIS therapy of human pancreatic cancer xenografts resulted in a significant reduction in tumor volume and increased survival time of the treated mice compared with the control mice. MV-NIS efficiently infects human pancreatic tumor cells and results in sufficient radioiodine uptake to enable noninvasive serial imaging and quantitation of the intensity, distribution, and time course of NIS gene expression. MV-NIS also shows oncolytic activity in human pancreatic cancer xenografts: Tumor growth is reduced and survival is increased in mice treated with the virus.

Characterization of small-molecule inhibitors of the sodium iodide symporter

The sodium/iodide symporter (NIS) mediates the active transport of iodide from the bloodstream into thyrocytes. NIS function is strategic for the diagnosis and treatment of various thyroid diseases. In addition, a promising anti-cancer strategy based on targeted NIS gene transfer in non-thyroidal cells is currently developed. However, only little information is available concerning the molecular mechanism of NIS-mediated iodide translocation. Ten small molecules have recently been identified using a high-throughput screening method for their inhibitory effect on iodide uptake of NIS-expressing mammalian cells. In the present study, we analyzed these compounds for their rapid and reversible effects on the iodide-induced current in NIS-expressing Xenopus oocytes. Four molecules almost completely inhibited the iodide-induced current; for three of them the effect was irreversible, for one compound the initial current could be fully re-established after washout. Three molecules showed a rapid inhibitory effect of about 75%, half of which was reversible. Another three compounds inhibited the iodide-induced current from 10 to 50%. Some molecules altered the membrane conductance by themselves, i.e. in the absence of iodide. For one of these molecules the observed effect was also found in water-injected oocytes whereas for some others the iodide-independent effect was associated with NIS expression. The tested molecules show a surprisingly high variability in their possible mode of action, and thus are promising tools for further functional characterization of NIS on a molecular level, and they could be useful for medical applications.

Mitogen-Activated Protein Kinase Signaling Enhances Sodium Iodide Symporter Function and Efficacy of Radioiodide Therapy in Nonthyroidal Cancer Cells

Although the success of sodium/iodide symporter (NIS) gene-based cancer therapy is critically dependent on the level of radioiodide accumulation attained, recent evidence indicates that successful therapy relies not solely on NIS amount but also crucially on its functional activity. In this study, we investigated the role of kinase-linked signaling on the regulation of NIS function in cancer cells. T47D human breast cancer and PC-12 rat pheochromocytoma cells were transduced with the human NIS genes via an adenoviral vector. Cells were measured for (125)I uptake, and the effects of activation or inhibition of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathways were evaluated. Membrane localization of NIS was evaluated by biotinylation-immunoblotting and confocal microscopy. (131)I-mediated cancer cell killing was evaluated by clonogenic assays. NIS function was acutely reduced by short stimulation with the PKC activator phorbol 12-myristate 13-acetate and increased by its inhibition with staurosporine or prolonged phorbol 12-myristate 13-acetate exposure. Surprisingly, epidermal growth factor (EGF) caused a strong dose-dependent augmentation of radioiodide transport, accompanied by extracellular signal-regulated kinase (ERK)-1/2 activation. Both effects were completely abrogated by specific MAP kinase kinase (MEK) inhibitors, which also reduced basal NIS function. Hence, radioiodide uptake levels could differ 24-fold, depending on ERK activity. Biotinylation-immunoblotting and confocal microscopy revealed that EGF increases plasma membrane-localized NIS without affecting total cellular levels. EGF stimulation was sufficient to enhance the killing effect of (131)I on the cancer cells. Thus, PKC and ERK signaling play important roles in the regulation of NIS function, and control of these signaling pathways may help enhance the efficacy of radioiodide cancer therapy.

Molecular Characterization of V59E NIS, a Na+/I− Symporter Mutant that Causes Congenital I− Transport Defect

I(-) is actively transported into thyrocytes via the Na+/I(-) symporter (NIS), a key glycoprotein located on the basolateral plasma membrane. The cDNA encoding rat NIS was identified in our laboratory, where an extensive structure/function characterization of NIS is being conducted. Several NIS mutants have been identified as causes of congenital I(-) transport defect (ITD), including V59E NIS. ITD is characterized by low thyroid I(-) uptake, low saliva/plasma I(-) ratio, hypothyroidism, and goiter and may cause mental retardation if untreated. Studies of other ITD-causing NIS mutants have revealed valuable information regarding NIS structure/function. V59E NIS was reported to exhibit as much as 30% of the activity of wild-type NIS. However, this observation was at variance with the patients' phenotype of total lack of activity. We have thoroughly characterized V59E NIS and studied several amino acid substitutions at position 59. We demonstrated that, in contrast to the previous report, V59E NIS is inactive, although it is properly targeted to the plasma membrane. Glu and all other charged amino acids or Pro at position 59 also yielded nonfunctional NIS proteins. However, I(-) uptake was rescued to different degrees by the other substitutions. Although the Km values for Na+ and I(-) were not altered in these active mutants, we found that the structural requirement for NIS function at position 59 is a neutral, helix-promoting amino acid. This result suggests that the region that contains V59 may be involved in intramembrane helix-helix interactions during the transport cycle without being in direct contact with the substrates.

Small‐Molecule Inhibitors of Sodium Iodide Symporter Function

The Na(+)/I(-) symporter (NIS) mediates iodide uptake into thyroid follicular cells. Although NIS has been cloned and thoroughly studied at the molecular level, the biochemical processes involved in post-translational regulation of NIS are still unknown. The purpose of this study was to identify and characterize inhibitors of NIS function. These small organic molecules represent a starting point in the identification of pharmacological tools for the characterization of NIS trafficking and activation mechanisms. The screening of a collection of 17,020 druglike compounds revealed new chemical inhibitors with potencies down to 40 nM. Fluorescence measurement of membrane potential indicates that these inhibitors do not act by disrupting the sodium gradient. They allow immediate and total iodide discharge from preloaded cells in accord with a specific modification of NIS activity, probably through distinct mechanisms.

Abstract 1767: Feasibility of In Vivo Cardiac Stem Cell Tracking, by SPECT and PET Imaging, Using the Sodium-iodide Symporter as Reporter Gene

Background. Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo . Aim. To develop a reporter gene that permits in vivo stem cell labeling. We examined the sodium-iodide symporter (NIS), a protein that is not expressed in the heart, but promotes cellular uptake of 99m Tc or 124 I, thus permitting cell tracking by SPECT or PET imaging, respectively. Methods. The human NIS gene ( h NIS) was expressed in rat cardiac derived stem cells (rCDCs) using lentivirus driven by the CAG or CMV promoter. NIS function in transduced cells was confirmed by in vitro 99m Tc uptake. Eleven rats were injected with 1 or 2 million rCDCs intramyocardially immediately after LAD ligation; 6 with CMV-NIS and 5 with CAG-NIS cells. Dual isotope SPECT imaging was performed on a small animal SPECT/CT system, using 99m Tc for cell detection and 201 Tl for myocardial delineation, 24 hrs after cell injection. PET was performed on a small animal PET scanner using 124 I for cell tracking and 13 NH 3 for myocardial delineation, 48hrs after cell injection. Contrast Ratio (CR) was defined as [(signal in the cells)-(signal in blood pool)]/signal in blood pool. High resolution ex vivo SPECT scans of explanted hearts (n=3) were obtained to confirm that in vivo signal was derived from the cell injection site. The presence of h NIS mRNA was confirmed in injected hearts after animal sacrifice (n=2), by real-time RT-PCR. Results. NIS expression in rCDCs did not affect cell viability/proliferation (p=0.718, ctr vs NIS). In vitro 99m Tc uptake was 6.0±0.9% vs 0.07±0.05, without and with perchlorate (specific NIS blocker), respectively. NIS-transduced rCDCs were easily visualized as spots of 99m Tc or 124 I uptake within a perfusion deficit in the SPECT and PET images. CR was considerably higher when cells were transduced by the CMV-NIS virus in comparison to the CAG-NIS virus (70±40% vs 28±29%, p=0.085). Ex vivo small animal SPECT imaging confirmed that in vivo 99m Tc signals were localized to the injection sites. PCR confirmed the presence of h NIS mRNA in injected hearts. Conclusion. NIS expression allows non invasive in vivo stem cell tracking in the myocardium, using both SPECT and PET. This reporter gene has great potential for translation in future clinical applications.

In vivo Radioiodide Imaging and Treatment of Pancreatic Cancer Xenografts after MUC1 Promoter-Driven Expression of the Human Sodium-Iodide Symporter

Introduction and Aims: Mucin 1 (MUC1) is a transmembrane glycoprotein that is overexpressed in many tumor types, including breast, pancreatic, and ovarian cancer. The aim of this study was to create a construct containing sodium-iodide symporter (NIS) under the control of the 0.8-kb MUC1 promoter to infect pancreatic cancer cells both in vitro and in vivo, to investigate the potential for radioiodide imaging and ablation of this disease. Methodology: We amplified the 797-bp MUC1 promoter by two-step nested PCR. Subsequently, a replication-deficient adenoviral construct was created containing the MUC1 promoter followed by the human NIS gene. Iodide uptake assays and immunofluorescence were used to confirm NIS expression and function. Pancreatic cancer xenografts in mice were infected with Ad/MUC1/NIS and then imaged and treated using radioiodide. Results: A 23- and 15.5-fold increase in iodide uptake was observed in Ad/MUC1/NIS-infected MUC1-positive Capan-2 and SW1990 cells with no significant increase observed in MUC1-negative Hela cells or in cells infected with the control virus. The in vivo study showed a clear image of Ad/MUC1/NIS-infected tumor xenografts using 125I. Administration of a therapeutic dose of 131I resulted in a regression in size to 76 ± 15% of their original volume, whereas control tumors continued to increase in size to >200% of their original volume. Conclusions: These results show that the 0.8-kb MUC1 promoter was successfully used to drive human NIS-targeted expression in pancreatic cancer cells, and Ad/ MUCI/NIS-mediated radiotherapy can make pancreatic cancer xenografts in mice shrinking. This could potentially have applications for both imaging and therapy in other MUC1-positive tumors. Copyright © 2007 S. Karger AG, Basel and IAP

Amino Acid Residues in Transmembrane Segment IX of the Na+/I– Symporter Play a Role in Its Na+ Dependence and Are Critical for Transport Activity

The Na+/I- symporter (NIS) is a key plasma membrane glycoprotein that mediates Na+-dependent active I- transport in the thyroid, lactating breast, and other tissues. The OH group of the side chain at position 354 in transmembrane segment (TMS) IX of NIS has been demonstrated to be essential for NIS function, as revealed by the study of the congenital I- transport defect-causing T354P NIS mutation. TMS IX has the most beta-OH group-containing amino acids (Ser and Thr) of any TMS in NIS. We have thoroughly characterized the functional significance of all Ser and Thr in TMS IX in NIS, as well as of other residues in TMS IX that are highly conserved in other transporters of the SLC5A protein family. Here we show that five beta-OH group-containing residues (Thr-351, Ser-353, Thr-354, Ser-356, and Thr-357) and Asn-360, all of which putatively face the same side of the helix in TMS IX, plus Asp-369, located in the membrane/cytosol interface, play key roles in NIS function and seem to be involved in Na+ binding/translocation.

Expression of the Na+/l-symporter (NIS) is markedly decreased or absent in gastric cancer and intestinal metaplastic mucosa of Barrett esophagus

BackgroundThe sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates iodide (I-) transport in the thyroid, lactating breast, salivary glands, and stomach. Whereas NIS expression and regulation have been extensively investigated in healthy and neoplastic thyroid and breast tissues, little is known about NIS expression and function along the healthy and diseased gastrointestinal tract.MethodsThus, we investigated NIS expression by immunohistochemical analysis in 155 gastrointestinal tissue samples and by immunoblot analysis in 17 gastric tumors from 83 patients.ResultsRegarding the healthy Gl tract, we observed NIS expression exclusively in the basolateral region of the gastric mucin-producing epithelial cells. In gastritis, positive NIS staining was observed in these cells both in the presence and absence of Helicobacter pylori. Significantly, NIS expression was absent in gastric cancer, independently of its histological type. Only focal faint NIS expression was detected in the direct vicinity of gastric tumors, i.e., in the histologically intact mucosa, the expression becoming gradually stronger and linear farther away from the tumor. Barrett mucosa with junctional and fundic-type columnar metaplasia displayed positive NIS staining, whereas Barrett mucosa with intestinal metaplasia was negative. NIS staining was also absent in intestinalized gastric polyps.ConclusionThat NIS expression is markedly decreased or absent in case of intestinalization or malignant transformation of the gastric mucosa suggests that NIS may prove to be a significant tumor marker in the diagnosis and prognosis of gastric malignancies and also precancerous lesions such as Barrett mucosa, thus extending the medical significance of NIS beyond thyroid disease.